Closed State Structure of the Pore Revealed by Uncoupled Shaker K+ Channel

Closed State Structure of the Pore Revealed by Uncoupled Shaker K+ Channel | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article Closed State Structure of the Pore Revealed by Uncoupled Shaker K+ Channel Francisco Bezanilla, Yichen Liu, Carlos Bassetto Jr., Gustavo Contreras, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6406486/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Voltage gated potassium (Kv) channels play key roles in physiological processes, from cellular excitability to immune response and are among the most important pharmaceutical targets 1 . Despite recent advances in the structural determination of Kv channels, the closed state structure of strictly coupled Kv1 family remains elusive. Here, we captured the structure of Shaker potassium channel with a closed pore by uncoupling its voltage sensor domains from the pore domains. Structural determination of the uncoupled I384R mutant by single particle cryoEM revealed a fully closed pore in the presence of activated, non-relaxed voltage sensors. Putative conformational transitions estimated from a fully open pore domain indicates a “roll and turn” movement along the length of the pore-forming S6 helices, in sharp contrast to canonical gating models based on limited movements of S6 2–4 . These rotational and translational movement place two hydrophobic residues, one at inner cavity and the other at the bundle crossing region, directly at the permeation pathway, limiting the pore radius to less than 1 Å. Surprisingly, the selectivity filter was captured in a noncanonical state, partially expanded at G446, unlike previously described dilated 5 or pinched 6 filter conformations. Based on the present data we propose a reinterpretation of the mechanism of activation gating for strictly coupled Kv1 channels and the strictly coupled interactions that underlie different functional states. Biological sciences/Biophysics/Permeation and transport Biological sciences/Neuroscience/Ion channels in the nervous system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Main Structural studies on a variety of Kv channels have provided a great deal of information on the conformational landscape of the channels as a whole. However, the vast majority of the structures correspond to activated or inactivated conformations, since they were obtained in the nominal absence of an electric field. In “strictly coupled” Kv channels, the electromechanical coupling (EMC) between the voltage sensing domain (VSD) and the pore domain (PD) is obligatory: channel opening or closure requires activation or deactivation of the voltage sensors, respectively 7 – 9 . This is in contrast to a typical allosteric coupling, as in BK 10 , HCN 11 or hERG 12 , 13 channels for instance. While this tight coupling ensures a remarkably low “leak” potassium conductance at rest, it has also hindered the structural understanding of the gating mechanisms, since at 0 mV the voltage sensors typically populate the activated (Up) conformation, with the pore domain displaying an open inner bundle gate. As a result, the current gating model of strictly gated Kv channels, such as the Kv1 and Kv2 families, relies primarily on bacteria potassium channel structures 2 and electrophysiological data 3 , 4 . A conserved isoleucine I384 in the S4-S5 linker region was identified as pivotal for the electromechanical coupling in Kv1 family 14 . Here, we report the closed structure of the pore in a strictly coupled Kv1 channel, Shaker K + channel, by disrupting the coupling between the pore and the voltage sensors from mutations at I384. Single channel, ionic current, gating current and fluorescence measurements demonstrated that mutations to the side chain of I384 directly affect the strength of electromechanical coupling. In the most extreme case of I384R, the VSDs become completely uncoupled from the PD: the pore remained closed within a large range of voltages (-120mV to 180mV) while the VSDs activate/deactivate independently. Single-particle cryo-EM structure of the uncoupled channel revealed a collapsed permeation pathway, consistent with a fully closed conformation. Site-directed fluorimetry measurements utilizing a fluorescent unnatural amino acid (UAA) strongly supports our structural observations. Structural rearrangements were also observed in the selectivity filter (SF) and the voltage sensors likely underlying the structural basis for activation-inactivation coupling and hysteresis of the VSDs, respectively. Modifying the canonical “hinge” model, we propose a “roll and turn” gating model for strictly coupled Kv channels and a molecular mechanism of interactions among different conformational states. Conserved isoleucine controls electromechanical coupling in Kv1 family A systematic mutagenesis survey of the S4-S5 linker, a region that has been demonstrated to be important for EMC 15 – 17 , led to the identification of a single residue that is able to tune the coupling strength between VSDs and PD. The conserved isoleucine 384 14 is located at the N-terminus end of the S4-S5 linker and forms elaborate interactions with the intracellular end of the pore-forming S6 helix 18 (Fig. 1 A, B). Replacing I384 with smaller residues such as alanine or cysteine, strengthened the already efficient electromechanical coupling (Fig. 1 C and Extended Data Fig. 1 ). I384C, for instance, activated in more negative voltages with much steeper voltage dependency, as illustrated by its conductance-voltage (GV) curve. More importantly, channel activation closely followed the voltage sensor movement, measured in the gating-charge-voltage (QV) curve (Fig. 1 D). While in wild type (WT) channels the difference between the V 1/2 for QV (measured with the nonconductive W434F mutant 19 and GV curve was > 25mV (Fig. 1 E, F, Extended Data Fig. 1 , and Extended Data Tables 1–2), that difference was less than 7.5mV in I384C, a hallmark for strengthened electromechanical coupling. On the other hand, mutating I384 to glutamate, asparagine or leucine led to severe uncoupling between the VSD and PD (Extended Data Fig. 1 ). This is characterized, in the case of I384L (Fig. 1 G), by large GV curve shifts (> 70mV) to more depolarized potentials, when compared to WT Shaker, together with a shallower slope in the GV curve (Fig. 1 H). Strikingly, while the GV was shifted to the right, the QV curve of I384L was shifted in the opposite direction (Fig. 1 H). Indeed, similar changes in the current kinetics and GV curves were also seen in hKv1.2 and hKv1.3 with mutations at equivalent position (I316 position in hKv1.2 and I386 position in hKv1.3), suggesting that I384’s critical role in the electromechanical coupling is conserved among strictly coupled Kv channels (Extended Data Fig. 2 ). Given the large V 1/2 gap between GV and QV in I384L, gating currents were easily resolved in the presence of ionic currents (Fig. 1 G inset). To confirm that no additional slow component in the VSD movements was present in I384L that is responsible for the opening of the pore, tetramethyl rhodamine (TMR) was introduced at A359C in the extracellular loops of the VSD so that the movement of the voltage sensors would be evaluated via its fluorescence signal 20 – 22 . The fluorescence records showed no additional component in the VSD movement, and the voltage-dependent fluorescence (FV) curve fully overlaps on the QV curve (Fig. 1 I, J). This result demonstrates that our gating current measurement reflects the true movement of VSDs. To confirm that the observed effects were indeed a consequence of impaired coupling rather than due to changes in single channel properties, we performed noise analysis and single channel recordings 23 , 24 in I384L mutant. Noise analysis demonstrated that at 195mV, the maximum opening probability was less than 70% (Extended Data Fig. 3 ) and single channel recordings showed that the unitary conductance level in I384L (Fig. 1 K, ~ 10 pS, Extended Data Tables 3–4) was similar to the WT (~ 12 pS) 24 , but channel flickering was increased (Fig. 1 K and Extended Data Fig. 3 ). While in I384L, the impairment in the coupling was severe, we discovered another mutant that completely uncouples the voltage sensors from the pore, I384R. Despite the presence of K + ions, I384R showed no ionic conduction, and only gating current could be recorded (Fig. 1 L). The gating current itself activated at more hyperpolarized voltages compared to WT (Fig. 1 M) and was kinetically faster compared to the WT with W434F background (Fig. 1 N), as if an energetic load was taken off from the voltage sensors. This is also consistent with left-shifted QV curves seen in other uncoupled mutants such as I384N, I384L and I384E (Extended Data Fig. 1 , and Extended Data Tables 1–2). In I384R, our recordings showed no discernible K + current from − 120mV to + 180mV, demonstrating the exceptional structural stability of the closed pore. Additionally, a left-shifted QV curve allows the VSDs to transition more easily to the up conformation. As a result, at 0mV, I384R possesses a stably closed pore and four activated VSDs with little structural heterogeneity. This represents an appealing target for structural investigation. Closed state structure revealed by a completely uncoupled channel We expressed, purified and solved the structure of Shaker-IR-I384R, by single-particle cryo-EM. The structure was globally resolved to 3.5 Å, with clear densities for all the transmembrane helices (Fig. 2 A, B, Extended Data Figs. 4 and 5 ). Similar to previously determined structures, the uncoupled channel assembled as a domain-swapped homotetramer (Fig. 2 A, B) 18 . However, unlike the strictly coupled WT channel structure, captured with an open pore, the uncoupled channel clearly displayed a collapsed permeation pathway. (Fig. 2 C, D). Compared to the open conformation of Shaker, the pore-forming S6 helices in the closed pore underwent a “roll and turn” movement, where a translational movement brings the backbone of the S6 helices closer together (Fig. 2 C), and a rotational movement places hydrophobic residues I470 and V474 directly into the pore. These form the two narrowest points of the closed pore (Fig. 2 D). Pore radius calculations showed a closed permeation pathway with radius less than ~ 1 Å 25 , leaving ion conduction an impossibility (Fig. 2 E, F). Unexpectedly, the rotating-in of the I470 residue led to a total collapse of the water-filled inner cavity underneath the selectivity filter (Fig. 2 E, F). Previous gating models predict a more limited “hinge-like” movement for the channel activation where a kink is created in the middle of the S6 helices around the conserved PVP motif and the intracellular half of the helices crosses or separates to close or open the channel 26 – 31 . To validate our structural observations, we set out to measure the local conformational changes around the position 470 in the WT channel utilizing a fluorescent unnatural amino acid probe. ANAP is comparable in size to a tryptophan, and its fluorescence changes according to the hydrophobicity of its local environment (Fig. 2 G) 32 , 33 . Utilizing the amber stop codon suppression method 34 , we incorporated ANAP in a site-specific manner at the 470 position and recorded the ionic current and fluorescence signal simultaneously from I470ANAP (Fig. 2 H, I). As channels opened, a fast transient fluorescence change was observed at the start of the depolarizing pulse and a slower one at the beginning of the hyperpolarizing pulse (Fig. 2 I, highlighted with dashed line). The transient nature of the fluorescence signal seems to suggest a rotational movement where the different environments are sampled before reaching its final state, consistent with our structural observations. In the negative voltage range (< -40 mV), where the channel does not open, no fast transient fluorescent signal was observed, demonstrating that the fluorescence signal at I470 is only observed when the channel opens. These results are fully consistent with the idea that the structure of the uncoupled channel most likely represents a true closed state of the pore and suggest interactions between the bundle crossing region and the selectivity filter. A tripartite interaction pocket essential for Electromechanical Coupling Globally, I384R does not cause a kink in the S4-S5 linker or a local movement at the “elbow” region as was seen before in the resting bacteria Nav channels structures 35 . Instead, we see a lateral, translational movement along the whole length of the S4-S5 linker towards the pore (Fig. 3 A). Closer inspection revealed a tripartite interaction pocket among the N-terminus end of the S4-S5 linker, S6 helix in the same subunit and the C-terminus end the S4-S5 linker helix from the adjacent subunit (Fig. 3 B). In the strictly coupled WT structure, I384 was securely lodged in a hydrophobic pocket formed by F484 and Y485 within the same subunit in the S6 helix (Fig. 3 B – gray color). The hydroxyl group of Y485, on the other hand, interacted intimately with R394 and E395 in the S4-S5 linker from the adjacent subunit, establishing a structural coupling between the pore and the S4-S5 linker (Fig. 3 C – gray color). In the uncoupled I384R structure, however, these interactions are all abolished. The full positive charge of the introduced arginine at position 384 forces itself out of the hydrophobic pocket where the side chain swings towards the adjacent S4-S5 linker. This conformational rearrangement pushes the adjacent R394, facing towards the pore previously, away from the S6 helix and allows for the formation of a salt bridge between R384 and E395 (Fig. 3 B, C – red color). These newly formed interactions allows for a closer interaction among the S4-S5 linkers, creating a tight collar around the S5 and S6 segments stabilizing the closed state, and abolish the previous interactions with Y485 in the S6 helices. The observered disruption of the tripartite interactions likely underlies the structural basis for the uncoupling mechanism of I384R, similar to what was shown physiologically elsewhere 36 , 37 . Voltage-sensing domain captured in its activated but not relaxed state Gating current measurements demonstrated that at 0mV, the QV curve of I384R had reached its maximum, suggesting that all the voltage sensors had activated. However, since there are multiple intermediate states for the VSDs, the electrophysiological data cannot unequivocally define whether the voltage sensors reach the fully activated state in the uncoupled channel or even if they move in a similar way as WT channels. To address this, we compared the VSD structures in I384R and the WT (Fig. 3 D). Clear densities were resolved for all gating charges in S4 (R362, R365, R368, R371) 38 , 39 as well as the key residues that form the hydrophobic plug in S2 (I287, F290) 40 – 42 and the countercharge (E283) (Fig. 3 E). The I384R structure shows that all four gating charges have moved pass the hydrophobic plug in the uncoupled conformation and are accessible to the extracellular solution (Fig. 3 F), as seen in the WT structure. This suggests that in the uncoupled channel, voltage sensors move similarly to the WT channels to reach the fully activated state. However, unlike the WT, voltage sensors in I384R do not appear to enter the relaxed state, a conformation that has been observed in Kv, Nav, Cav channels and voltage sensitive phosphatases (VSP) and is driven by prolonged depolarization 43 – 48 . In WT channels with the W434F background, holding