PIP2 activation of the cardiac IKs potassium channel

PIP2 activation of the cardiac IKs potassium 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 Article PIP 2 activation of the cardiac I Ks potassium channel Jianmin Cui, Lu Zhao, Xianjin Xu, Chenxi Cui, Rui Duan, Ali Kermani, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7609003/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The I Ks channel complex, composed of the voltage-gated potassium channel KCNQ1 and its regulatory subunit KCNE1, is essential for the termination of cardiac action potentials. The function of KCNQ1 and I Ks requires PIP 2 , and its depletion abolishes channel opening. Previous studies revealed that KCNQ1 adopts both bent and straight conformations and can bind two PIP 2 molecules: one adjacent to VSD (V-PIP 2 ), and the other at the VSD-pore interface (C-PIP 2 ). Here we show that the two PIP 2 perform essential yet distinct roles: V-PIP 2 enables the bent-to-straight transition, whereas C-PIP 2 mediates VSD-pore coupling and stabilizes the straight conformation. Structure-function analysis and molecular dynamic simulations show that VSD activation elevates the V-PIP 2 site and weakens the CaM-VSD interaction, permitting the conformational shift from the bent, intermediate open (IO) state associated with KCNQ1 to the straight, I Ks -exclusive activated open (AO) state, which is further stabilized by C-PIP 2 . Leveraging this mechanism, we developed a compound CA1, which selectively targets the V-PIP 2 site and modulates I Ks channel activity without affecting KCNQ1, offering a novel and promising conceptional path for specific and safe antiarrhythmic therapeutics. Biological sciences/Biophysics/Membrane biophysics/Ion transport Physical sciences/Engineering/Biomedical engineering V-PIP2 C-PIP2 KCNQ1 IKs anti-arrhythmia therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Main Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is an important signaling molecule that regulates the function of a variety of membrane proteins 1-3 including many of ion channels 4,5 . Among PIP 2 -regulated ion channels, the family of KCNQ1-5 potassium (K + ) channels plays a crucial role in regulating membrane excitability in the brain, heart and epithelium 6-9 . PIP 2 is required for KCNQ channel function, and channel activity diminishes when membrane PIP 2 levels are reduced 7,10,11 . Thus, investigating PIP 2 -dependent activation of KCNQ channels could yield valuable insights into the physiological effects and molecular mechanisms of PIP 2 regulation in ion channels. We recently solved the structure of KCNQ1 in association with its regulatory subunit, KCNE1, and PIP 2 molecules ( Fig. S1b ) 11 , providing a structural foundations for understanding mechanisms of the PIP 2 -dependent activation of KCNQ1 channels. KCNQ1 channels are voltage-gated K + (Kv) channels 12-14 . KCNQ1, together with the regulatory subunit KCNE1, forms the slow-delayed rectifier potassium (I Ks ) channels in the heart and inner ear, which are crucial for regulating heart rhythm and supporting K + recycling in the endolymph 15-17 . When associated with the regulatory subunit KCNE3, KCNQ1 is constitutively active at physiological voltages, functioning as a K + transporter to maintain ionic homeostasis in epithelial and endothelial cells 18,19 . Malfunctions of KCNQ1 caused by drugs, mutations, and single nucleotide polymorphisms are linked to arrhythmia, deafness, atrial fibrillation and type-2 diabetes mellitus 12,20-22 . The voltage-dependent gating of K v channels involves three fundamental processes, voltage sensor domain (VSD) activation, VSD-pore coupling, and pore opening. The VSD of Kv channels, such as Shaker and KCNQ1, can exist in three resolvable states: resting (R), intermediate (I) and activated (A). Upon membrane depolarization the VSD of these channels transition from the R state to the A state via the I state 23-25 . However, in Shaker channels, the pore conducts only at the activated open (AO) state when the VSD is in the A state 23,26 . In contrast, in KCNQ1, the pore opens when the VSD is in either the I or the A state, resulting in the IO (intermediate open) and AO states 14,24,27,28 ( Fig. S1a ). The IO and AO states exhibit drastically different properties in their voltage dependence, activation kinetics, current amplitude, ion permeability, and pharmacology 13,14,24 . The IO and AO states of KCNQ1 are differentially regulated by various KCNE subunits, which exhibit distinct tissue distributions 15,16,18,19 . This differential regulation enables KCNQ1 + KCNE channels to function across diverse tissues, supporting a wide range of physiological functions. For instance, both KCNE1 and KCNE3 selectively shift the VSD activation to the I state at more negative voltages 18 . However, KCNE1 suppresses IO but enhances AO of KCNQ1, making the channel open exclusively in the AO state, with a more positively shifted voltage dependence, slower activation kinetics, and larger current amplitudes 24,28 . These features are essential for terminating cardiac action potential. In contrast, KCNE3 does not suppress IO, allowing KCNQ1 + KCNE3 channels open at more negative voltages and remain constitutively open at physiological voltages to maintain K + homeostasis in the epithelium 18,19 . The structural basis underlying the IO and AO states in KCNQ1 remains unclear. KCNQ1 channels adopt a homo-tetrameric assembly, where each subunit contains six transmembrane α-helices S1-S6, with S1-S4 forming the VSD and S5 and S6 forming the central pore 29,30 . Each KCNQ1 subunit interacts with a calmodulin (CaM) molecule mainly through its cytosolic helix A (HA) and helix B (HB) ( Fig. S1b ). Our previous structural studies revealed two distinct conformations of the cytosolic domain in KCNQ1 channels; straight and bent 29,30 . In the straight conformation, HA forms a continuous helix with the transmembrane helix S6, positioning CaM deep in the cytosol, away from the membrane-spanning domain of KCNQ1. The bent conformation is facilitated by a structural kink at the “RQKH” motif, located between helix S6 and HA. This kink allows HA to adopt an upward-bent orientation, enabling CaM to undergo rotational displacement to interact with the cytosolic S2-S3 linker (S2S3L). Our previous functional studies demonstrated that mutations disrupting the S2S3L-CaM interface shift KCNQ1 channel properties from predominantly IO to characteristics resembling the AO state. Based on these findings, we proposed that the IO state likely adopts the bent conformation, while the AO state corresponds to the straight conformation, and that disrupting S2S3L-CaM interactions promotes the transition from the IO to the AO state 31 . Our prior studies demonstrated that PIP 2 is