the channels at 0 mV for extended periods of time shifts the QV curve almost 20 mV to more negative potentials when compared to holding at -90 mV (Fig. 3 G). This relaxed state was not observed in the uncoupled channel. Holding I384R at 0mV for > 30 s did not cause significant shifts in the QV curve, suggesting that the voltage sensors in I384R did not enter in the relaxed state, at least at this voltage (Fig. 3 H). Since the cryo-EM structures were captured at 0 mV, the physiological evidence would then argue that the structure of VSDs in the open channel represented the relaxed state while in the uncoupled channel, the VSDs likely resided in the non-relaxed state. Structurally speaking, the major difference between VSDs in the WT and I384R lies mostly in the lateral displacement of the S4 and S4-S5 linker alpha helixes. In I384R, the S4-S5 linkers displayed a considerable lateral shift, particularly at the C-terminus end of the helix, 4.1 Å away from the WT structure (Fig. 3 I). This movement was transduced to the N-terminus end of S4-S5 linker and the S4 helixes, dragging them 1.7 Å and 1.4 Å away from the open state structure, respectively. Since entry into the relaxed state has been associated with opening the pore 14 , it is possible that the observed helical displacement in I384 formed the structural basis for the relaxed state of the VSDs. Noncanonical conformation of the selectivity filter and the decreased volume of the closed state channel One surprising observation of the I384R closed state structure comes from the selectivity filter conformation. Instead of the now classical linear coordination of 4 K + ion densities seen in the conductive filter (Fig. 4 B) 18 , 49 , only 2 putative bound K + ions, at the S2 and S4 sites of the SF were resolved (Fig. 4 A). It is intriguing to see that the S3 K + was absent in the structure, since generally, the S3 position typically displays the strongest K + occupancy in K channel structural determinations 50 . In our case, the coulombic density suggests that occupancy was similar for these two positions with a slightly higher occupancy at S4 position (Extended Data Fig. 6). Structurally, two small twists were observed at the S1 and S4 binding site when compared to the WT structure (Fig. 4 C). The carbonyl group of the last glycine in the TVGYG selectivity filter, G446, flipped away from the pore, directly altering the binding site at S1 position (Fig. 4 D). A similar twist was also observed at the bottom of the selectivity filter at the T442 position, which might account for the slight shift of the K + ion at the S4 position compared to the WT. Another intriguing observation in the closed state structure was the decrease of the protein volume in the transmembrane region. When comparing the WT with I384R structures, we noticed that the protein expanded laterally in the open state (Fig. 4 E). This expansion was due to the translational movement of S4-S5 linker and the S6 helices. Area calculations with CHARMM_GUI show an asymmetric increase of the cross-section area of the open channel compared to the closed one (Fig. 4 F) 51 . The most significant expansion happened around the I470 and V474 region, where the cross section of the open channel increased by almost 10% (Extended Data Fig. 7). This expansion in volume might be the underlying mechanism of the reported mechanosensitivity of the Kv1 channels 52 , 53 . Discussion Electromechanical coupling in voltage-gated ion channels and its energetics Most voltage-gated channels share two basic functional modules: the voltage sensing domain and the pore domain. Electromechanical coupling describes the communication between these two modules. In the present study, we identified a tripartite pocket that we argue is essential for electromechanical coupling in the Shaker potassium channel, a strictly coupled channel. Structurally, interactions among Y485, F484, I384, E395 and R394 establish the functional connectivity between the pore and the S4-S5 linker. These intersubunit interactions likely contribute to the cooperativity of the voltage sensors and the pore opening as well 54 . It has been demonstrated previously that reducing the side chain volume at positions Y485 and F484 in the S6 leads to shallower and right-shifted GV curves, 14 , 55 , 56 , similar to what we show here in I384E, I384L and I384N. Mutagenesis experiments and thermodynamic cycle analysis among E395, R394 and Y485 have confirmed their energetic coupling and demonstrated their importance for electromechanical coupling 36 , 37 , 57 . The identified tripartite pocket is thus likely responsible for the efficient transfer of the movement from the voltage sensor to the pore seen in the Kv channels. In strictly coupled voltage-gated channels, the opening of the pore requires the activation of the voltage sensors. Understanding the energetics of this coupling is of fundamental importance to define the nature of EMC. It has long been debated whether it is energetically favorable for the pore to stay in the open or the closed state. In other words, are the voltage sensors doing work to “pull” the channel open or to “push” to keep the pore closed. While some computational work suggests the pore prefers to stay open in the absence of an external energy bias 58 , our results argue otherwise. In the I384 mutants, all the uncoupled mutants (I384L, I384E and I384N) show a right shifted GV curve and a left shifted Q-V curve, an expected behavior if an energetic load to the sensor is decreased. In the partially uncoupled mutants, these results indicate that the pore is now less firmly coupled to the VSD, making it more difficult to open for a given charge movement, preferring to stay in the closed state. In an uncoupled mutant, it is expected that the movement of the voltage sensor is independent of pore opening, therefore the left-shift of the QV curve observed in such mutant would reflect the energy required to open the pore. However, in the case of the uncoupled I384R mutant, the presence of four salt bridges between R384 and E395 introduces additional stabilization of the preopen state, a bias that is not expected to be present in the normal operation of the channel. While the newly formed interactions stabilize the closed state of the pore, they are likely to influence the voltage sensor movements as well, given the intimate connection between the voltage sensors and the S4-S5 linkers. The QV of I384R is shifted leftwards compared to the WT, indicating a lessened energy load, which was estimated to be at 3.41 ± 2.25 kcal/mol using the V median approach 59 . While it is tempting to conclude that this would be the energy for the pore opening, the presence of the aforementioned salt bridges invalidates this assumption. Most likely, the energy estimated has not only contributions of salt bridges, but also all the other energetic components present in the gating process. Kv channel structure captured with a closed pore Among the uncoupling mutants studied, I384R is the most extreme case, where only gating currents are seen. This is quite different from the classical W434F mutant, even though they both have minimal ionic conductance. W434F mutant speeds up the C-type inactivation and stabilized the channel in the inactivated state 60 . Thus, I384R represented an ideal candidate to probe the conformation of the inner bundle gate, and we were able to capture the closed pore structure of Shaker potassium channel by disrupting the electromechanical coupling (Fig. 1 , 2 ). It is worth pointing out that this uncoupled pore most likely represented the closed state, instead of an inactivated state, another nonconductive state in Kv channels. It is the EMC that was altered by substituting isoleucine at position 384, as indicated by the relative shifts in QV and GV curves (Fig. 1 & Extended Data Fig. 1 ). There are informative differences in the behavior of the gating currents measured in mutants W434F and I384R. In I384R, the gating current was significantly faster. This is consistent with severing the electromechanical coupling between PD and VSD, as the sensor is free to move in the absence of a mechanical “load”. In this case we are not tampering with the PD itself and therefore, the structure of the closed pore likely represents the true closed state of the pore in the WT channels. In contrast, gating currents in W434F, are a reflection of the VSD movement under a physiological load. The suppression of ionic currents is derived from effects downstream to the activation gating. A surprising finding from the closed pore structure is the large degree of conformational changes happening along the entire length of the S6 helices. In contrast to the simple hinge-like movement seen in prokaryotes 30 , 61 . Inner gate opening in eukaryotic strictly coupled Kv channels appears to be more complex, with both, a lateral movement of the PVP motif plus an additional rotational movement at the inner cavity of pore. This type of movement leads to the collapse of the inner cavity in the closed state, where hydrophobic I470 rotates and points directly into the permeation pathway. Our fluorescence experiments with ANAP directly demonstrated that a conformational change happens near position 470 as the channels open and close, supporting the structural observation. This is also consistent with early pharmacological studies with tetraethyl ammonium (TEA) ion. TEA exerts its blocking effects by entering the inner cavity of the channel in the open state and needs to be expelled out the channel before the pore closure can happen, a foot in the door effect 62 . Mutating I470 to a smaller residue like cysteine, allows TEA to stay in the inner cavity in the closed state 63 . Conformation of selectivity filter in closed and open state The selectivity filter in the I384R closed state structure was captured in what appears to be a noncanonical conformation. Current SF conformation is different from the typical conductive filter where four bound K + ions orderly occupy the S1-S4 positions. It is also dissimilar to alternative “dilated” inactivated filter where the dilation of the top part of the filter abolish S1 and S2 binding sites (Extended Data Fig. 8A) 5 or the “pinched” inactivated filtered captured in prokaryotic Kv channel (Extended Data Fig. 8B). In I384R, two bound K + ions were found at S2 and S4 position in the absence of any expansion at Y445 position or pinching at G444 6 (Extended Data Fig. 8). It is then possible that as the channel opens, a conformational change around the SF occurs. Fluorescence measurements seem to be in line with this hypothesis; TMR labeled channels at T449 position, around the selectivity filter region, gave rise to a fluorescence change when the channels opened and the FV curve followed very closed the GV curve 20 . In Nav channel, similar phenomenon has been suggested in the fast inactivated state as well 64 . It is possible that the S6 movements during activation are allosterically communicated to the selectivity filter, triggering the transition from the noncanonical conformation to the typical conductive conformation 65 . However, further investigation is certainly required before a definitive conclusion can be reached. Isoleucine 470 as a shared structural element for both activation and inactivation Pore radius calculation identified I470 in the inner cavity as the upper gate in closed state structure. This is a very interesting observation because I470 has been mostly associated with the C-type inactivation of Kv channels, previously. Physiological experiments mutating I470, together with T449, demonstrated that the inactivated state was rendered conductive by the double mutations 66 , and computationally, long-time scale simulation also observed a coincidence of I470 movement and closure of the permeation pathway in the inactivated channels 67 . Moreover, structural results from Kv4.2 identified the role of the equivalent isoleucine in inactivation mechanisms 68 . In the case of Shaker K + channel, the inactivation state is entered after the activation of the channel and the open inactivated state is the most dominant inactivated state in the channel 69 – 71 . It appears that I470 is a shared structural element for both activation and inactivation and could serve as the link for the interplay of these two functional states. Mechanosensitivity of Kv channel gating In the closed state structure, we saw a collapsed inner cavity in the center of the protein which led to a global shrinkage of the protein volume. Compared to the open state structure, the protein occupied less area on the membrane in the closed state. It is worth noting that the open state structure was solved in lipid nanodisc and the current closed state structure was solved in detergents. The difference in structural determination method may result in an underestimation of the actual change in protein volume between closed and open state since it has been reported that reconstitution of proteins into nanodisc could introduce additional lateral forces from the lipid bilayer to the proteins 72 . Therefore, it is possible that the actual cross section area of the channel in the closed state would be smaller and the actual expansion from closed to open would be more significant in a physiological condition. One prediction coming from the observed decrease of the protein volume is that to open the channel, additional energy is required to push against the lateral pressure from the membrane and the amount of the lateral pressure will modulate the gating of the channel, giving rise to some mechanosensitivity to the channel. Some physiological experiments support this prediction. Excised patch clamp experiments have demonstrated that the GV curve of Shaker-IR-WT channels shifted to the left, opening more easily, with increased suction in the pipette 52 , 73 , 74 . Additionally, mechanical modulation on the gating of the channel seems to be mostly limited to the change in the V 1/2 , which mostly reflects changes in steady-state energy level, without altering the number of charges that move. It is possible that the transition from the closed to the open state requires a global expansion of the protein which could serve as the molecular basis of the mechanosensitivity in strictly coupled Kv channels and possible other members of voltage gated channels, such as Nav channels 75 . Activation mechanism for strictly gated Kv channels The present experimental results argue for a new gating model in strictly gated Kv channels (Fig. 5 ). At very hyperpolarizing voltage, the channels occupy the deepest closed state, where all four VSDs are in the down state. In this deep closed state, the permeation pathway is gated by I470 in the inner cavity as well as V474 at the bundle crossing region. Upon depolarization, asynchronous activation of the VSDs moves the channels into an ensemble of intermediate closed states, where some but not all VSDs are activated. The movement of the S4 in the VSDs creates a direct pull on the S4-S5 linker. However, this pull does not provide enough energy to open the channel and the VSDs are stabilized in the non-relaxed state. When all four voltage sensors move up, the channels enter a transient pre-open state, which is represented by the structure of the uncoupled channel. In this pre-open state, all the VSDs populate the activated (up) conformation which provides enough energy into the linkers to drive the pore into its open conformation. As part of a last concerted movement. S4-S5 linkers expand, allowing the opening of the pore and the relaxation of the VSDs. During opening, the S6 undergoes a roll and turn movement that rotates away the upper and lower activation gate, creating a water-filled permeation pathway. This movement is allosterically communicated to the selectivity filter leading to a small local conformation change, allowing for the selective permeation of the K + ions and predisposing itself for C-type inactivation. This allosteric communication forms the structural and molecular basis for interaction among different functional states. Methods Site-directed mutagenesis and cRNA in-vitro synthesis Shaker zH4 K + channel with fast inactivation removed (∆6–46 - Shaker-IR 76 )in pBSTA vector, flanked by β-globin sequences, was used in this study for all the physiological experiments. Point mutations were generated using mismatched mutagenic primers. The PCR product was digested in DpnI to remove the template and was used to transform the XL-gold ultra-competent cells. After ampicillin resistance screening, plasmids were purified from the colonies using standard miniprep protocols. Purified plasmids were sent for either Sanger (Genomics Facility at University of Chicago) or nanopore (Plasmidsaurus) sequencing to confirm the introduction of the point mutation and the absence of off-target mutations. cRNA was then transcribed in vitro from the linearized plasmids (T7 RNA expression kit; Ambion Invitrogen, Thermo Fisher Scientific, Waltham, MA). Xenopus laevis oocyte preparation and channel expression Ovaries of Xenopus laevis were purchased from XENOPUS1 (Dexter, Michigan). The follicular membrane was removed using collagenase type II (Worthington Biochemical Corporation) 2 mg/mL with bovine serum albumin at 1mg/mL (BSA). After defolliculation, stage V–VI oocytes were then selected and microinjected with 5-100 ng cRNA. Injected oocytes were incubated at 18°C for 1–5 days in SOS solution (in mM: 100 NaCl, 5 KCl, 2 CaCl 2 , 0.1 EDTA, and 10 HEPES at pH 7.4) supplemented with 50 µg/mL. Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich. Cut-open voltage clamp on Xenopus laevis oocytes Macroscopic ionic and gating current were recorded using cut-open voltage clamp technique 77 . Micropipettes filled with 3M CsCl or NaCl, with resistance between 0.4 and 0.8 MΩ were used to measure the internal voltage of the oocytes. Current data were online filtered at 20 kHz with a low-pass 4 pole Bessel filter and sampled by a 16-bit A/D (USB-1604; Measurement Computing, Norton, MA) converter at 1 MHz. All experiments were conducted at room temperature (~ 17 ºC). For ionic current experiments, unless otherwise stated, were conducted in external solution consisted of in mM: 12 K methylsulfonate (MES), 108 N-methyl-D-glucamine (NMG) MES, 2 Ca MES, 10 HEPES, 0.1 EDTA, pH = 7.4 and internal solution consisted of in mM: 120 K MES, 10 HEPES, 2 EGTA, pH = 7.4. The capacitive transient was manually compensated with a dedicated circuit and in some cases, further removed by an online P/-4 protocol with a holding voltage of either − 80 or -90 mV 78 . For gating current experiments, all experiments were conducted in external solutions consisted of in mM: 120 NMG MES, 2 Ca MES, 10 HEPES, 0.1 EDTA, pH = 7.4 and in internal solution consisted of in mM: 120 NMG MES, 2 Ca MES, 10 HEPES, 2 EGTA, pH = 7.4. The gating current was recorded with W434F background with the exception of I384R, which by itself produced no discernable ionic current. Single channel recordings and noise analysis Single channel recordings and noise analysis were performed on excised inside-out Xenopus Laevis oocyte patches. Briefly, the cells were injected with 5ng of RNA and maintained at 12 C° the day before the experiment. To remove the vitelline membrane, oocytes were incubated for 5 minutes in a hypertonic solution (SOS solution supplemented with 300 mM sucrose). This procedure shrank the oocyte separating the plasma membrane from the vitelline membrane, making the mechanical removal of the vitelline membrane easier without compromising the integrity of the cell. After removal of the vitelline membrane, oocytes were immediately and gently washed three times with intracellular solution containing in mM: 120KMES, 2EGTA, 10HEPES, pH = 7.40. They were placed in a recording chamber onto an inverted microscope. Current was recorded with an Axopatch 200B patch-clamp amplifier. The pipette solution consisted of, in mM, 120KMES, 2KCl, 2CaMES, 10HEPES, 0.1 EDTA, pH = 7.40. The resistances of the tips were between 13 ~ 17 MΩ for single channel experiments and 6 ~ 10 MΩ for noise analysis experiment. To reduce stray capacitance, the tips of the pipette were covered by Sylgard 184 (Dow Corning Corporation). Current was filtered with a digital 8 pole Bessel filter set at 10kHz (3384 Krohn-Hlite). For noise analysis, we applied hundreds of depolarizing voltage pulses. The ionic currents elicited were averaged to obtain the mean. The variance and the mean were obtained using our Analysis software. For single channels, we recorded several current traces from voltage pulses to + 140mV. The histograms were obtained using Analysis. Unnatural amino acid incorporation and voltage-clamp fluorimetry To incorporate ANAP, we utilized the amber suppression technique. Briefly, the oocytes were injected with a mixture of ANAP-synthetase, ANAP methylester, ANAP-tRNA, Xenopus release factor 1 with D55E mutation and messenger RNA encoding the channel with the amber stop codon introduced at I470 position 32 , 33 , 79 . The oocytes were incubated in the dark for 3 to 5 days prior to recording. The voltage clamp fluorometry setup and the filter set used in the study was similar to what was previously described 21 , 34 . However, instead of using a photo diode, a photomultiplier was used to maximize the sensitivity of the signal, while allowing a decrease of the excitation light, decreasing photobleaching. The fluorophore was excited with a LED at 365nm (Thor lab M365L3). TMR labeling was done similarly as previous described 20 . Briefly, oocytes were incubated in 1mM DTT for 15 minutes. Three washes were administered before transferring the cells to the labeling solution with 20 µM Tetramethylrhodamine-5-Maleimide (Thermo Fisher T6027) in a depolarizing solution (in Mm: 120 KMES, 2 CaMes, 10HEPES, 0.1EDTA, pH = 7.40) for 30 minutes. Data Analysis Ionic current was taken by the steady-state current level and converted to conductance using the following relationship: $$\:G\left(V\right)=\frac{I}{V-{V}_{rev}}\:\left(1\right)$$ where, I is the ionic current in steady state, V is the membrane voltage, and V rev is the reversal potential for the conducting ion. The GV curves were then fitted using a two-state model given by the equation: $$\:G\left(V\right)=\frac{1}{1+\text{exp}\left(-\frac{zF}{RT}\left(V-{V}_{1/2}\right)\:\right)}\:\left(2\right)$$ where z is the apparent charge expressed in units of elementary charge ( \(\:{e}_{0}\) ), V is the voltage and V 1/2 is the voltage of half-maximal conductance. R is the ideal gas constant; T is the temperature in Kelvin and F is the Faraday constant. The gating charge was obtained by integrating the on and off-gating currents. They were plotted against the voltage to obtain the QV curves. For the analysis of the normalized QV curves, we used a two-state model fitting equivalent to the one in Eq. 2 to fit the gating from the I384E, I384L, I384N, and I384R mutants. The equation was the following: $$\:Q\left(V\right)=\frac{1}{1+\text{exp}\left(-\frac{zF}{RT}\left(V-{V}_{1/2}\right)\:\right)}\:\left(3\right)$$ For WT, I384A, I384C, we used a three-state model fitting given by the following equation: $$\:Q\left(V\right)=N\frac{{z}_{2}+\:{z}_{1}\left(1+\left(\text{e}\text{x}\text{p}\frac{{z}_{2}F}{RT}\:\left({V}_{2}-V\right)\right)\right)}{1+\text{e}\text{x}\text{p}\left(\frac{{z}_{2}F}{RT}\:\left({V}_{2}-V\right)\right)\left(1+\text{e}\text{x}\text{p}\left(\frac{{z}_{1}F}{RT}\:\left({V}_{1}-V\right)\right)\right)}\:\left(4\right)$$ where N, z 1 , z 2 , V 1 and V 2 are the number of channels, the charges associated and equilibrium voltages for the first and second transition, respectively. For nonstationary noise analysis, the mean variance data were fitted using a parabolic equation as follows: $$\:{\sigma\:}^{2}=i-\frac{}^{2}}{N}\:\left(5\right),$$ where \(\:{\sigma\:}^{2}\) is the variance, \(\:i\) is the single-channel current, \(\:\) is the mean current, and \(\:N\) is the number of channels in the patch. We can estimate the maximal open probability ( \(\:{Po}_{max}\) ) by knowing the maximum mean current ( \(\:{I}_{max}\) ) using the following equation: $$\:{Po}_{max}=\frac{{}_{max}}{Ni}\:\left(6\right).$$ For single channel analysis data, we obtained all-points histogram binned at 0.05 pA using our Analysis software. The data was then fitted using a mixture of k Gaussian Distributions as follows 80 : $$\:f\left(x\right)=\sum\:_{i=1}^{k}{a}_{i}{f}_{i}\left(y\right)\left(7\right),$$ Where \(\:{a}_{i}\) and \(\:\:{f}_{i}\left(y\right)\) , are, respectively, the relative areas of the components and the Gaussian equation described by: $$\:{f}_{i}\left(y\right)=\frac{1}{{\sigma\:}_{i}\sqrt{2\pi\:}}{e}^{-\frac{{\left({n}_{i}\right)}^{2}}{2}}\:\left(8\right),$$ And $$\:{n}_{i}=\frac{y-{\mu\:}_{i}}{{\sigma\:}_{i}}\:\left(9\right)$$ Where \(\:{\mu\:}_{i}\) and \(\:{\sigma\:}_{i}\) are the mean and the standard deviation of the individual component i. Data is presented as Mean ± SEM. For noise analysis, ionic and gating currents experiments we used at least 3 oocytes from different batches. For single channels, we recorded several oocytes and only for presentation purposes show the representative traces from one cell. Expression and purification of mutant shaker potassium channel Shaker-IR-I384R was subcloned into a modified pEG BacMam vector containing a C-terminal HRV 3C protease site, an eGFP tag and an 8×-His tag utilizing the 5′ NotI and 3′ XbaI restriction sites. The Bacmid plasmid was generated using the Bac-to-Bac system which was then used to transfect the Sf9 insect cells with Cellfectin (Thermo Fisher). After 4 to 7 days of incubation at 27°C, P 0 virus was then collected after removing the remaining cell and debris. P 0 was then amplified to produce P 1 and P 2 virus. 2L of HEK293S GnTI − cells were infected with 200mL of P 2 virus. After 24 h of shaking incubation at 37°C, sodium butyrate was added to the cells at a final concentration of 10mM and the culture was transferred to 30°C. Cells were collected 48 h after infection. The cell pellets were then washed with phosphate-buffered saline (PBS) at pH 7.4, collected by centrifugation, flash-frozen and stored at − 80°C for later purification. All purification steps were conducted at 4°C. Frozen cell pellets were thawed in water bath and homogenized with a Dounce homogenizer in suspension buffer (150mM KCl, 2mM TCEP, 1mM EDTA, 50mM TRIS, Pierce protease inhibitor tablet (Thermo Fisher Scientific) PH 7.5). Resuspended cells culture were supplemented with DDM:CHS (10:1) at 1% final concentration (m. / v.) and were extracted for 2 hours with gentle agitation. The cell debris was pelleted by ultracentrifugation for 1 hr at 40000 rpm in Ti45 rotor (Beckman Coulter). The supernatant was collected and incubated with 2 ml CNBR-activated Sepharose beads (GE Healthcare) coupled with 4 mg high-affinity GFP nanobodies purified in-house for 3 hours. The Sepharose beads were then rinsed three times 10 column volume with the suspension buffer supplemented with 0.1% DDM:CHS (10:1), 0.05% DDM:CHS + 0.02% GDN, 0.02% GDN respectively. Then beads were incubated with HRV 3C protease overnight to release the purified channels. The next day, 3 column volume of suspension buffer was used to elute the beads. Eluted solution was concentrated with Amicon Ultra Centrifugal Filter unit (Millipore) with 100kDa cutoff to approximately 1mL and loaded onto a Superose6 (10 × 300 mm) gel filtration column (GE Healthcare) and separated with suspension buffer supplemented with 0.02% GDN. Cryo-EM sample preparation and data acquisition Samples purified from size-exclusion chromatography were pooled and concentrated to around 0.7 mg/mL measured with a nanodrop machine. 3.5 µL concentrated protein was applied to glow-discharged (30 s, 20 W Solarus Plasma Cleaner) Quantifoil grids (R 1.2/1.3 Au 300 mesh). The grids were blotted for 3s with blot-force 3 in a FEI Vitrobot Mark IV (Thermo Fisher) chamber with 100% humidity at room temperature before plunge froze in liquid ethane. Clipped grids were subsequently loaded onto a Titan Krios microscope. Single-particle movies were acquired using a K3 direct electron detector in super-resolution mode, coupled with a 20 eV GIF energy filter. Data collection was performed at a nominal magnification of ×81,000, corresponding to a pixel size of 0.5315 Å, binned by 2 during acquisition. The total electron dose was calibrated to 60 e−/Ų, distributed across 50 frames per movie. Single-particle cryo-EM analysis All steps for structure determination were performed using CryoSPARC 81 , including motion correction and contrast transfer function (CTF) estimation. A subset of 2,000 particles was initially picked and classified in 2D to generate templates for template-based particle picking. Approximately 4,500,000 initial particles were picked and subjected to multiple rounds of 2D classification. From these, 180,000 particles were selected to generate three ab initio models with C1 symmetry. Particles from the best class (~ 116,000) were then processed for 3D refinement with C4 symmetry, yielding a nominal resolution of 5.7 Å. We performed 3D refinement using non-uniform refinement algorithm 82 , which consistently yields best result in our data. A subsequent 3D classification was performed using the best-refined model from the previous round and a junk model generated ab initio . The best class from this step was further refined using C4 symmetry, resulting in a 3.55 Å nominal resolution. After identifying a subset of ~ 80,000 particles for final refinement, Reference-Based Motion Correction was applied to estimate per-particle movement trajectories and empirical dose weights. A final 3D refinement step was performed, followed by postprocessing using a tighter mask and C4 symmetry enforcement. Local resolution was calculated using CryoSPARC. Validation was done using MOLprobity 83 . Model building and structural refinement The Shaker-IR model (PDB 7SIP) was used as a template to build atomic models for the Shaker-IR-I384R mutants into our density map. All structural models were constructed using unsharpened maps. Initial models were generated through iterative rounds of manual model building in COOT 84 and real-space refinement in Phenix 85 . Final refined atomic models were obtained using interactive flexible fitting in ISOLDE 86 . All structural analyses and figure generation were performed using UCSF ChimeraX 87 , 88 . Declarations Acknowledgements We would like to thank Gethiely Gasparini and Hlafira Polishchuk for oocytes preparation, DNA mutation and RNA preparation. 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UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021). Goddard, T. D. et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018). Additional Declarations There is NO Competing Interest. Supplementary Files ExtendedDataFinal.docx Extended data Cite Share Download PDF Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6406486","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":446448933,"identity":"7515667b-fc72-4bda-88c6-82e1f54ff89e","order_by":0,"name":"Francisco Bezanilla","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYBAC9hlgSkKGj4H5AANjgwRUnA23Fp4bEC08bAxsCSRpYQBq4TEAaoGJ49Mi3Xz4w88dFjxs7Ge+SQAZ8vz9iw8wfCg7jFuLzLEEw94zQIfx5G6TBDIMZ9x4lsA44xxuLfYSOQYJvG0gv+Ruk2Zsk0hguHHGgJm3DY8tEvkfDv4FaeF/8wysRR6k5S9eLTmMzWBbJHLYwFoMzvcYMDPi0yJzzJhZFqzlmbFlb5uE4cYbbAkHe86l49Yi3fz449u2Ojl+/uSHN3621cnLnT988MGPMmucWrAAYAgcIEU9EPCTqmEUjIJRMAqGOwAAQfRPcpNyaVIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-6663-7931","institution":"University of Chicago","correspondingAuthor":true,"prefix":"","firstName":"Francisco","middleName":"","lastName":"Bezanilla","suffix":""},{"id":446448934,"identity":"cb837059-baa1-45df-8cbb-480dd972c2da","order_by":1,"name":"Yichen Liu","email":"","orcid":"https://orcid.org/0000-0003-0774-6932","institution":"University of Chicago","correspondingAuthor":false,"prefix":"","firstName":"Yichen","middleName":"","lastName":"Liu","suffix":""},{"id":446448935,"identity":"0aad2e17-ea06-4f27-bd61-ddaeb254cd00","order_by":2,"name":"Carlos Bassetto Jr.","email":"","orcid":"https://orcid.org/0000-0002-7012-5699","institution":"University of Texas at San Antonio","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"","lastName":"Bassetto","suffix":"Jr."},{"id":446448936,"identity":"f689a152-0fbb-4676-8de9-c5a61e5d5dee","order_by":3,"name":"Gustavo Contreras","email":"","orcid":"","institution":"University of Chicago","correspondingAuthor":false,"prefix":"","firstName":"Gustavo","middleName":"","lastName":"Contreras","suffix":""},{"id":446448937,"identity":"c2a3097f-e8f0-4e90-9274-9c504a823dcf","order_by":4,"name":"Eduardo Perozo","email":"","orcid":"https://orcid.org/0000-0001-7132-2793","institution":"University of Chicago","correspondingAuthor":false,"prefix":"","firstName":"Eduardo","middleName":"","lastName":"Perozo","suffix":""}],"badges":[],"createdAt":"2025-04-08 22:05:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6406486/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6406486/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-65124-0","type":"published","date":"2025-11-19T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82312516,"identity":"3b45a08a-9088-4c87-95c6-be9c4fb7eb39","added_by":"auto","created_at":"2025-05-09 02:21:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":693784,"visible":true,"origin":"","legend":"\u003cp\u003eI384 residue controls the electromechanical coupling in Shaker potassium channel. \u003cstrong\u003eA)\u003c/strong\u003e Overall structure of Shaker potassium channel (PDB:7SIP). Shaker potassium channel is a homotetramer assembled in a domain-swapped manner. Each subunit has two major function domains, the \u003cu\u003ev\u003c/u\u003eoltage \u003cu\u003es\u003c/u\u003eensing \u003cu\u003ed\u003c/u\u003eomain (VSD) and \u003cu\u003ep\u003c/u\u003eore-forming \u003cu\u003ed\u003c/u\u003eomain (PD). Two function domains are connected by a short helix, the S4-S5 linker. \u003cstrong\u003eB)\u003c/strong\u003e Location of coupling controlling residue I384. I384 is surrounded by residues located in the S6 of the contiguous subunit. \u003cstrong\u003eC)\u003c/strong\u003e Representative ionic current traces from I384C. Inset in shows the comparison of the tail current between I384C in black and WT in red hyperpolarizing to the same voltage. Note the difference in time scale. Clearly the deactivation is much slower in I384C. \u003cstrong\u003eD)\u003c/strong\u003e GV and QV curves for I384C. The GV is much sharper and follows much closer the QV curve. \u003cstrong\u003eE)\u003c/strong\u003e Representative traces of WT Shaker channel. \u003cstrong\u003eF)\u003c/strong\u003e GV and QV curves for WT. \u003cstrong\u003eG)\u003c/strong\u003e Representative traces of I384L. The inset highlights the gating current resolved at the beginning of depolarizing pulse. GV and QV curves for I384L. The GV is significantly displaced to the right and is shallower. For \u003cstrong\u003eC)\u003c/strong\u003e, \u003cstrong\u003eE)\u003c/strong\u003e, \u003cstrong\u003eG)\u003c/strong\u003e, prepulse and returning pulse are both -120 mV and ∆V = 10mV. For \u003cstrong\u003eD)\u003c/strong\u003e, \u003cstrong\u003eF)\u003c/strong\u003e, \u003cstrong\u003eH)\u003c/strong\u003e, the data were fitted with a two-state model (details in method section) for easy visualization and calculation of change in ∆V\u003csub\u003e1/2\u003c/sub\u003e. Fitting results could be found in Extended Data Tables 1-2. \u003cstrong\u003eI) \u003c/strong\u003eIonic current and fluorescence traces from TMR-labeled at position A359C in I384L mutant background. Note there is no slow component in the fluorescence traces. \u003cstrong\u003eJ)\u003c/strong\u003e Comparison of GV, QV and FV curves of I384L. The FV curve is essentially overlapping with the QV curve. \u003cstrong\u003eK) \u003c/strong\u003eOne example of single channel currents. The single channel conductance is not significantly altered but flickering behaviors increased.\u0026nbsp; \u003cstrong\u003eL) \u003c/strong\u003eRepresentative traces of I384R. Even in the presence of high K\u003csup\u003e+\u003c/sup\u003e concentration in and outside of the cell no ionic current is detected, and only gating current could be seen. \u003cstrong\u003eM) \u003c/strong\u003eQV curve for I384R. Compared to the WT, the QV is shifted more than 30mV to the left. \u003cstrong\u003eN\u003c/strong\u003e) Time constants of on-gating kinetics of I384R and WT with W434F background. Clearly the gating current in I384 is faster. All the data is shown as Mean ± SEM. N = 3-6 independent experiments.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6406486/v1/9be8c500cd3bc156617b599b.png"},{"id":82312517,"identity":"3b32c3ae-e6ab-4f7e-bc2d-1c7c3f091af8","added_by":"auto","created_at":"2025-05-09 02:21:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":929362,"visible":true,"origin":"","legend":"\u003cp\u003eCryo-EM structure of the completely uncoupled I384R channel, captured in a closed state \u003cstrong\u003eA) \u003c/strong\u003eSide view of I384R structure. The model is shown overlapping with the electron density, with the intracellular T1 domain omitted. Global resolution is 3.5 Å, with clear densities for all the transmembrane helices. \u003cstrong\u003eB) \u003c/strong\u003eIntracellular view of I384R. Like the previous solved structure, the channel adopts a domain swapped arrangement. \u003cstrong\u003eC)\u003c/strong\u003e Structure of the pore in I384R (red) compared to the open state (gray, PDB:7SIP) from the side view. A rotational movement at the upper part of the S6 helices (highlighted by arrows) posits two hydrophobic residues, I470 and V474 from the side direct into the permeation pathway. \u003cstrong\u003eD)\u003c/strong\u003e Intracellular view of the pore in I384R (red) and WT (gray). In addition to the rotational movement at the extracellular end of the S6 helices, a translational movement towards the pore is observed in the closed state structure, further constraining the pore. \u003cstrong\u003eE)\u003c/strong\u003e the radius profile of open state pore from the WT channel. The inner cavity and bundle crossing are both open. Dashed vertical line indicates the approximate size of hydrated K\u003csup\u003e+\u003c/sup\u003e ion. \u003cstrong\u003eF)\u003c/strong\u003e Radius profile of pore in the uncoupled channel. The pore shows two constriction sites, one at I470 in the inner cavity and the other at V474 in the conserved “PVP” motif. The Narrowest point of the pore is less than ~1 Å, indicated by the dashed red line. Clearly the pore is captured in the closed state. \u003cstrong\u003eG)\u003c/strong\u003e ANAP is a fluorescent unnatural amino acid that is sensitive to hydrophobicity of its local environment. With the filter set used in this work, more hydrophobic environment would lead to an increase in fluorescence signal. \u003cstrong\u003eH)\u003c/strong\u003e Representative ionic traces from I470ANAP. ANAP is incorporated at the 470 position in a site-specific manner through amber stop codon suppression. Robust ionic current could be recorded from I470ANAP. \u003cstrong\u003eI)\u003c/strong\u003e Fluorescence signal from I470ANAP. A transient signal could be seen among voltages where the channel opens (above -40 mV). A slightly slower signal is seen at the repolarizing pulse as well (highlighted by red dashed line). At more negative voltages however, no such signal could be resolved. It seems the observed fluorescence signal is associated with the opening and closing of the channel.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6406486/v1/3237353c9dffd03ea349a320.png"},{"id":82312407,"identity":"0ab49e39-844e-4764-92f9-d7fb60dbd450","added_by":"auto","created_at":"2025-05-09 02:13:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":647808,"visible":true,"origin":"","legend":"\u003cp\u003eA tripartite interaction pocket for electromechanical coupling and the structure of fully activated but not relaxed voltage sensors. \u003cstrong\u003eA) \u003c/strong\u003eIntracellular view of the uncoupled structure (red) and coupled structure (gray) with their S4-S5 linkers highlighted in colors. In the uncoupled structure, S4-S5 linkers undergo a translational movement towards the pore, forming a much tighter collar around the pore-forming S6 helices. \u003cstrong\u003eB), C)\u003c/strong\u003e In the coupled structure (gray), Y445 at the bottom of the S6 helix interacts with E395 and R394 in the C-terminus end of the S4-S5 linker from the adjacent subunit. I384 from the same subunit is lodged in a hydrophobic pocket formed by F484 and Y485. In the uncoupled structure (red), however, R384 jumps out of the hydrophobic pocket and forms a direct salt bridge with E395 in the adjacent unit, which also repulses the R394 away from facing the S6 helix, abolishing completely the interactions with Y445. \u003cstrong\u003eD) \u003c/strong\u003eSide view of the VSD of the uncoupled channel (red) and the WT channel (gray). \u003cstrong\u003eE) \u003c/strong\u003eElectron density for the VSD in I384R. All the side chain of the gating charges can be clearly resolved. \u003cstrong\u003eF)\u003c/strong\u003eComparison of the WT and I384R voltage sensors. In both cases, all for gating charges (R362, R365, R368 and R371) have moved passed the hydrophobic plug around I287 and F290 region. Both VSDs are in the fully activated state. \u003cstrong\u003eG), H) \u003c/strong\u003eHysteresis of the VSDs in WT and I384R, respectively. Holding at 0mV for prolonged time (30s) leads to a ~20mV shift of the QV curve to the left in the WT channels. However, in the uncoupled channel I384R, no such dramatic shift was observed. It seems at 0 mV, the VSDs in I384R does not enter into the relaxed state. \u003cstrong\u003eI)\u003c/strong\u003e Helical movements in S4 due to the S4-S5 linker. The tight conformation of the S4-S5 linker shifted the C terminus of the S4-S5 linker for 4.1 Å. This shift is transduced to the N terminus end of the linker and the S4 helix as well, shifting them 1.7 Å and 1.4 Å, respectively.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6406486/v1/d970d924312bff95db65e1eb.png"},{"id":82312409,"identity":"8c36cf00-b495-47fc-bb08-3371cc2d6012","added_by":"auto","created_at":"2025-05-09 02:13:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":698681,"visible":true,"origin":"","legend":"\u003cp\u003eNoncanonical conformation of the selectivity filter and global decrease in protein volume in the closed state. \u003cstrong\u003eA)\u003c/strong\u003e The SF captured in closed state. The atomic model is overlaid with the density map. Clearly there only exist two K\u003csup\u003e+\u003c/sup\u003e in the SF. \u003cstrong\u003eB) \u003c/strong\u003eConductive SF captured in the open state. (PDB:7SIP). \u003cstrong\u003eC)\u003c/strong\u003e Overlay of the SF in the closed (red) and open state (gray). In the closed state, K\u003csup\u003e+\u003c/sup\u003e ions are seen bound at S2 and S4 position. \u003cstrong\u003eD) \u003c/strong\u003eDifference in SF between open and closed state structure. The carbonyl on the backbone of G446 flipped away from the permeation pathway, abolishing the S1 binding site on the top of the selectivity filter. A similar twist is seen at the bottom of the SF at T442 position, resulting in small displacement of the threonine side chain. \u003cstrong\u003eE) \u003c/strong\u003eThe channel is more expanded in the open state (gray) compared to the closed state (red). The expansion can be seen in the VSDs as well as the pore. \u003cstrong\u003eF)\u003c/strong\u003e Cross section area calculation utilizing CHARMM_GUI.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6406486/v1/b16f1d2bcec8e20a629f94a8.png"},{"id":82312966,"identity":"ab73e81c-b75e-4365-b894-4cc88a8f4da6","added_by":"auto","created_at":"2025-05-09 02:29:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":302802,"visible":true,"origin":"","legend":"\u003cp\u003eProposed activation mechanism and interactions with different functional states in Kv1 channels. The progression to activation is from left to right. In the deepest closed state of the channel, all the VSDs are down, and the SF likely resides in the noncanonical state (①). The closed pore is sealed closed by hydrophobic I470 and V474 (①, ②). Upon depolarization, VSDs transit to active but not-relaxed state (③). The upward movement of the VSDs creates a pull on the S4-S5 linker, yet since not all VSDs are up, the energetic input is not enough to open the pore (④). In the last closed state before opening, or the pre-open state, all four VSDs move up (⑤), creating enough pull on the S4-S5 linkers (⑥). In the case of I384R, the channel is most likely stabilized in this pre-open state with newly introduced salt bridges. From the pre-open state, the last concerted movement happens and the S6 helices undergo a roll and turn movement, opening up the permeation path and expanding the channel laterally (⑧). At the same time as the pore opens, the lateral movement of the S4-S5 linker drives the VSDs into a different state, the relaxed state (⑦).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6406486/v1/a1d2e4a588f9c79dc0744a1d.png"},{"id":96354841,"identity":"4c47e210-2fb4-4c1b-9037-8c5cf085d74c","added_by":"auto","created_at":"2025-11-20 08:11:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4051423,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6406486/v1/f86a65af-b9d7-4b25-ad2c-3b4d02b01587.pdf"},{"id":82312412,"identity":"9972178a-70bd-499b-b8e0-6bbdf6f3c9bf","added_by":"auto","created_at":"2025-05-09 02:13:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3931322,"visible":true,"origin":"","legend":"Extended data","description":"","filename":"ExtendedDataFinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-6406486/v1/809a418695f22b360d36be24.