required for VSD-pore coupling in KCNQ1 in both the IO and AO states 7,10 . Without PIP 2 , the VSD activates but fails to induce pore opening. Functional and mutagenesis studies indicated a PIP 2 binding site at the interface between the VSD and the pore, essential for KCNQ1 channel opening 7 . However, our previous structural analyses of KCNQ1 and KCNQ1 + KCNE3 did not clearly show PIP 2 binding at the VSD-pore interface but instead revealed a PIP 2 -binding site adjacent to the VSD 30 . We recently solved the structure of the KCNQ1-KCNE1 complex, which revealed not only the PIP 2 -binding site near the VSD, but also an additional PIP 2 site at the VSD-pore interface, yielding a total of eight PIP 2 molecules in the channel complex 11 ( Fig. S1b ). In this study we solved the structure of KCNQ1 in an I state. We examined the structural and functional properties of the two PIP 2 -binding sites in KCNQ1. These results, together with the identification of a compound, CA1, that selectively binds to one of PIP 2 sites and specifically enhances the activation of KCNQ1 only in the presence of KCNE1, support that the IO and AO states adopt the bent and straight conformations, respectively. Through electrophysiological experiments and structural characterization of KCNQ1 channel at the I state, we found that the first PIP 2 molecule located near the VSD and termed V-PIP 2 for its role in VSD function ( Fig. S1b ), drives the conformational shift from IO to AO during VSD activation. VSD activation to the A state alters the conformation of V-PIP 2 and its binding site ( Fig. 4a ), detaching CaM from the VSD and promoting the transition from bent to straight. The second PIP 2 molecule, designated C-PIP 2 , was revealed in our recently determined KCNQ1-KCNE1 structure at the VSD-pore interface, promoting VSD-pore coupling to enable channel opening in both IO and AO states while stabilizing the straight conformation ( Figs. 4g, S1b ) 11 . VSD-pore coupling determines the channel’s ability to open, whereas the IO-to-AO transition is critical for the function of I Ks channels in the heart. Therefore, both PIP 2 molecules are vital for the physiological functions of KCNQ1 channels. Conformations of KCNQ1 in various functional states Voltage dependent gating of KCNQ1 involves two measurable steps of VSD activation to the I and A states, both of which trigger pore opening to the IO and AO ( Fig. S1a ). Previous structural studies indicate that KCNQ1 may adopt a bent conformation in the resting-closed (RC) state 32 and a straight conformation in the AO state 11,30 . Then, what is the conformation, bent or straight, in the IO state? To address this question, we trapped the VSD of KCNQ1 in I state by introducing E160R/R231E (E1R/R2E) mutations 24,33 ( Fig. 1a ). The structure of KCNQ1-E1R/R2E in I state shares high similarity with KCNQ1-PIP 2 structure (PDB: 9VEN), and both adopt a bent conformation: the S6 and HA helices form a helix-loop-helix structure. The R237 (R4) residue of the S4 helix points to the gate charge transfer center formed by F167, E170 and D202, while R231 (R2) and H240 (H5) of S4 helix sit within the gate charge transfer center in the resting state (PDB: 8SIN) and activated state (PDB: 9VE1), respectively. This supports that the structure is captured in the I state, with the S4 is ~5 Å lower than in the A state 34 . The ion conductance pathway plotted using HOLE program shows that the pore radius (~1 Å at the narrowest point) is too small for hydrated potassium to pass, suggesting that the structure may represent an intermediate-closed (IC) state. In the structure, one PIP 2 molecule is observed at the V-PIP 2 site, which is mainly formed by the S2S3L, S3 and S4-S5 linker (S4S5L) ( Figs. 1a-c ). In functional studies, while the KCNQ1-E1R/R2E channels showed constitutive macroscopic currents independent of voltage 24,33 , single-channel recordings exhibited flickering openings with low open probability 28 . These results are consistent with the KCNQ1-E1R/R2E structure being in the IC conformation. Then, does the IO state adopt the bent conformation? In KCNQ1 channel structures, each subunit binds a CaM molecule at the cytosolic domain 11,29,30,32,34 . In the bent conformation, CaM interacts with the S2S3L on the cytosolic side of the VSD ( Fig. 1a ). During VSD activation (R →I →A), if the channel remains bent, with S2S3L and CaM likely interacting differently before reaching the straight A state, these S2S3L-CaM interactions would influence activation of the IO state more than to the AO state. To test this hypothesis, we performed mutational scanning in both CaM (D94 and D96) and S2S3L (C180, R181, S182, K183, Y184, L191 and R192) as well as in S3 (R195 and K196) to evaluate the function of individual mutations and their pairs in the activation of KCNQ1 and I Ks , respectively ( Figs. S2-6 ). Four pairs of these mutations are noteworthy: KCNQ1-C180D in S2S3L paired with CaM-D94K and R195Q in S3 paired with CaM-D94K ( Figs. 1e, S3a, S4d ), and KCNQ1-Y184W and L191D in S2S3L, each paired with CaM-D96R ( Figs. 1d, e, S6b ). Among these four pairs of mutations, three double mutations (KCNQ1-C180D and CaM-D94K, KCNQ1-R195Q and CaM-D94K, KCNQ1-Y184W and CaM-D96R) caused near complete loss of current compared to single mutations in KCNQ1 or CaM alone, whereas the KCNQ1-L191D and CaM-D96R double mutation pair significantly recued the current of KCNQ1-L191D mutation ( Figs. 1e, S6b ). Such drastic changes in current amplitude by the double mutations suggests that these paired residues may interact during channel opening. The four mutation pairs specifically affect KCNQ1, as their double mutations in I Ks produced current amplitudes similar to those of single KCNQ1 or CaM mutations ( Figs. 1d, e, S3a, S4d, S6b ). It is unlikely that these double mutations reduce surface expression, as evidenced by the robust currents exhibited by the mutant I Ks complexes, although it is known that KCNQ1 and KCNE1 traffic to the plasma membrane via independent pathways in both cardiac myocytes and Xenopus oocytes 35 . Since KCNQ1 opens predominantly to the IO state and I Ks opens exclusively to the AO state, these results support that the channel adopts a bent conformation in the IO state and a straight conformation in the AO state. Further support for the correlation between the conformation and functional state is shown below. C-PIP 2 binding is essential for channel activation In the KCNQ1-E1R/R2E structure, no obvious C-PIP 2 was observed. Is C-PIP 2 important for both the IO and AO states, and what role does C-PIP 2 play? The KCNQ1-KCNE1 structure reveals that C-PIP 2 interacts with residues in the S4S5 linker (V255 and F256), S5 (R259, Q260, L262 and L263) of one KCNQ1 subunit, and S6-HA (Q359, K362, and