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Closed State Structure of the Pore Revealed by Uncoupled Shaker K+ Channel","fulltext":[{"header":"Main","content":"\u003cp\u003eStructural studies on a variety of Kv channels have provided a great deal of information on the conformational landscape of the channels as a whole. However, the vast majority of the structures correspond to activated or inactivated conformations, since they were obtained in the nominal absence of an electric field. In \u0026ldquo;strictly coupled\u0026rdquo; Kv channels, the electromechanical coupling (EMC) between the voltage sensing domain (VSD) and the pore domain (PD) is obligatory: channel opening or closure requires activation or deactivation of the voltage sensors, respectively \u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. This is in contrast to a typical allosteric coupling, as in BK \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, HCN \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e or hERG \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e channels for instance. While this tight coupling ensures a remarkably low \u0026ldquo;leak\u0026rdquo; potassium conductance at rest, it has also hindered the structural understanding of the gating mechanisms, since at 0 mV the voltage sensors typically populate the activated (Up) conformation, with the pore domain displaying an open inner bundle gate. As a result, the current gating model of strictly gated Kv channels, such as the Kv1 and Kv2 families, relies primarily on bacteria potassium channel structures \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and electrophysiological data \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA conserved isoleucine I384 in the S4-S5 linker region was identified as pivotal for the electromechanical coupling in Kv1 family \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Here, we report the closed structure of the pore in a strictly coupled Kv1 channel, Shaker K\u003csup\u003e+\u003c/sup\u003e channel, by disrupting the coupling between the pore and the voltage sensors from mutations at I384. Single channel, ionic current, gating current and fluorescence measurements demonstrated that mutations to the side chain of I384 directly affect the strength of electromechanical coupling. In the most extreme case of I384R, the VSDs become completely uncoupled from the PD: the pore remained closed within a large range of voltages (-120mV to 180mV) while the VSDs activate/deactivate independently. Single-particle cryo-EM structure of the uncoupled channel revealed a collapsed permeation pathway, consistent with a fully closed conformation. Site-directed fluorimetry measurements utilizing a fluorescent unnatural amino acid (UAA) strongly supports our structural observations. Structural rearrangements were also observed in the selectivity filter (SF) and the voltage sensors likely underlying the structural basis for activation-inactivation coupling and hysteresis of the VSDs, respectively. Modifying the canonical \u0026ldquo;hinge\u0026rdquo; model, we propose a \u0026ldquo;roll and turn\u0026rdquo; gating model for strictly coupled Kv channels and a molecular mechanism of interactions among different conformational states.\u003c/p\u003e\n\u003ch3\u003eConserved isoleucine controls electromechanical coupling in Kv1 family\u003c/h3\u003e\n\u003cp\u003eA systematic mutagenesis survey of the S4-S5 linker, a region that has been demonstrated to be important for EMC \u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, led to the identification of a single residue that is able to tune the coupling strength between VSDs and PD. The conserved isoleucine 384 \u003csup\u003e14\u003c/sup\u003e is located at the N-terminus end of the S4-S5 linker and forms elaborate interactions with the intracellular end of the pore-forming S6 helix \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Replacing I384 with smaller residues such as alanine or cysteine, strengthened the already efficient electromechanical coupling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). I384C, for instance, activated in more negative voltages with much steeper voltage dependency, as illustrated by its conductance-voltage (GV) curve. More importantly, channel activation closely followed the voltage sensor movement, measured in the gating-charge-voltage (QV) curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). While in wild type (WT) channels the difference between the V\u003csub\u003e1/2\u003c/sub\u003e for QV (measured with the nonconductive W434F mutant \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and GV curve was \u0026gt;\u0026thinsp;25mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, F, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and Extended Data Tables\u0026nbsp;1\u0026ndash;2), that difference was less than 7.5mV in I384C, a hallmark for strengthened electromechanical coupling. On the other hand, mutating I384 to glutamate, asparagine or leucine led to severe uncoupling between the VSD and PD (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This is characterized, in the case of I384L (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), by large GV curve shifts (\u0026gt;\u0026thinsp;70mV) to more depolarized potentials, when compared to WT Shaker, together with a shallower slope in the GV curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Strikingly, while the GV was shifted to the right, the QV curve of I384L was shifted in the opposite direction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Indeed, similar changes in the current kinetics and GV curves were also seen in hKv1.2 and hKv1.3 with mutations at equivalent position (I316 position in hKv1.2 and I386 position in hKv1.3), suggesting that I384\u0026rsquo;s critical role in the electromechanical coupling is conserved among strictly coupled Kv channels (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the large V\u003csub\u003e1/2\u003c/sub\u003e gap between GV and QV in I384L, gating currents were easily resolved in the presence of ionic currents (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG inset). To confirm that no additional slow component in the VSD movements was present in I384L that is responsible for the opening of the pore, tetramethyl rhodamine (TMR) was introduced at A359C in the extracellular loops of the VSD so that the movement of the voltage sensors would be evaluated via its fluorescence signal \u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The fluorescence records showed no additional component in the VSD movement, and the voltage-dependent fluorescence (FV) curve fully overlaps on the QV curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, J). This result demonstrates that our gating current measurement reflects the true movement of VSDs. To confirm that the observed effects were indeed a consequence of impaired coupling rather than due to changes in single channel properties, we performed noise analysis and single channel recordings \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e in I384L mutant. Noise analysis demonstrated that at 195mV, the maximum opening probability was less than 70% (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and single channel recordings showed that the unitary conductance level in I384L (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, ~\u0026thinsp;10 pS, Extended Data Tables\u0026nbsp;3\u0026ndash;4) was similar to the WT (~\u0026thinsp;12 pS) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, but channel flickering was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhile in I384L, the impairment in the coupling was severe, we discovered another mutant that completely uncouples the voltage sensors from the pore, I384R. Despite the presence of K\u003csup\u003e+\u003c/sup\u003e ions, I384R showed no ionic conduction, and only gating current could be recorded (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). The gating current itself activated at more hyperpolarized voltages compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM) and was kinetically faster compared to the WT with W434F background (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN), as if an energetic load was taken off from the voltage sensors. This is also consistent with left-shifted QV curves seen in other uncoupled mutants such as I384N, I384L and I384E (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and Extended Data Tables\u0026nbsp;1\u0026ndash;2). In I384R, our recordings showed no discernible K\u003csup\u003e+\u003c/sup\u003e current from \u0026minus;\u0026thinsp;120mV to +\u0026thinsp;180mV, demonstrating the exceptional structural stability of the closed pore. Additionally, a left-shifted QV curve allows the VSDs to transition more easily to the up conformation. As a result, at 0mV, I384R possesses a stably closed pore and four activated VSDs with little structural heterogeneity. This represents an appealing target for structural investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClosed state structure revealed by a completely uncoupled channel\u003c/h2\u003e \u003cp\u003eWe expressed, purified and solved the structure of Shaker-IR-I384R, by single-particle cryo-EM. The structure was globally resolved to 3.5 \u0026Aring;, with clear densities for all the transmembrane helices (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B, Extended Data Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Similar to previously determined structures, the uncoupled channel assembled as a domain-swapped homotetramer (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B) \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, unlike the strictly coupled WT channel structure, captured with an open pore, the uncoupled channel clearly displayed a collapsed permeation pathway. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Compared to the open conformation of Shaker, the pore-forming S6 helices in the closed pore underwent a \u0026ldquo;roll and turn\u0026rdquo; movement, where a translational movement brings the backbone of the S6 helices closer together (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and a rotational movement places hydrophobic residues I470 and V474 directly into the pore. These form the two narrowest points of the closed pore (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Pore radius calculations showed a closed permeation pathway with radius less than ~\u0026thinsp;1 \u0026Aring; \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, leaving ion conduction an impossibility (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). Unexpectedly, the rotating-in of the I470 residue led to a total collapse of the water-filled inner cavity underneath the selectivity filter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). Previous gating models predict a more limited \u0026ldquo;hinge-like\u0026rdquo; movement for the channel activation where a kink is created in the middle of the S6 helices around the conserved PVP motif and the intracellular half of the helices crosses or separates to close or open the channel \u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo validate our structural observations, we set out to measure the local conformational changes around the position 470 in the WT channel utilizing a fluorescent unnatural amino acid probe. ANAP is comparable in size to a tryptophan, and its fluorescence changes according to the hydrophobicity of its local environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG) \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Utilizing the amber stop codon suppression method \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, we incorporated ANAP in a site-specific manner at the 470 position and recorded the ionic current and fluorescence signal simultaneously from I470ANAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, I). As channels opened, a fast transient fluorescence change was observed at the start of the depolarizing pulse and a slower one at the beginning of the hyperpolarizing pulse (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, highlighted with dashed line). The transient nature of the fluorescence signal seems to suggest a rotational movement where the different environments are sampled before reaching its final state, consistent with our structural observations. In the negative voltage range (\u0026lt; -40 mV), where the channel does not open, no fast transient fluorescent signal was observed, demonstrating that the fluorescence signal at I470 is only observed when the channel opens. These results are fully consistent with the idea that the structure of the uncoupled channel most likely represents a true closed state of the pore and suggest interactions between the bundle crossing region and the selectivity filter.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eA tripartite interaction pocket essential for Electromechanical Coupling\u003c/h3\u003e\n\u003cp\u003eGlobally, I384R does not cause a kink in the S4-S5 linker or a local movement at the \u0026ldquo;elbow\u0026rdquo; region as was seen before in the resting bacteria Nav channels structures \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Instead, we see a lateral, translational movement along the whole length of the S4-S5 linker towards the pore (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Closer inspection revealed a tripartite interaction pocket among the N-terminus end of the S4-S5 linker, S6 helix in the same subunit and the C-terminus end the S4-S5 linker helix from the adjacent subunit (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In the strictly coupled WT structure, I384 was securely lodged in a hydrophobic pocket formed by F484 and Y485 within the same subunit in the S6 helix (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB \u0026ndash; gray color). The hydroxyl group of Y485, on the other hand, interacted intimately with R394 and E395 in the S4-S5 linker from the adjacent subunit, establishing a structural coupling between the pore and the S4-S5 linker (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC \u0026ndash; gray color). In the uncoupled I384R structure, however, these interactions are all abolished. The full positive charge of the introduced arginine at position 384 forces itself out of the hydrophobic pocket where the side chain swings towards the adjacent S4-S5 linker. This conformational rearrangement pushes the adjacent R394, facing towards the pore previously, away from the S6 helix and allows for the formation of a salt bridge between R384 and E395 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C \u0026ndash; red color). These newly formed interactions allows for a closer interaction among the S4-S5 linkers, creating a tight collar around the S5 and S6 segments stabilizing the closed state, and abolish the previous interactions with Y485 in the S6 helices. The observered disruption of the tripartite interactions likely underlies the structural basis for the uncoupling mechanism of I384R, similar to what was shown physiologically elsewhere \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eVoltage-sensing domain captured in its activated but not relaxed state\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e \u003cp\u003eGating current measurements demonstrated that at 0mV, the QV curve of I384R had reached its maximum, suggesting that all the voltage sensors had activated. However, since there are multiple intermediate states for the VSDs, the electrophysiological data cannot unequivocally define whether the voltage sensors reach the fully activated state in the uncoupled channel or even if they move in a similar way as WT channels. To address this, we compared the VSD structures in I384R and the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Clear densities were resolved for all gating charges in S4 (R362, R365, R368, R371) \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e as well as the key residues that form the hydrophobic plug in S2 (I287, F290) \u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and the countercharge (E283) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The I384R structure shows that all four gating charges have moved pass the hydrophobic plug in the uncoupled conformation and are accessible to the extracellular solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), as seen in the WT structure. This suggests that in the uncoupled channel, voltage sensors move similarly to the WT channels to reach the fully activated state.