R366) of a neighboring KCNQ1 subunit, and the KCNE1 (F57, I61, L63, S64, R67, S68, K69, and K70) subunit ( Fig. 2a ). First, we mutated each C-PIP 2 -interacting residue in both KCNQ1 and KCNE1 to alanine (Ala) or neutralized charged amino acids (glutamine Q or asparagine N) and assessed the function of the mutant channels. All mutations, except KCNE1-F57A, significantly reduced I Ks current amplitudes ( Figs. 2b, c, S7 ). The similar effect of KCNE1-K70N was reported previously 10 . The C-PIP 2 site mutations also reduced KCNQ1 current amplitude in the absence of KCNE1 ( Figs. 2b, c, S7 ). These results are consistent with observations made when membrane PIP 2 levels were reduced 10,11 , indicating that these mutations diminish PIP 2 binding to the KCNQ1 channel and that C-PIP 2 is critical for the conductance of KCNQ1 and I Ks . Importantly, all these mutations caused only minor shifts of the voltage-dependent activation, with the conductance-voltage (G-V) relationship deviating from the wild-type (WT) less than 25 mV ( Figs. 2d, S7 ). This is consistent with the mechanism whereby C-PIP 2 binding is crucial for coupling of VSD movements and pore opening but not for VSD activation 7 . Among the exanimated mutations, KCNQ1-I263A, Q359A, K362A and R366A produced less pronounced reductions in KCNQ1 current amplitude than the reduction observed in the I Ks current ( Figs. 2c, S8a-d ). These results indicate that the C-PIP 2 binding sites in KCNQ1 may alter in the absence of KCNE1 32 . Notably, residues Q359, K362 and R366 within or around the “RQKH” motif, which is the hinge for the bent and straight conformation switch ( Fig. 2e ), interact with C-PIP 2 primarily in the presence of KCNE1 ( Figs. 2c, S8b-d ). Thus, the binding of C-PIP 2 to these residues may help KCNE1 stabilize the straight conformation 7,10 , while the disruption of the “RQKH” motif from it α-helical structure into a loop may alter C-PIP 2 binding. To verify this notion, we evaluated the structure of the C-PIP 2 binding sites in the bent conformation. Since C-PIP 2 binding was not observed in any available structures of KCNQ1 in the bent conformation, we performed molecular docking of C-PIP 2 to the bent conformation of KCNQ1 ( Fig. 2f ). The results revealed that the residues I263, Q359, K362, and R366 no longer interact with C-PIP 2 ( Figs. 2f, g ). These findings align with the functional data showing that mutations of these residues reduce KCNQ1 current amplitudes to a lesser extent than I Ks ( Figs. 2c, S8a-d ). Molecular docking further revealed that, in the bent conformation, residues T264, K358 and Q361 interacted with the C-PIP 2 , which are not part of the C-PIP 2 site in the I Ks structure. Mutation of these residues reduced KCNQ1 current amplitudes to a greater extent than I Ks ( Figs. 2g-i, S8e, f ). These results indicate that the opening of KCNQ1 channel is associated with the bent conformation, whereas the opening of I Ks channel is associated with the straight conformation, and the C-PIP 2 site alters between the two conformations. Our previous findings indicated that both KCNQ1 and I Ks require PIP 2 for channel function, but the EC 50 of PIP 2 dose-response for KCNQ1 was over 100-fold greater than that for I Ks , suggesting a higher PIP 2 affinity for I Ks 10 . The differences in C-PIP 2 binding sites within KCNQ1 across different states, combined with the role of KCNE1 in facilitating C-PIP 2 binding, explain the ﹥100-fold increase in PIP 2 sensitivity observed in I Ks channels compared to KCNQ1 alone. This mechanism also accounts for the observation that no PIP 2 is resolved in the C-site in the bent conformation due to its low affinity. V-PIP 2 binding is essential for channel activation to AO but not to IO In the structure of KCNQ1-E1R/R2E, V-PIP 2 interacts with residues in the S2S3L (R181, K183, and Y184), the S3 (K196 and I198) and S4S5L (Q244, W248, and R249) ( Figs. 1a, 4a ). In the structure of KCNQ1-KCNE1 (PDB: 9VEI), V-PIP 2 additionally interacts with the N-terminus S0 (Y111 and R116) of KCNQ1, as well as with KCNE1 (E72 and H73) ( Fig. 3a ). We mutated these V-PIP 2 interacting residues to A or N/Q using a mutation scanning. Most mutations in KCNQ1 (R181, K183, Y184, K196, and R249) did not reduce but rather increased KCNQ1 current amplitudes ( Figs. 3c, S9c-f, i ), with the exceptions of Q244N and W248A ( Figs. 3c, S9g, h ), which are critical for VSD-pore coupling 25 . Y111A and R116A reduced KCNQ1 current amplitudes ( Figs. 3c, S9a, b ); however, these residues did not interact with V-PIP 2 in the bent conformation and the IO state, indicating that the effects of Y111A and R116A on KCNQ1 current amplitudes may not directly disrupt V-PIP 2 binding. On the other hand, most of the mutations significantly reduced the current amplitudes of I Ks except for the KCNQ1-R181Q + KCNE1 and KCNQ1-K183N + KCNE1 ( Figs. 3c, S9c, d ). Although these two mutations enhanced I Ks current amplitudes, the enhancement was significantly smaller compared to the enhanced KCNQ1 current amplitudes of the same mutant. All mutations induce small GV shifts in both KCNQ1 (less than 15 mV) and I Ks (less than 25 mV) ( Figs. 3d, S9 ), indicating that the mutations did not modify current amplitudes by altering voltage dependence. Additionally, we observed that mutations at the V-PIP 2 sites did not significantly affect the current amplitudes of KCNQ1 + KCNE3 ( Figs. 3e, f ), whereas mutations at the C-PIP 2 sites drastically reduced the current amplitudes of KCNQ1 + KCNE3 ( Fig. S10 ). Thus, these results suggest that V-PIP 2 is essential for the AO state but not the IO state, as both KCNQ1 and KCNQ1 + KCNE3 primarily open to the IO state, whereas I Ks exclusively opens to the AO state 24,25,28,34 . To validate this mechanism, we introduced some of the V-PIP 2 site mutations into the mutant KCNQ1-S338F and KCNQ1-F351A, which were previously established as opening only in the IO and AO state, respectively 24,28 . Notably, these mutations either had no impact (Y184, K196, I198, Q244, and R249) or enhanced KCNQ1-S338F current amplitudes (K183), while significantly reducing KCNQ1-F351A current amplitudes ( Figs. 3g, h, S11a-d ), supporting the notion that V-PIP 2 is specifically required for the AO state, but not the IO state. By contrast, KCNQ1-R259Q, which affects C-PIP 2 binding, reduced the current amplitudes of both KCNQ1-S338F and KCNQ1-F351A ( Fig. S11e ). V-PIP 2 modulates IO to AO transition Comparing V-PIP 2 and its binding site in the structures of KCNQ1-E1R/R2E and KCNQ1-KCNE1 ( Fig. 4a ), notable differences were observed in the interaction network of PIP 2 . In the I state, the head group of PIP 2 contacts S4, S4S5L, S2S3L and CaM. Upon further activation of the VSD, S4 moves upward, followed by S4S5L and