\u003c/p\u003e \u003cp\u003eHowever, unlike the WT, voltage sensors in I384R do not appear to enter the relaxed state, a conformation that has been observed in Kv, Nav, Cav channels and voltage sensitive phosphatases (VSP) and is driven by prolonged depolarization \u003csup\u003e\u003cspan additionalcitationids=\"CR44 CR45 CR46 CR47\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In WT channels with the W434F background, holding the channels at 0 mV for extended periods of time shifts the QV curve almost 20 mV to more negative potentials when compared to holding at -90 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). This relaxed state was not observed in the uncoupled channel. Holding I384R at 0mV for \u0026gt;\u0026thinsp;30 s did not cause significant shifts in the QV curve, suggesting that the voltage sensors in I384R did not enter in the relaxed state, at least at this voltage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Since the cryo-EM structures were captured at 0 mV, the physiological evidence would then argue that the structure of VSDs in the open channel represented the relaxed state while in the uncoupled channel, the VSDs likely resided in the non-relaxed state. Structurally speaking, the major difference between VSDs in the WT and I384R lies mostly in the lateral displacement of the S4 and S4-S5 linker alpha helixes. In I384R, the S4-S5 linkers displayed a considerable lateral shift, particularly at the C-terminus end of the helix, 4.1 \u0026Aring; away from the WT structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). This movement was transduced to the N-terminus end of S4-S5 linker and the S4 helixes, dragging them 1.7 \u0026Aring; and 1.4 \u0026Aring; away from the open state structure, respectively. Since entry into the relaxed state has been associated with opening the pore \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, it is possible that the observed helical displacement in I384 formed the structural basis for the relaxed state of the VSDs.\u003c/p\u003e\n\u003ch3\u003eNoncanonical conformation of the selectivity filter and the decreased volume of the closed state channel\u003c/h3\u003e\n\u003cp\u003eOne surprising observation of the I384R closed state structure comes from the selectivity filter conformation. Instead of the now classical linear coordination of 4 K\u003csup\u003e+\u003c/sup\u003e ion densities seen in the conductive filter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, only 2 putative bound K\u003csup\u003e+\u003c/sup\u003e ions, at the S2 and S4 sites of the SF were resolved (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). It is intriguing to see that the S3 K\u003csup\u003e+\u003c/sup\u003e was absent in the structure, since generally, the S3 position typically displays the strongest K\u003csup\u003e+\u003c/sup\u003e occupancy in K channel structural determinations \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. In our case, the coulombic density suggests that occupancy was similar for these two positions with a slightly higher occupancy at S4 position (Extended Data Fig.\u0026nbsp;6). Structurally, two small twists were observed at the S1 and S4 binding site when compared to the WT structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The carbonyl group of the last glycine in the TVGYG selectivity filter, G446, flipped away from the pore, directly altering the binding site at S1 position (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). A similar twist was also observed at the bottom of the selectivity filter at the T442 position, which might account for the slight shift of the K\u003csup\u003e+\u003c/sup\u003e ion at the S4 position compared to the WT.\u003c/p\u003e \u003cp\u003eAnother intriguing observation in the closed state structure was the decrease of the protein volume in the transmembrane region. When comparing the WT with I384R structures, we noticed that the protein expanded laterally in the open state (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). This expansion was due to the translational movement of S4-S5 linker and the S6 helices. Area calculations with CHARMM_GUI show an asymmetric increase of the cross-section area of the open channel compared to the closed one (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. The most significant expansion happened around the I470 and V474 region, where the cross section of the open channel increased by almost 10% (Extended Data Fig.\u0026nbsp;7). This expansion in volume might be the underlying mechanism of the reported mechanosensitivity of the Kv1 channels \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eElectromechanical coupling in voltage-gated ion channels and its energetics\u003c/h2\u003e \u003cp\u003eMost voltage-gated channels share two basic functional modules: the voltage sensing domain and the pore domain. Electromechanical coupling describes the communication between these two modules. In the present study, we identified a tripartite pocket that we argue is essential for electromechanical coupling in the Shaker potassium channel, a strictly coupled channel. Structurally, interactions among Y485, F484, I384, E395 and R394 establish the functional connectivity between the pore and the S4-S5 linker. These intersubunit interactions likely contribute to the cooperativity of the voltage sensors and the pore opening as well \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. It has been demonstrated previously that reducing the side chain volume at positions Y485 and F484 in the S6 leads to shallower and right-shifted GV curves, \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, similar to what we show here in I384E, I384L and I384N. Mutagenesis experiments and thermodynamic cycle analysis among E395, R394 and Y485 have confirmed their energetic coupling and demonstrated their importance for electromechanical coupling \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The identified tripartite pocket is thus likely responsible for the efficient transfer of the movement from the voltage sensor to the pore seen in the Kv channels.\u003c/p\u003e \u003cp\u003eIn strictly coupled voltage-gated channels, the opening of the pore requires the activation of the voltage sensors. Understanding the energetics of this coupling is of fundamental importance to define the nature of EMC. It has long been debated whether it is energetically favorable for the pore to stay in the open or the closed state. In other words, are the voltage sensors doing work to “pull” the channel open or to “push” to keep the pore closed. While some computational work suggests the pore prefers to stay open in the absence of an external energy bias \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, our results argue otherwise. In the I384 mutants, all the uncoupled mutants (I384L, I384E and I384N) show a right shifted GV curve and a left shifted Q-V curve, an expected behavior if an energetic load to the sensor is decreased. In the partially uncoupled mutants, these results indicate that the pore is now less firmly coupled to the VSD, making it more difficult to open for a given charge movement, preferring to stay in the closed state.\u003c/p\u003e \u003cp\u003eIn an uncoupled mutant, it is expected that the movement of the voltage sensor is independent of pore opening, therefore the left-shift of the QV curve observed in such mutant would reflect the energy required to open the pore. However, in the case of the uncoupled I384R mutant, the presence of four salt bridges between R384 and E395 introduces additional stabilization of the preopen state, a bias that is not expected to be present in the normal operation of the channel. While the newly formed interactions stabilize the closed state of the pore, they are likely to influence the voltage sensor movements as well, given the intimate connection between the voltage sensors and the S4-S5 linkers. The QV of I384R is shifted leftwards compared to the WT, indicating a lessened energy load, which was estimated to be at 3.41 ± 2.25 kcal/mol using the V\u003csub\u003emedian\u003c/sub\u003e approach \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. While it is tempting to conclude that this would be the energy for the pore opening, the presence of the aforementioned salt bridges invalidates this assumption. Most likely, the energy estimated has not only contributions of salt bridges, but also all the other energetic components present in the gating process.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eKv channel structure captured with a closed pore\u003c/h3\u003e\n\u003cp\u003eAmong the uncoupling mutants studied, I384R is the most extreme case, where only gating currents are seen. This is quite different from the classical W434F mutant, even though they both have minimal ionic conductance. W434F mutant speeds up the C-type inactivation and stabilized the channel in the inactivated state \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Thus, I384R represented an ideal candidate to probe the conformation of the inner bundle gate, and we were able to capture the closed pore structure of Shaker potassium channel by disrupting the electromechanical coupling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It is worth pointing out that this uncoupled pore most likely represented the closed state, instead of an inactivated state, another nonconductive state in Kv channels. It is the EMC that was altered by substituting isoleucine at position 384, as indicated by the relative shifts in QV and GV curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026amp; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). There are informative differences in the behavior of the gating currents measured in mutants W434F and I384R. In I384R, the gating current was significantly faster. This is consistent with severing the electromechanical coupling between PD and VSD, as the sensor is free to move in the absence of a mechanical “load”. In this case we are not tampering with the PD itself and therefore, the structure of the closed pore likely represents the true closed state of the pore in the WT channels. In contrast, gating currents in W434F, are a reflection of the VSD movement under a physiological load. The suppression of ionic currents is derived from effects downstream to the activation gating.\u003c/p\u003e \u003cp\u003eA surprising finding from the closed pore structure is the large degree of conformational changes happening along the entire length of the S6 helices. In contrast to the simple hinge-like movement seen in prokaryotes \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Inner gate opening in eukaryotic strictly coupled Kv channels appears to be more complex, with both, a lateral movement of the PVP motif plus an additional rotational movement at the inner cavity of pore. This type of movement leads to the collapse of the inner cavity in the closed state, where hydrophobic I470 rotates and points directly into the permeation pathway. Our fluorescence experiments with ANAP directly demonstrated that a conformational change happens near position 470 as the channels open and close, supporting the structural observation. This is also consistent with early pharmacological studies with tetraethyl ammonium (TEA) ion. TEA exerts its blocking effects by entering the inner cavity of the channel in the open state and needs to be expelled out the channel before the pore closure can happen, a foot in the door effect \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Mutating I470 to a smaller residue like cysteine, allows TEA to stay in the inner cavity in the closed state \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eConformation of selectivity filter in closed and open state\u003c/h3\u003e\n\u003cp\u003eThe selectivity filter in the I384R closed state structure was captured in what appears to be a noncanonical conformation. Current SF conformation is different from the typical conductive filter where four bound K\u003csup\u003e+\u003c/sup\u003e ions orderly occupy the S1-S4 positions. It is also dissimilar to alternative “dilated” inactivated filter where the dilation of the top part of the filter abolish S1 and S2 binding sites (Extended Data Fig.\u0026nbsp;8A) \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e or the “pinched” inactivated filtered captured in prokaryotic Kv channel (Extended Data Fig.\u0026nbsp;8B). In I384R, two bound K\u003csup\u003e+\u003c/sup\u003e ions were found at S2 and S4 position in the absence of any expansion at Y445 position or pinching at G444 \u003csup\u003e6\u003c/sup\u003e (Extended Data Fig.\u0026nbsp;8). It is then possible that as the channel opens, a conformational change around the SF occurs. Fluorescence measurements seem to be in line with this hypothesis; TMR labeled channels at T449 position, around the selectivity filter region, gave rise to a fluorescence change when the channels opened and the FV curve followed very closed the GV curve \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In Nav channel, similar phenomenon has been suggested in the fast inactivated state as well \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. It is possible that the S6 movements during activation are allosterically communicated to the selectivity filter, triggering the transition from the noncanonical conformation to the typical conductive conformation \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. However, further investigation is certainly required before a definitive conclusion can be reached.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIsoleucine 470 as a shared structural element for both activation and inactivation\u003c/h2\u003e \u003cp\u003ePore radius calculation identified I470 in the inner cavity as the upper gate in closed state structure. This is a very interesting observation because I470 has been mostly associated with the C-type inactivation of Kv channels, previously. Physiological experiments mutating I470, together with T449, demonstrated that the inactivated state was rendered conductive by the double mutations \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e, and computationally, long-time scale simulation also observed a coincidence of I470 movement and closure of the permeation pathway in the inactivated channels \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Moreover, structural results from Kv4.2 identified the role of the equivalent isoleucine in inactivation mechanisms \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. In the case of Shaker K\u003csup\u003e+\u003c/sup\u003e channel, the inactivation state is entered after the activation of the channel and the open inactivated state is the most dominant inactivated state in the channel \u003csup\u003e\u003cspan additionalcitationids=\"CR70\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e–\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. It appears that I470 is a shared structural element for both activation and inactivation and could serve as the link for the interplay of these two functional states.