S2S3L, a motion that appears to elevate the center of the PIP 2 -binding pocket. This motion weakens interactions between CaM and the rest of the V-PIP 2 site, facilitating the alteration of the V-PIP 2 site and repositioning of V-PIP 2 to form closer interactions with S2S3L ( Figs. 4a, S12 ). The upward movement of the PIP 2 -interaction network, the detachment of CaM from the V-PIP 2 site, and the alteration of V-PIP 2 binding are associated with the “bent-to-straight” conformational transition of the KCNQ1 channel. To validate the observed structural difference in V-PIP 2 binding between the I and A states, we employed small-molecule screening approaches 36 . We reasoned that a compound selectively binding to the V-PIP 2 site in the straight conformation would modulate the I Ks channel without affecting KCNQ1 alone in the I state. We screened the Available Chemical Database (ACD, Molecular Design Ltd.) by docking its compound to the V-PIP 2 binding sites of KCNQ1-KCNE1in the straight conformation. Docking results revealed that the compound CA1 ( Fig. S13a ), selectively binds to the straight conformation of the channel, with the binding site nearly overlapping with the V-PIP 2 site. In contrast, the binding pocket undergoes structural rearrangement in the bent conformation, preventing CA1 from binding ( Fig. 4b ). Our functional data showed that CA1 significantly enhanced the current amplitudes of I Ks and shifted its GV to more negative voltages ( Fig. 4c ). Mutations in the putative CA1-binding residues ( Fig. S13b ) attenuated the effects of CA1 ( Figs. 4d, S13d-o ), supporting our docking results for the CA1 binding site in the straight conformation of the I Ks channel, which opens exclusively to the AO state. Conversely, CA1 exhibited no enhancing effect on channels predominantly open to the IO state in the bent conformation, including KCNQ1 alone and the KCNQ1-KCNE3 complex ( Figs. 4d, e ). Notably, KCNQ1-KCNE3 could adopt a straight conformation and open in the AO state, albeit with a low probability 30 . Docking of CA1 onto the straight conformation of KCNQ1-KCNE3 showed that CA1 could bind to the same pocket ( Fig. S13c ). Thus, the absence of CA1-induced current augmentation in KCNQ1 + KCNE3 channels is consistent with the complex primarily conducting in the IO state via the bent conformation, which precludes CA1 binding. To support the above mechanism that the VSD movement to the A state and the change in V-PIP 2 trigger the bent to straight conformational switch, we performed molecular dynamics (MD) simulations by first constructing a hybrid starting model. In this model, the VSD and pore domains (residues 1–236) preceding the bending point (RQKH, residues 251–254), including the bound PIP 2 , were taken from the straight conformation, whereas the remaining regions were retained from the bent conformation. We then performed metadynamics simulations to accelerate sampling along the “bent-to-straight” transition. Representative snapshots are shown in Fig. 4f . Along the biased trajectory, CaM progressively dissociated from the VSD and rotated while KCNQ1 spontaneously adopted a straight configuration. These conformational changes align with our structures of the I and A states and support the mechanism in which upward S4 movement facilitates VSD activation and conformational changes in the V-PIP 2 binding site, displacing CaM and stabilizing a straight cytosolic conformation, thus promoting the AO state. To visualize the conformational transition from the bent conformation to the straight conformation, we also performed targeted MD simulations. Snapshots from the simulation are presented in Fig. S14 and Movie. S1 . During the transition, the S4 helix exhibits an upward movement compared to its position in the bent conformation ( Fig. S14a ). The phosphatidylinositol head group of V-PIP 2 undergoes a reorientation toward S2S3L, establishing contacts with this linker ( Fig S14b ). Concurrently, CaM gradually dissociates from the VSD. A model of the activation of KCNQ1 and I Ks by VSD, PIP 2 and CaM Based on our findings and prior studies, we propose a model for KCNQ1 and I Ks activation ( Fig. 5 ). In this model, V-PIP 2 interacts with the channel via a voltage-dependent mechanism to trigger the IO to AO transition, while C-PIP 2 is associated with the channel to mediate the VSD-pore coupling and stabilize the straight conformation. The recently published structure of KCNQ1 with the VSD in the RC state indicated that the channel adopts a bent conformation, which obstructs the V-PIP 2 site and prevents its binding 32 . Therefore, V-PIP 2 binds to the channel upon VSD activation to the I state, in both IC and IO, which adopt the bent conformation ( Fig. 5 ). V-PIP 2 is subsequently reoriented upon VSD further activation to the A state, triggering the switch from bent to straight, which culminates in the activating-closed (AC) and AO states ( Figs. 3, 4f ). C-PIP 2 binds to the channel in both the I and A states to facilitate the VSD-pore coupling during the IC-IO and AC-AO transitions, but whether C-PIP 2 binds to the RC state is not clear. In the I state, C-PIP 2 binds to the channel with a lower affinity than in the A state owing to the alteration of the C-PIP 2 binding sites during the “bent-to-straight” transition ( Fig. 2 ). The KCNE1 association inhibits the IO state and enhances the AO state ( Fig. S1a ) 24,25,28 . We propose that, even in association with KCNE1, the KCNQ1 subunit undergoes the same bent and straight transition as the VSD moves from the I state to the A state, based on the following observations. First, V-PIP 2 is essential for the activation of the KCNQ1 + KCNE1 channel ( Fig. 3 ), potentially by stabilizing the straight conformation. Second, C-PIP 2 in I Ks exhibits a state-dependent change in binding affinity; in the open state (AO, straight), the affinity is enhanced, and C-PIP 2 is resistant to digestion by the lipid phosphatase CiVSP. By contrast, the C-PIP 2 affinity decreases following channel deactivation at hyperpolarized voltages (IC and RC, bent), allowing for digestion by CiVSP 37,38 . The correspondence between RC, IC, and IO (all adopting bent conformation) and AO (adopting the straight conformation) is supported by structural and functional evidence from previous studies 24,25,28-31,34 and from this study. However, whether the AC state corresponds to the straight conformation is not supported by available data and remains an assumption in the model. This model accounts for many aspects of KCNQ1/ I Ks channel function and underscores the pivotal roles of PIP 2 in their gating mechanism. Compounds, such as CA1, which target the V-PIP 2 binding site modulate the activity of I Ks channel ( Fig. 4b-e ) and may specifically modify cardiac physiology without affecting other tissues, such as epithelium, due to the tissue-specific distribution of KCNE1. This feature potentially aids in the development of safe and effective antiarrhythmic therapies. Methods Constructs and mutagenesis Point mutations were introduced in KCNQ1, KCNE1, and CaM via overlap extension and high-fidelity polymerase chain reaction. DNA sequencing verified the existence of all introduced mutations. Mutant complementary RNA (cRNA) was synthesized using the mMessage T7 polymerase kit (Applied Biosystems-Thermo Fisher Scientific). cRNA stocks were stored at -80 ℃. Cryo-EM sample preparation and data collection Construct design, protein expression and purification, nanodisc reconstitution for KCNQ1-KCNE1 followed the same protocol as the recent study 11 . Briefly, N- and C-terminus loop of KCNQ1 are truncated for stability, resulting in a construct including residues 76-620. I145C and K41C mutations were introduced to KCNQ1 and KCNE1, respectively, for KCNQ1-KCNE1 stabilization and purification. To trap KCNQ1-VSD into I state, E160R (E1R) and R231E (R2E) were introduced to KCNQ1. Viruses of KCNQ1-I145C-E1R-R2E and KCNE1-K41C were added with volume ratio 1:1. Resolved structure lacks KCNE1, but CaM binds to KCNQ1 at a 1:1 stoichiometry, as an essential structural component. Quantifoil R1.2/1.3 (400 mesh) holey carbon gold grids were glow-discharged for 30-s. The concentrated protein sample was mixed with 120 mM Fos-Choline-8 at a volume ratio of 1:50 immediately before applying to the grid. 3.5 μL of ~2.5 mg/mL protein sample was applied to each grid, after 20-s waiting, which were double-blotted for 5-s under blot force -3 at 100% humidity and 16 ℃, then vitrified by plunging into liquid ethane cooled by liquid nitrogen using Vitrobot Mark IV (FEI). Dataset was acquired on 300 keV Titan Krios microscope (FEI) equipped with a K3 direct electron detector (Gatan) using EPU software with magnification of 130,000. Data collection was conducted in super-resolution mode with pixel size of 0.6485 Å. Images were recorded with a defocus range of −1.0 to −2.0 μm at a dose rate of 14.7 e/frame/s with images captured over 50 frames. Cryo-EM image processing and 3D reconstruction Image stacks were gain-normalized and corrected for beam-induced motion using MotionCor2 39 . The contrast transfer function parameters were estimated from motion-corrected summed images without dose-weighting using CTFFIND4 40 . All subsequent processing steps were performed on motion-corrected, dose-weighted summed images. Data processing was performed in CryoSPARC 41 . 2D classification was conducted to remove junk particles. Good particles were subjected to Ab-Initio Reconstruction and Heterogeneous Refinement with C1 symmetry. Correct conformational particles were sorted out and input to Non-Uniform Refinement to generate a final reconstruction map with C4 symmetry. Cryo-EM structural refinement and model building Initially, KCNQ1 bent (PDB 6UZZ) was docked into the cryo-EM map KCNQ1-I145C-E160R-R231E. Model was manually built in Coot 42 . PIP 2 molecule was generated as CIF file by the phenix.eLBOW 43 and imported as PT5. The structural model was iteratively refined using phenix.real_space_refine 44 with secondary structure restraints and checked in Coot. The quality of the structures was assessed using the MolProbity server 45 . The pore radii were calculated using HOLE 46 . Figures were created using PyMOL (The PyMOL Molecular Graphics System, Version 2.6.0 and 3.1.1, Schrödinger, LLC) and UCSF Chimera 47 . Molecular docking The interaction between C-PIP 2 and KCNQ1 in the bent conformation ( Figs. 2f, g ) was modeled using AutoDock Vina 48 . The docking box was set to include residues surrounding the “RQKH” motif, and the side chains of residues Q360, K354, K358, and Q361 at the C-PIP 2 binding site were treated as flexible during docking. It is noteworthy that PIP 2 has two long and highly flexible fatty acid chains, which pose challenges for molecular docking. To address this, we used a PIP 2 structure with shortened tails (each containing four carbon atoms) for docking. The PIP 2 structure was treated as flexible. The exhaustiveness parameter was increased from the default value of 8 to 64. A predicted model of the C-PIP 2 -KCNQ1 complex in the bent conformation is consistent with mutagenesis results ( Figs. 2g-i, S8e, f ). In silico compound screening The in silico screening strategy employed our recently developed template-guided docking method 36 as the search engine to screen a subset of the Available Chemical Database (ACD, Molecular Design Ltd.), consisting of approximately 10,000 compounds. Each compound in this subset carries two formal charges and was screened against the V-PIP 2 binding site in the KCNQ1-KCNE1 complex structure. For each compound in the chemical library, OMEGA2 (Version 3.0.1.2, OpenEye, Cadence Molecular Sciences, Santa Fe, NM, USA, http://www.eyesopen.com/) 49,50 was used to generate up to 200 conformers. These 3D conformers were then superimposed onto the co-bound V-PIP 2 (focused on the head group) using the 3D similarity calculation program SHAFTs 51 . Subsequently, the molecular docking program AutoDock Vina 48 was employed to refine the complex structures generated during the superposition step. Compounds were ranked using a hybrid scoring function implemented in our method. Briefly, the ranking score combines the 3D similarity between each compound and the template PIP 2 with the binding score of the compound at the V-PIP 2 site. Additional details of our template-guided docking method are provided in Ref. 36 . The predicted binding modes of the top-ranked compounds were further evaluated by visual inspection. Given that the V-PIP 2 binding site contains several positively charged residues (e.g., R181, K183, and K196), we prioritized compounds with negatively charged groups capable of forming salt bridges with these residues. As a result, compound CA1 was identified as an active binder of KCNQ1 + KCNE1 at the V-PIP 2 site in experimental assays ( Fig. 4c ). Metadynamics simulations We examined the conformational transition of the KCNQ1 channel from the bent to straight conformation without applying external steering forces, as is done in targeted MD (see the next subsection). Well-tempered metadynamics simulations were performed to accelerate this transition using Amber22 52 patched with PLUMED version 2.9.3 53 . Simulations were carried out using a single subunit of KCNQ1 in complex with CaM. The initial hybrid structure was constructed by combining the VSD and pore domains (residues 1–236) preceding the bending point (“RQKH”, residues 251–254), including the bound PIP₂, from the straight conformation, with the remaining regions taken from the bent