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMechanosensitivity of Kv channel gating\u003c/h2\u003e \u003cp\u003eIn the closed state structure, we saw a collapsed inner cavity in the center of the protein which led to a global shrinkage of the protein volume. Compared to the open state structure, the protein occupied less area on the membrane in the closed state. It is worth noting that the open state structure was solved in lipid nanodisc and the current closed state structure was solved in detergents. The difference in structural determination method may result in an underestimation of the actual change in protein volume between closed and open state since it has been reported that reconstitution of proteins into nanodisc could introduce additional lateral forces from the lipid bilayer to the proteins \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Therefore, it is possible that the actual cross section area of the channel in the closed state would be smaller and the actual expansion from closed to open would be more significant in a physiological condition.\u003c/p\u003e \u003cp\u003eOne prediction coming from the observed decrease of the protein volume is that to open the channel, additional energy is required to push against the lateral pressure from the membrane and the amount of the lateral pressure will modulate the gating of the channel, giving rise to some mechanosensitivity to the channel. Some physiological experiments support this prediction. Excised patch clamp experiments have demonstrated that the GV curve of Shaker-IR-WT channels shifted to the left, opening more easily, with increased suction in the pipette \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. Additionally, mechanical modulation on the gating of the channel seems to be mostly limited to the change in the V\u003csub\u003e1/2\u003c/sub\u003e, which mostly reflects changes in steady-state energy level, without altering the number of charges that move. It is possible that the transition from the closed to the open state requires a global expansion of the protein which could serve as the molecular basis of the mechanosensitivity in strictly coupled Kv channels and possible other members of voltage gated channels, such as Nav channels \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eActivation mechanism for strictly gated Kv channels\u003c/h2\u003e \u003cp\u003eThe present experimental results argue for a new gating model in strictly gated Kv channels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). At very hyperpolarizing voltage, the channels occupy the deepest closed state, where all four VSDs are in the down state. In this deep closed state, the permeation pathway is gated by I470 in the inner cavity as well as V474 at the bundle crossing region. Upon depolarization, asynchronous activation of the VSDs moves the channels into an ensemble of intermediate closed states, where some but not all VSDs are activated. The movement of the S4 in the VSDs creates a direct pull on the S4-S5 linker. However, this pull does not provide enough energy to open the channel and the VSDs are stabilized in the non-relaxed state. When all four voltage sensors move up, the channels enter a transient pre-open state, which is represented by the structure of the uncoupled channel. In this pre-open state, all the VSDs populate the activated (up) conformation which provides enough energy into the linkers to drive the pore into its open conformation. As part of a last concerted movement. S4-S5 linkers expand, allowing the opening of the pore and the relaxation of the VSDs. During opening, the S6 undergoes a roll and turn movement that rotates away the upper and lower activation gate, creating a water-filled permeation pathway. This movement is allosterically communicated to the selectivity filter leading to a small local conformation change, allowing for the selective permeation of the K\u003csup\u003e+\u003c/sup\u003e ions and predisposing itself for C-type inactivation. This allosteric communication forms the structural and molecular basis for interaction among different functional states.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e "},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eSite-directed mutagenesis and cRNA\u003c/b\u003e \u003cb\u003ein-vitro\u003c/b\u003e \u003cb\u003esynthesis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eShaker zH4 K + channel with fast inactivation removed (∆6–46 - Shaker-IR \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e)in pBSTA vector, flanked by β-globin sequences, was used in this study for all the physiological experiments. Point mutations were generated using mismatched mutagenic primers. The PCR product was digested in DpnI to remove the template and was used to transform the XL-gold ultra-competent cells. After ampicillin resistance screening, plasmids were purified from the colonies using standard miniprep protocols. Purified plasmids were sent for either Sanger (Genomics Facility at University of Chicago) or nanopore (Plasmidsaurus) sequencing to confirm the introduction of the point mutation and the absence of off-target mutations. cRNA was then transcribed \u003cem\u003ein vitro\u003c/em\u003e from the linearized plasmids (T7 RNA expression kit; Ambion Invitrogen, Thermo Fisher Scientific, Waltham, MA).\u003c/p\u003e\u003cp\u003e \u003cb\u003eXenopus laevis\u003c/b\u003e \u003cb\u003eoocyte preparation and channel expression\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOvaries of \u003cem\u003eXenopus laevis\u003c/em\u003e were purchased from XENOPUS1 (Dexter, Michigan). The follicular membrane was removed using collagenase type II (Worthington Biochemical Corporation) 2 mg/mL with bovine serum albumin at 1mg/mL (BSA). After defolliculation, stage V–VI oocytes were then selected and microinjected with 5-100 ng cRNA. Injected oocytes were incubated at 18°C for 1–5 days in SOS solution (in mM: 100 NaCl, 5 KCl, 2 CaCl\u003csub\u003e2\u003c/sub\u003e, 0.1 EDTA, and 10 HEPES at pH 7.4) supplemented with 50 µg/mL. Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich.\u003c/p\u003e\u003cp\u003e \u003cb\u003eCut-open voltage clamp on\u003c/b\u003e \u003cb\u003eXenopus laevis\u003c/b\u003e \u003cb\u003eoocytes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMacroscopic ionic and gating current were recorded using cut-open voltage clamp technique \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. Micropipettes filled with 3M CsCl or NaCl, with resistance between 0.4 and 0.8 MΩ were used to measure the internal voltage of the oocytes. Current data were online filtered at 20 kHz with a low-pass 4 pole Bessel filter and sampled by a 16-bit A/D (USB-1604; Measurement Computing, Norton, MA) converter at 1 MHz. All experiments were conducted at room temperature (~ 17 ºC). For ionic current experiments, unless otherwise stated, were conducted in external solution consisted of in mM: 12 K methylsulfonate (MES), 108 N-methyl-D-glucamine (NMG) MES, 2 Ca MES, 10 HEPES, 0.1 EDTA, pH = 7.4 and internal solution consisted of in mM: 120 K MES, 10 HEPES, 2 EGTA, pH = 7.4. The capacitive transient was manually compensated with a dedicated circuit and in some cases, further removed by an online P/-4 protocol with a holding voltage of either − 80 or -90 mV \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. For gating current experiments, all experiments were conducted in external solutions consisted of in mM: 120 NMG MES, 2 Ca MES, 10 HEPES, 0.1 EDTA, pH = 7.4 and in internal solution consisted of in mM: 120 NMG MES, 2 Ca MES, 10 HEPES, 2 EGTA, pH = 7.4. The gating current was recorded with W434F background with the exception of I384R, which by itself produced no discernable ionic current.\u003c/p\u003e\u003ch2\u003eSingle channel recordings and noise analysis\u003c/h2\u003e\u003cp\u003eSingle channel recordings and noise analysis were performed on excised inside-out \u003cem\u003eXenopus Laevis\u003c/em\u003e oocyte patches. Briefly, the cells were injected with 5ng of RNA and maintained at 12 C° the day before the experiment. To remove the vitelline membrane, oocytes were incubated for 5 minutes in a hypertonic solution (SOS solution supplemented with 300 mM sucrose). This procedure shrank the oocyte separating the plasma membrane from the vitelline membrane, making the mechanical removal of the vitelline membrane easier without compromising the integrity of the cell. After removal of the vitelline membrane, oocytes were immediately and gently washed three times with intracellular solution containing in mM: 120KMES, 2EGTA, 10HEPES, pH = 7.40. They were placed in a recording chamber onto an inverted microscope. Current was recorded with an Axopatch 200B patch-clamp amplifier. The pipette solution consisted of, in mM, 120KMES, 2KCl, 2CaMES, 10HEPES, 0.1 EDTA, pH = 7.40. The resistances of the tips were between 13 ~ 17 MΩ for single channel experiments and 6 ~ 10 MΩ for noise analysis experiment. To reduce stray capacitance, the tips of the pipette were covered by Sylgard 184 (Dow Corning Corporation). Current was filtered with a digital 8 pole Bessel filter set at 10kHz (3384 Krohn-Hlite).\u003c/p\u003e\u003cp\u003eFor noise analysis, we applied hundreds of depolarizing voltage pulses. The ionic currents elicited were averaged to obtain the mean. The variance and the mean were obtained using our Analysis software. For single channels, we recorded several current traces from voltage pulses to + 140mV. The histograms were obtained using Analysis.\u003c/p\u003e\u003ch2\u003eUnnatural amino acid incorporation and voltage-clamp fluorimetry\u003c/h2\u003e\u003cp\u003eTo incorporate ANAP, we utilized the amber suppression technique. Briefly, the oocytes were injected with a mixture of ANAP-synthetase, ANAP methylester, ANAP-tRNA, \u003cem\u003eXenopus\u003c/em\u003e release factor 1 with D55E mutation and messenger RNA encoding the channel with the amber stop codon introduced at I470 position \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. The oocytes were incubated in the dark for 3 to 5 days prior to recording. The voltage clamp fluorometry setup and the filter set used in the study was similar to what was previously described \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, instead of using a photo diode, a photomultiplier was used to maximize the sensitivity of the signal, while allowing a decrease of the excitation light, decreasing photobleaching. The fluorophore was excited with a LED at 365nm (Thor lab M365L3). TMR labeling was done similarly as previous described \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Briefly, oocytes were incubated in 1mM DTT for 15 minutes. Three washes were administered before transferring the cells to the labeling solution with 20 µM Tetramethylrhodamine-5-Maleimide (Thermo Fisher T6027) in a depolarizing solution (in Mm: 120 KMES, 2 CaMes, 10HEPES, 0.1EDTA, pH = 7.40) for 30 minutes.\u003c/p\u003e\u003ch2\u003eData Analysis\u003c/h2\u003e\u003cp\u003eIonic current was taken by the steady-state current level and converted to conductance using the following relationship:\u003c/p\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:G\\left(V\\right)=\\frac{I}{V-{V}_{rev}}\\:\\left(1\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ewhere, \u003cem\u003eI\u003c/em\u003e is the ionic current in steady state, \u003cem\u003eV\u003c/em\u003e is the membrane voltage, and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003erev\u003c/em\u003e\u003c/sub\u003e is the reversal potential for the conducting ion. The GV curves were then fitted using a two-state model given by the equation:\u003c/p\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:G\\left(V\\right)=\\frac{1}{1+\\text{exp}\\left(-\\frac{zF}{RT}\\left(V-{V}_{1/2}\\right)\\:\\right)}\\:\\left(2\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cem\u003ez\u003c/em\u003e is the apparent charge expressed in units of elementary charge (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{e}_{0}\\)\u003c/span\u003e\u003c/span\u003e), \u003cem\u003eV\u003c/em\u003e is the voltage and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003e1/2\u003c/em\u003e\u003c/sub\u003e is the voltage of half-maximal conductance. \u003cem\u003eR\u003c/em\u003e is the ideal gas constant; \u003cem\u003eT\u003c/em\u003e is the temperature in Kelvin and \u003cem\u003eF\u003c/em\u003e is the Faraday constant.\u003c/p\u003e\u003cp\u003eThe gating charge was obtained by integrating the on and off-gating currents. They were plotted against the voltage to obtain the QV curves.\u003c/p\u003e\u003cp\u003eFor the analysis of the normalized QV curves, we used a two-state model fitting equivalent to the one in Eq.\u0026nbsp;2 to fit the gating from the I384E, I384L, I384N, and I384R mutants. The equation was the following:\u003c/p\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:Q\\left(V\\right)=\\frac{1}{1+\\text{exp}\\left(-\\frac{zF}{RT}\\left(V-{V}_{1/2}\\right)\\:\\right)}\\:\\left(3\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor WT, I384A, I384C, we used a three-state model fitting given by the following equation:\u003c/p\u003e\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:Q\\left(V\\right)=N\\frac{{z}_{2}+\\:{z}_{1}\\left(1+\\left(\\text{e}\\text{x}\\text{p}\\frac{{z}_{2}F}{RT}\\:\\left({V}_{2}-V\\right)\\right)\\right)}{1+\\text{e}\\text{x}\\text{p}\\left(\\frac{{z}_{2}F}{RT}\\:\\left({V}_{2}-V\\right)\\right)\\left(1+\\text{e}\\text{x}\\text{p}\\left(\\frac{{z}_{1}F}{RT}\\:\\left({V}_{1}-V\\right)\\right)\\right)}\\:\\left(4\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cem\u003eN, z\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003ez\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e \u003cem\u003eand V\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e are the number of channels, the charges associated and equilibrium voltages for the first and second transition, respectively.\u003c/p\u003e\u003cp\u003eFor nonstationary noise analysis, the mean variance data were fitted using a parabolic equation as follows:\u003c/p\u003e\u003cdiv id=\"Eque\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Eque\" name=\"EquationSource\"\u003e\n$$\\:{\\sigma\\:}^{2}=i\u0026lt;I\u0026gt;-\\frac{\u0026lt;{I\u0026gt;}^{2}}{N}\\:\\left(5\\right),$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\sigma\\:}^{2}\\)\u003c/span\u003e\u003c/span\u003e is the variance, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:i\\)\u003c/span\u003e\u003c/span\u003e is the single-channel current, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\u0026lt;I\u0026gt;\\)\u003c/span\u003e\u003c/span\u003e is the mean current, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:N\\)\u003c/span\u003e\u003c/span\u003e is the number of channels in the patch. We can estimate the maximal open probability (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Po}_{max}\\)\u003c/span\u003e\u003c/span\u003e) by knowing the maximum mean current (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{I}_{max}\\)\u003c/span\u003e\u003c/span\u003e) using the following equation:\u003c/p\u003e\u003cdiv id=\"Equf\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equf\" name=\"EquationSource\"\u003e\n$$\\:{Po}_{max}=\\frac{{\u0026lt;I\u0026gt;}_{max}}{Ni}\\:\\left(6\\right).