conformation. The system was embedded in a POPC bilayer and solvated with TIP3P water and 0.15 M NaCl using the CHARMM-GUI server 54 . PIP 2 structures were optimized at the HF/6-31G** level using the Gaussian 16 package 55 , followed by single-point energy calculations at the B3LYP/cc-pVTZ level to obtain the electrostatic potential (ESP). Restrained ESP (RESP) charges were derived from these calculations for use in force field parameterization. Protein parameters were assigned using the AMBER ff14SB force field 56 , and ligand parameters were generated using the general AMBER force field 2 (GAFF2) 57 . The collective variable (CV) was defined as the RMSD of KCNQ1 relative to the straight conformation, and well-tempered metadynamics biasing was applied along this CV to enhance sampling of the “bent-to-straight” transition. Targeted molecular dynamics (TMD) simulations TMD simulations were performed to visualize the conformational transition of the KCNQ1 channel from the IO state (initial structure) to the AO state (target structure). TMD applies a biasing potential that drives the system from an initial to a target conformation by minimizing the root-mean-square deviation (RMSD) between selected atoms of the current and target structures over the course of the simulation. To reduce system complexity and computational cost, we performed the simulations using a single subunit of KCNQ1 in complex with CaM in an implicit solvent system, rather than the full tetrameric complex embedded in a lipid bilayer. This simplification is justified because both the IO and AO conformations have been experimentally determined, and the major structural differences occur at the single-chain level. Our TMD simulation goal was to visualize the conformational changes within one KCNQ1-CaM unit. In our setup, the Cα atoms of the AO state conformation were defined as the target reference. A force constant of 100 kJ·mol⁻¹·nm⁻² was applied to drive the transition. The initial and target structures were pre-aligned prior to simulation to remove overall translational and rotational differences. Artificial rotational motion was further removed every 10 simulation steps to ensure smooth convergence toward the target. TMD simulations were carried out using the Amber22 software suite patched with PLUMED version 2.9.3. Ion channel expression in Xenopus oocytes Stage Ⅴ or Ⅵ oocytes were procured from Xenopus laevis via laparotomy, in accordance with the protocol approved by the Washington University Animal Studies Committee (protocol #24-0405). Oocytes were subjected to digestion with collagenase (0.5 mg/ml, sigma-Aldrich, St. Louis) and subsequently injected with channel cRNAs using a Nanoject (Drummond, Broomall). Each oocyte was injected an identical amount of cRNAs corresponding to either WT or mutant KCNQ1. In tests using KCNE1 or CaM co-expression, KCNE1 and CaM cRNAs were co-injected at mass ratios of 4:1 (KCNQ1: KCNE1) and 1:1 (KCNQ1: CaM), respectively. Injected oocytes were incubated in ND96 solution [96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 5 mM Hepes, 2.5 mM CH 3 COCO 2 Na, and 1: 100 penicilin-streptomycin (pH 7.6)] at 18 ℃ for 3-5 days before recording. Two-electrode voltage clamp (TEVC) Microelectrodes were fabricated using thin wall borosilicate glass (B150-117-10) and a micropipette puller (P-1000, Sutter Instrument, Novato, CA). The pipette resistance ranged from 0.5 to 3 MΩ when filled with a 3 M KCl solution and immerged in ND96 solution, which comprises 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 5 mM Hepes, and 2.5 mM CH 3 COCO 2 Na at pH 7.6. The experiments were recorded in ND96 solutions at room temperature. Whole-cell currents were recorded using a GeneClamp 500B amplifier (Axon Instruments, CA) driven by Patchmaster (HEKA, Holliston, MA). To prevent aliasing, the device applied a low-pass filter to the measured currents at 2 kHz. The testing voltages were from -100 mV to +60 mV (without KCNE1) or +80 mV (with KCNE1 or KCNQ1-F351A) with 10 mV increments and then returned to -40 mV for measuring the tail currents. The holding potential is -80 mV for all electrophysiology recordings. In compound CA1 application experiments, CA1 was initially dissolved in Dimethyl Sulfoxide (DMSO) to make a 10 mM stock solution, and 1 μL of this stock was added to 1000 μL bath solution (ND96) to achieve a final concentration of 10 μM using a manual pipette. The recording chamber was thoroughly rinsed with 70% ethanol and the deionized water following each experiment involving CA1 administrations. All the electrophysiological recordings were repeated with at least two different batches of oocyte. Electrophysiological data analysis Data were analyzed using MATLAB (MathWorks, MA) and Sigmaplot (SPSS) software. The G-V relationship calculation involved normalizing the instantaneous tail currents subsequent to test pulses to the maximum current. The G-V relationship was fitted using a single Boltzmann equation in the form of G(V) = (1 + exp (- V s ( V - V 1/2 )) -1 where V represents the test pulse voltage, V 1/2 represents the half-activation voltage, and V s regulates the steepness of the Boltzmann equation. V s is related to RT/zF , where R denotes the gas constant, T represents the temperature, z signfies the equivalent valence, and F indicates the Faraday constant. The current amplitude comparison was determined using the steady-state current amplitude at the end of the four-seconds test pulse. Statistical analysis Data was presented as mean ± standard error of mean (SEM), with n specifying the number of independent experiments. Statistical analysis, including t-test and one-way ANOVA, were performed using SPSS. Statistical significance was designated as “*” for p ≦ 0.05, “**” for p ≦ 0.005, and “***” for p ≦0.0005; “NS.” represents no statistical difference. Declarations Acknowledgements This work was supported by the National Institute of Health (NIH) grant R01HL155398 and R01HL166628 for Dr. Jianmin Cui and Dr. Xiaoqin Zou. NIH 1R35GM136409 and 2R35GM136409 for Dr. Xiaoqin Zou. American Lebanese Syrian Associated Charities (ALSAC), President Young Professional (PYP) from National University of Singapore, MOE Tier 1 grant A-8002958-00-00 and NIH R00HL143037 to Dr. Ji Sun. We are grateful to Cadence Molecular Science (Santa Fe, NM, USA) for providing the OMEGA2 program and the OEChem Python toolkit (http://www.eyeopen.com/) Author Contributions LZ, JShi, LH, and JC performed mutagenesis and voltage clamp studies. XX, DR, and XZ performed MD simulation and in silico docking, CC, AK and JSun performed cryo-EM data process and analyzed the structure. LZ, XX, CC, RD, XZ, JSun, and JC wrote the manuscript with the input from all authors. Competing Interests The authors declare no conflict interest. Additional Information Supplementary Information is available for this paper. Correspondence and requests for materials should be addressed to Lu Zhao ( [email protected] ), Jianmin Cui ( [email protected] ), Xiaoqin Zou ( [email protected] ), and Ji Sun ( [email protected] ). References Czech, M. P. PIP2 and PIP3: complex roles at the cell surface. Cell 100, 603-606 (2000). Martin, T. F. PI (4, 5) P2 regulation of surface membrane traffic. Current opinion in cell biology 13, 493-499 (2001). McLaughlin, S., Wang, J., Gambhir, A. & Murray, D. PIP2 and proteins: interactions, organization, and information flow. Annual review of biophysics and biomolecular structure 31, 151-175 (2002). Suh, B.-C. & Hille, B. Regulation of ion channels by phosphatidylinositol 4, 5-bisphosphate. Current opinion in neurobiology 15, 370-378 (2005). 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Supplementary Files FigureS13.tif Figure S13-The effects of CA1 on KCNQ1-mutation + KCNE1-WT and mutation channels FigureS8.tif Figure S8-Electrophysiological recordings of additional C-PIP2 binding residues in KCNQ1 FigureS10.tif Figure S10-Electrophysiological recordings of C-PIP2 binding residues co-expressed with KCNE3 FigureS5.tif Figure S5-Mutation scanning of KCNQ1-S2S3L (residues: C180 to K183) with CaM-D96 MovieS1.mp4 Supplementary Movie 1 FigureS14.tif Figure S14-Conformation transition from the bent conformation to the straight conformation in the presence of V-PIP2 FigureS11.tif Figure S11-Electrophysiological recordings of V-PIP2 binding residues in the KCNQ1S338F and KCNQ1F351A backgrounds FigureS1.tif Figure S1-KCNQ1 gating and structure models FigureS4.tif Figure S4-Mutation scanning of KCNQ1-S2S3L and S3 (residues: R190 to K196) with CaM-D94 FigureS6.tif Figure S6-Fig. S6 Mutation scanning of KCNQ1-S2S3L and S3 (residues: R190 to K196) with CaM-D96 FigureS3.tif Figure S3-Mutation scanning of KCNQ1-S2S3L (residues: C180 to Y184) with CaM-D94 FigureS9.tif Figure 9-Electrophysiological recordings of V-PIP2 binding residues in KCNQ1 and KCNE1 FigureS2.tif Figure S2-Putative interactions between KCNQ1-VSD and CaM FigureS7.tif Figure S7-Electrophysiological recordings of C-PIP2 binding residues in both KCNQ1 and KCNE1 FigureS12.tif Figure S12-Structural comparison of the V-PIP2 binding site on KCNQ1 between the straight and bent conformations Supplementaryinformation.docx Supplementary information Cite Share Download PDF Status: Under Review 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. 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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-7609003","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":530301342,"identity":"fffb5dad-5ca6-49c4-99ce-0af3a65f6456","order_by":0,"name":"Jianmin Cui","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqklEQVRIiWNgGAWjYBAC9gYGhgMfwCwg5iFGCyNQy8EZINXMpGhh5iFNS/sZw8O2bYft7ZkZGB+8bSNGS0+OweHctsOJPcwMzIZzidIygweoZdvhBKDD2KR5idZiue2wPVAL+2+itAiCtDBuO8wIdBgbM1FapHnSCg72/ktP7DnM2Cw55xwRWvjYD2/+8OOMtT17e/PBD2/KiNCCBEBRNApGwSgYBaOAOgAA2zEwWA0TP7cAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-9198-6332","institution":"Washington University in Saint Louis","correspondingAuthor":true,"prefix":"","firstName":"Jianmin","middleName":"","lastName":"Cui","suffix":""},{"id":530301343,"identity":"2df194f2-474a-4dad-9212-9b9f5a163d71","order_by":1,"name":"Lu Zhao","email":"","orcid":"https://orcid.org/0000-0003-3533-7693","institution":"Washington University in St. Louis","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Zhao","suffix":""},{"id":530301344,"identity":"980a283d-5b50-462b-876c-469b5436e383","order_by":2,"name":"Xianjin Xu","email":"","orcid":"","institution":"University of Missouri – Columbia","correspondingAuthor":false,"prefix":"","firstName":"Xianjin","middleName":"","lastName":"Xu","suffix":""},{"id":530301345,"identity":"fe53cff2-4b1b-4ea2-b2aa-c910b048b4b3","order_by":3,"name":"Chenxi Cui","email":"","orcid":"https://orcid.org/0000-0001-9698-4494","institution":"National University of Singapore","correspondingAuthor":false,"prefix":"","firstName":"Chenxi","middleName":"","lastName":"Cui","suffix":""},{"id":530301346,"identity":"54c21d66-068d-4f73-b07e-9e7dd25edf00","order_by":4,"name":"Rui Duan","email":"","orcid":"","institution":"University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Duan","suffix":""},{"id":530301347,"identity":"d4e16b24-3518-45a6-8279-bd1bf9a7caa2","order_by":5,"name":"Ali Kermani","email":"","orcid":"","institution":"St Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Kermani","suffix":""},{"id":530301348,"identity":"a45cb575-0602-45b0-8d0b-0d0244eceb52","order_by":6,"name":"Jingyi Shi","email":"","orcid":"","institution":"Washington University in St. Louis","correspondingAuthor":false,"prefix":"","firstName":"Jingyi","middleName":"","lastName":"Shi","suffix":""},{"id":530301349,"identity":"1d2fc2c8-de7a-4197-938e-ca41d54f1c1d","order_by":7,"name":"Lu Han","email":"","orcid":"","institution":"Washington University in Saint Louis","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Han","suffix":""},{"id":530301350,"identity":"63a82ef7-0d80-4d03-a587-e3b2c22ff342","order_by":8,"name":"Ji Sun","email":"","orcid":"https://orcid.org/0000-0002-9302-3177","institution":"St Jude Children's Rsearch Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ji","middleName":"","lastName":"Sun","suffix":""},{"id":530301351,"identity":"6efbd5d5-7423-49c8-b95b-842c177f095f","order_by":9,"name":"Xiaoqin Zou","email":"","orcid":"https://orcid.org/0000-0003-0637-8648","institution":"University of Missouri-Columbia","correspondingAuthor":false,"prefix":"","firstName":"Xiaoqin","middleName":"","lastName":"Zou","suffix":""}],"badges":[],"createdAt":"2025-09-13 18:25:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7609003/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7609003/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94010746,"identity":"b50cd711-2f2a-4666-8310-7ad898329385","added_by":"auto","created_at":"2025-10-21 10:16:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5411644,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConformations of KCNQ1 in various functional states\u003c/strong\u003e. (\u003cstrong\u003ea\u003c/strong\u003e) Cryo-EM map and structure model of KCNQ1-E160R/R231E IC state in the presence of PIP\u003csub\u003e2\u003c/sub\u003e. KCNQ1, PIP\u003csub\u003e2\u003c/sub\u003e and CaM are colored in blue, black and orange, respectively. (\u003cstrong\u003eb\u003c/strong\u003e) Comparison of voltage sensors between KCNQ1-RC state, KCNQ1-IC state and KCNQ1-AO state. Only the helical regions of S2-S4 and S4S5L are shown for clarity. The c-alphas pf the positive charged (or polar) resides on S4 are shown in yellow spheres with stick side chains, and the gating charge transfer center residues F167 (green), E170 and D202 are shown as sticks. (\u003cstrong\u003ec\u003c/strong\u003e) The ion conductance path and core radius for KCNQ1 bent conformation (PDB: 6uzz), KCNQ1 with PIP\u003csub\u003e2\u003c/sub\u003e IC bent conformation, and KCNQ1-KCNE1 with PIP\u003csub\u003e2\u003c/sub\u003e AO straight conformation, respectively. Figures are plotted using the HOLE program. (\u003cstrong\u003ed\u003c/strong\u003e) Representative current traces of one interaction pairs between KCNQ1-S2S3L and CaM: KCNQ1-Y184W and CaM-D96R, displayed without (upper) and with (lower) KCNE1 co-expression. (\u003cstrong\u003ee\u003c/strong\u003e) Current amplitude comparison (+40 mV without KCNE1, +80 mV with KCNE1) of all four identified interaction pairs, shown without (upper) and with (lower) KCNE1. The color of bars was the same as the current traces. The holding potential is -80 mV for all electrophysiology recordings. All error bars are SEM (n ≧ 10).