$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor single channel analysis data, we obtained all-points histogram binned at 0.05 pA using our Analysis software. The data was then fitted using a mixture of \u003cem\u003ek\u003c/em\u003e Gaussian Distributions as follows \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e:\u003c/p\u003e\u003cdiv id=\"Equg\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equg\" name=\"EquationSource\"\u003e\n$$\\:f\\left(x\\right)=\\sum\\:_{i=1}^{k}{a}_{i}{f}_{i}\\left(y\\right)\\left(7\\right),$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{a}_{i}\\)\u003c/span\u003e\u003c/span\u003e and\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:{f}_{i}\\left(y\\right)\\)\u003c/span\u003e\u003c/span\u003e, are, respectively, the relative areas of the components and the Gaussian equation described by:\u003c/p\u003e\u003cdiv id=\"Equh\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equh\" name=\"EquationSource\"\u003e\n$$\\:{f}_{i}\\left(y\\right)=\\frac{1}{{\\sigma\\:}_{i}\\sqrt{2\\pi\\:}}{e}^{-\\frac{{\\left({n}_{i}\\right)}^{2}}{2}}\\:\\left(8\\right),$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnd\u003c/p\u003e\u003cdiv id=\"Equi\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equi\" name=\"EquationSource\"\u003e\n$$\\:{n}_{i}=\\frac{y-{\\mu\\:}_{i}}{{\\sigma\\:}_{i}}\\:\\left(9\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\mu\\:}_{i}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\sigma\\:}_{i}\\)\u003c/span\u003e\u003c/span\u003e are the mean and the standard deviation of the individual component \u003cem\u003ei.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eData is presented as Mean ± SEM. For noise analysis, ionic and gating currents experiments we used at least 3 oocytes from different batches. For single channels, we recorded several oocytes and only for presentation purposes show the representative traces from one cell.\u003c/p\u003e\u003cp\u003e \u003cb\u003eExpression and purification of mutant shaker potassium channel\u003c/b\u003e \u003c/p\u003e\u003cp\u003eShaker-IR-I384R was subcloned into a modified pEG BacMam vector containing a C-terminal HRV 3C protease site, an eGFP tag and an 8×-His tag utilizing the 5′ NotI and 3′ XbaI restriction sites. The Bacmid plasmid was generated using the Bac-to-Bac system which was then used to transfect the Sf9 insect cells with Cellfectin (Thermo Fisher). After 4 to 7 days of incubation at 27°C, P\u003csub\u003e0\u003c/sub\u003e virus was then collected after removing the remaining cell and debris. P\u003csub\u003e0\u003c/sub\u003e was then amplified to produce P\u003csub\u003e1\u003c/sub\u003e and P\u003csub\u003e2\u003c/sub\u003e virus. 2L of HEK293S GnTI\u003csup\u003e−\u003c/sup\u003e cells were infected with 200mL of P\u003csub\u003e2\u003c/sub\u003e virus. After 24 h of shaking incubation at 37°C, sodium butyrate was added to the cells at a final concentration of 10mM and the culture was transferred to 30°C. Cells were collected 48 h after infection. The cell pellets were then washed with phosphate-buffered saline (PBS) at pH 7.4, collected by centrifugation, flash-frozen and stored at − 80°C for later purification.\u003c/p\u003e\u003cp\u003eAll purification steps were conducted at 4°C. Frozen cell pellets were thawed in water bath and homogenized with a Dounce homogenizer in suspension buffer (150mM KCl, 2mM TCEP, 1mM EDTA, 50mM TRIS, Pierce protease inhibitor tablet (Thermo Fisher Scientific) PH 7.5). Resuspended cells culture were supplemented with DDM:CHS (10:1) at 1% final concentration (m. / v.) and were extracted for 2 hours with gentle agitation. The cell debris was pelleted by ultracentrifugation for 1 hr at 40000 rpm in Ti45 rotor (Beckman Coulter). The supernatant was collected and incubated with 2 ml CNBR-activated Sepharose beads (GE Healthcare) coupled with 4 mg high-affinity GFP nanobodies purified in-house for 3 hours. The Sepharose beads were then rinsed three times 10 column volume with the suspension buffer supplemented with 0.1% DDM:CHS (10:1), 0.05% DDM:CHS + 0.02% GDN, 0.02% GDN respectively. Then beads were incubated with HRV 3C protease overnight to release the purified channels. The next day, 3 column volume of suspension buffer was used to elute the beads. Eluted solution was concentrated with Amicon Ultra Centrifugal Filter unit (Millipore) with 100kDa cutoff to approximately 1mL and loaded onto a Superose6 (10 × 300 mm) gel filtration column (GE Healthcare) and separated with suspension buffer supplemented with 0.02% GDN.\u003c/p\u003e\u003ch2\u003eCryo-EM sample preparation and data acquisition\u003c/h2\u003e\u003cp\u003eSamples purified from size-exclusion chromatography were pooled and concentrated to around 0.7 mg/mL measured with a nanodrop machine. 3.5 µL concentrated protein was applied to glow-discharged (30 s, 20 W Solarus Plasma Cleaner) Quantifoil grids (R 1.2/1.3 Au 300 mesh). The grids were blotted for 3s with blot-force 3 in a FEI Vitrobot Mark IV (Thermo Fisher) chamber with 100% humidity at room temperature before plunge froze in liquid ethane. Clipped grids were subsequently loaded onto a Titan Krios microscope. Single-particle movies were acquired using a K3 direct electron detector in super-resolution mode, coupled with a 20 eV GIF energy filter. Data collection was performed at a nominal magnification of ×81,000, corresponding to a pixel size of 0.5315 Å, binned by 2 during acquisition. The total electron dose was calibrated to 60 e−/Ų, distributed across 50 frames per movie.\u003c/p\u003e\u003ch2\u003eSingle-particle cryo-EM analysis\u003c/h2\u003e\u003cp\u003eAll steps for structure determination were performed using CryoSPARC \u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e, including motion correction and contrast transfer function (CTF) estimation. A subset of 2,000 particles was initially picked and classified in 2D to generate templates for template-based particle picking. Approximately 4,500,000 initial particles were picked and subjected to multiple rounds of 2D classification. From these, 180,000 particles were selected to generate three \u003cem\u003eab initio\u003c/em\u003e models with C1 symmetry. Particles from the best class (~ 116,000) were then processed for 3D refinement with C4 symmetry, yielding a nominal resolution of 5.7 Å. We performed 3D refinement using non-uniform refinement algorithm \u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e, which consistently yields best result in our data. A subsequent 3D classification was performed using the best-refined model from the previous round and a junk model generated \u003cem\u003eab initio\u003c/em\u003e. The best class from this step was further refined using C4 symmetry, resulting in a 3.55 Å nominal resolution. After identifying a subset of ~ 80,000 particles for final refinement, Reference-Based Motion Correction was applied to estimate per-particle movement trajectories and empirical dose weights. A final 3D refinement step was performed, followed by postprocessing using a tighter mask and C4 symmetry enforcement. Local resolution was calculated using CryoSPARC. Validation was done using MOLprobity \u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eModel building and structural refinement\u003c/h2\u003e\u003cp\u003eThe Shaker-IR model (PDB 7SIP) was used as a template to build atomic models for the Shaker-IR-I384R mutants into our density map. All structural models were constructed using unsharpened maps. Initial models were generated through iterative rounds of manual model building in COOT \u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e and real-space refinement in Phenix \u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e. Final refined atomic models were obtained using interactive flexible fitting in ISOLDE \u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e. All structural analyses and figure generation were performed using UCSF ChimeraX \u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e,\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eWe would like to thank Gethiely Gasparini and Hlafira Polishchuk for oocytes preparation, DNA mutation and RNA preparation. We would like to thank Dr. Chris Ahern and Dr. Jason Galpin for helping with the ANAP experiments We would also like to thank the staff at the cryo-EM core in the University of Chicago for their assistance. Supported by NIH grants R01-GM030376,\u0026nbsp;R01-GM150272 and\u0026nbsp;National Science Foundation Award QuBBE QLCI (NSF OMA-2121044).\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eAuthors Contribution\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eC.B. and Y.L. performed electrophysiological experiments.\u003c/p\u003e\n\u003cp\u003eY.L. and G.C. performed cryoEM experiments.\u003c/p\u003e\n\u003cp\u003eY.L., C.B. and G.C. analyzed the data.\u003c/p\u003e\n\u003cp\u003eY.L., C.B., and F.B. conceptualized the project.\u003c/p\u003e\n\u003cp\u003eY.L., C.B., G.C., E.P. and F.B. interpreted the data.\u003c/p\u003e\n\u003cp\u003eY.L. and C.B. wrote the original draft.\u003c/p\u003e\n\u003cp\u003eY.L., C.B., G.C, E.P. and F.B edited and reviewed the paper\u003c/p\u003e\n\u003cp\u003eE.P. and F.B. supervised the project and oversaw funding acquisition.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJan, L. Y. \u0026amp; Jan, Y. N. Voltage-gated potassium channels and the diversity of electrical signalling. J. Physiol. 590, 2591\u0026ndash;2599 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoyle, D. A. \u003cem\u003eet al.\u003c/em\u003e The structure of the potassium channel: molecular basis of K\u0026thinsp;+\u0026thinsp;conduction and selectivity. Science 280, 69\u0026ndash;77 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTombola, F., Pathak, M. M. \u0026amp; Isacoff, E. Y. How Does Voltage Open an Ion Channel? Annu. Rev. Cell Dev. Biol. 22, 23\u0026ndash;52 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolmgren, M., Shin, K. S. \u0026amp; Yellen, G. The activation gate of a voltage-gated K\u0026thinsp;+\u0026thinsp;channel can be trapped in the open state by an intersubunit metal bridge. Neuron 21, 617\u0026ndash;621 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStix, R. \u003cem\u003eet al.\u003c/em\u003e Eukaryotic Kv channel Shaker inactivates through selectivity filter dilation rather than collapse. Sci. Adv. 9, (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCuello, L. G., Jogini, V., Cortes, D. M. \u0026amp; Perozo, E. Structural mechanism of C-type inactivation in K(+) channels. Nature 466, 203\u0026ndash;208 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIslas, L. D. \u0026amp; Sigworth, F. J. Voltage Sensitivity and Gating Charge in Shaker and Shab Family Potassium Channels. J. Gen. Physiol. 114, 723\u0026ndash;742 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHirschberg, B., Rovner, A., Lieberman, M. \u0026amp; Patlak, J. Transfer of twelve charges is needed to open skeletal muscle Na\u0026thinsp;+\u0026thinsp;channels. J. Gen. 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Putative receptor for the cytoplasmic inactivation gate in the Shaker K\u0026thinsp;+\u0026thinsp;channel. \u003cem\u003eNat.\u003c/em\u003e 1991 \u003cem\u003e3536339\u003c/em\u003e 353, 86\u0026ndash;90 (1991).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcCormack, K. \u003cem\u003eet al.\u003c/em\u003e A role for hydrophobic residues in the voltage-dependent gating of Shaker K\u0026thinsp;+\u0026thinsp;channels. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e 88, 2931\u0026ndash;2935 (1991).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSlesinger, P. A., Jan, Y. N. \u0026amp; Jan, L. Y. The S4\u0026ndash;S5 loop contributes to the ion-selective pore of potassium channels. Neuron 11, 739\u0026ndash;749 (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTan, X. F. \u003cem\u003eet al.\u003c/em\u003e Structure of the Shaker Kv channel and mechanism of slow C-type inactivation. Sci. Adv. 8, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerozo, E., MacKinnon, R., Bezanilla, F. \u0026amp; Stefani, E. Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K\u0026thinsp;+\u0026thinsp;channels. Neuron 11, 353\u0026ndash;358 (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCha, A. \u0026amp; Bezanilla, F. Characterizing Voltage-Dependent Conformational Changes in the ShakerK\u0026thinsp;+\u0026thinsp;Channel with Fluorescence. Neuron 19, 1127\u0026ndash;1140 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalstrup, T. \u0026amp; Blunck, R. Dynamics of internal pore opening in KV channels probed by a fluorescent unnatural amino acid. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e 110, 8272\u0026ndash;8277 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMannuzzu, L. M., Moronne, M. M. \u0026amp; Isacoff, E. Y. 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Protein Sci. 27, 14\u0026ndash;25 (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6406486/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6406486/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVoltage gated potassium (Kv) channels play key roles in physiological processes, from cellular excitability to immune response and are among the most important pharmaceutical targets\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Despite recent advances in the structural determination of Kv channels, the closed state structure of strictly coupled Kv1 family remains elusive. Here, we captured the structure of Shaker potassium channel with a closed pore by uncoupling its voltage sensor domains from the pore domains. Structural determination of the uncoupled I384R mutant by single particle cryoEM revealed a fully closed pore in the presence of activated, non-relaxed voltage sensors. Putative conformational transitions estimated from a fully open pore domain indicates a \u0026ldquo;roll and turn\u0026rdquo; movement along the length of the pore-forming S6 helices, in sharp contrast to canonical gating models based on limited movements of S6 \u003csup\u003e2\u0026ndash;4\u003c/sup\u003e. These rotational and translational movement place two hydrophobic residues, one at inner cavity and the other at the bundle crossing region, directly at the permeation pathway, limiting the pore radius to less than 1 \u0026Aring;. Surprisingly, the selectivity filter was captured in a noncanonical state, partially expanded at G446, unlike previously described dilated\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e or pinched\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e filter conformations. Based on the present data we propose a reinterpretation of the mechanism of activation gating for strictly coupled Kv1 channels and the strictly coupled interactions that underlie different functional states.\u003c/p\u003e","manuscriptTitle":"Closed State Structure of the Pore Revealed by Uncoupled Shaker K+ Channel","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-09 02:13:12","doi":"10.21203/rs.3.rs-6406486/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"69a14398-1fdf-405a-a999-cda72dac8471","owner":[],"postedDate":"May 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47521425,"name":"Biological sciences/Biophysics/Permeation and transport"},{"id":47521426,"name":"Biological sciences/Neuroscience/Ion channels in the nervous system"}],"tags":[],"updatedAt":"2025-11-20T08:11:30+00:00","versionOfRecord":{"articleIdentity":"rs-6406486","link":"https://doi.org/10.1038/s41467-025-65124-0","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-11-19 05:00:00","publishedOnDateReadable":"November 19th, 2025"},"versionCreatedAt":"2025-05-09 02:13:12","video":"","vorDoi":"10.1038/s41467-025-65124-0","vorDoiUrl":"https://doi.org/10.1038/s41467-025-65124-0","workflowStages":[]},"version":"v1","identity":"rs-6406486","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6406486","identity":"rs-6406486","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}