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/c573642d21cf3235ce3b9ee0.png"},{"id":94010791,"identity":"e7d9b055-5930-415a-a247-c91aefb04945","added_by":"auto","created_at":"2025-10-21 10:16:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":292896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC-PIP\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e binding is essential for channel activation\u003c/strong\u003e. (\u003cstrong\u003ea\u003c/strong\u003e) C-PIP\u003csub\u003e2\u003c/sub\u003e binding site in the KCNQ1-KCNE1-PIP\u003csub\u003e2\u003c/sub\u003e complex (PDB: 9VEI). KCNQ1, KCNE1 and PIP\u003csub\u003e2\u003c/sub\u003e are colored in blue, magenta and black, respectively. C-PIP\u003csub\u003e2\u003c/sub\u003e is shown with ball-and-stick model. Key residues are displayed. (\u003cstrong\u003eb\u003c/strong\u003e) Representative current recordings of KCNQ1-WT (left) and mutant KCNQ1-R259Q (middle) channels, displayed without (upper) and with (lower) KCNE1 co-expression. Right: G-V relationship comparison with WT channels. (\u003cstrong\u003ec\u003c/strong\u003e) Current amplitude comparison (+40 mV without KCNE1, +80 mV with KCNE1) of mutagenesis screens of all labeled C-PIP\u003csub\u003e2\u003c/sub\u003e binding sites in (\u003cstrong\u003ea\u003c/strong\u003e). The black bars show without KCNE1, and the red bars show with KCNE1. (\u003cstrong\u003ed\u003c/strong\u003e) V\u003csub\u003e1/2\u003c/sub\u003e of mutagenesis screens (black for without KCNE1, red for with KCNE1) of all labeled C-PIP\u003csub\u003e2\u003c/sub\u003e binding sites in (\u003cstrong\u003ea\u003c/strong\u003e). (\u003cstrong\u003ee\u003c/strong\u003e) Structure depiction of the straight conformation of one monomer of tetrameric KCNQ1-KCNE1-CaM complex (PDB: 9VEI) in the presence of C-PIP\u003csub\u003e2\u003c/sub\u003e. CaM and “RQKH” motif are colored in orange and green, respectively. (\u003cstrong\u003ef\u003c/strong\u003e) Molecular docking of C-PIP\u003csub\u003e2\u003c/sub\u003e into the bent conformation of KCNQ1-E1R/R2E. Secondary structure of the neighboring KCNQ1 subunit are labelled with S6’ and HA’. (\u003cstrong\u003eg\u003c/strong\u003e) Structure depiction of C-PIP\u003csub\u003e2\u003c/sub\u003e binding residues specific to KCNQ1 from molecular docking. (\u003cstrong\u003eh\u003c/strong\u003e) Representative current recordings of KCNQ1-WT (left) and mutant KCNQ1-T264A (middle) channels, displayed without (upper, black) and with (lower, red) KCNE1 co-expression. Right: G-V relationship comparison with WT channels. (\u003cstrong\u003ei\u003c/strong\u003e) Current amplitude comparison of C-PIP\u003csub\u003e2\u003c/sub\u003e binding residue mutations identified by docking in (\u003cstrong\u003eg\u003c/strong\u003e). Black: without KCNE1, red: with KCNE1. All error bars were SEM (n ≧ 10).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/eb5b5726975a9cbb80c79c41.png"},{"id":94010561,"identity":"66d0a4ea-9ea4-431a-9fde-a70cd2832450","added_by":"auto","created_at":"2025-10-21 10:16:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3055377,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eV-PIP\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e binding is essential for channel activation to AO but not to IO\u003c/strong\u003e. (\u003cstrong\u003ea\u003c/strong\u003e) V-PIP\u003csub\u003e2\u003c/sub\u003e binding site in the KCNQ1-KCNE1-PIP\u003csub\u003e2\u003c/sub\u003e complex (PDB: 9VEI). Key residues are displayed. (\u003cstrong\u003eb\u003c/strong\u003e) Representative current recordings of KCNQ1-WT (left) and mutant KCNQ1-Y184W (middle) channels, displayed without (upper, black) and with (lower, red) KCNE1 co-expression. Right: G-V relationship comparison with WT channels. (\u003cstrong\u003ec\u003c/strong\u003e) Current amplitude comparison of mutagenesis screens (+40 mV without KCNE1, +80 mV with KCNE1) of all labeled V-PIP\u003csub\u003e2\u003c/sub\u003e binding sites on (\u003cstrong\u003ea\u003c/strong\u003e). The black bars show without KCNE1, and the red bars show with KCNE1. (\u003cstrong\u003ed\u003c/strong\u003e) V\u003csub\u003e1/2\u003c/sub\u003e of mutagenesis screens (black bars: without KCNE1, red bars: with KCNE1) of all labeled V-PIP\u003csub\u003e2\u003c/sub\u003e binding sites in (\u003cstrong\u003ea\u003c/strong\u003e). (\u003cstrong\u003ee\u003c/strong\u003e) Representative current recordings of KCNQ1-WT + KCNE3-WT (black), mutant KCNQ1-Y184W (red) + KCNE3-WT and KCNQ1-K196Q (green) + KCNE3-WT channels. (\u003cstrong\u003ef\u003c/strong\u003e) The current amplitudes (at +40 mV) for the respective channels. The color of bars was the same as the current traces. (\u003cstrong\u003eg\u003c/strong\u003e) Representative current recordings of KCNQ1-S338F (upper, IO only) and\u003csub\u003e \u003c/sub\u003eKCNQ1-F351A\u003csub\u003e \u003c/sub\u003e(lower, AO only), and mutant KCNQ1-Y184W on KCNQ1-S338F and KCNQ1-F351A, respectively. Right: average G-V relationship comparison for the respective channels with KCNQ1-S338F (upper) and KCNQ1-F351A (lower), respectively. (\u003cstrong\u003eh\u003c/strong\u003e) Current amplitude comparison of selected V-PIP\u003csub\u003e2\u003c/sub\u003e mutants (+40 mV with KCNQ1-S338F, brown; +80 mV with KCNQ1-F351A, orange). The color of bars was the same as the current traces. All error bars are SEM (n ≧ 10).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/5dcf573c232e24e3159b26de.png"},{"id":94010691,"identity":"abeea4c4-8f70-4ff3-af44-8317059e3cf6","added_by":"auto","created_at":"2025-10-21 10:16:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7695987,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eV-PIP\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e modulates IO to AO transition\u003c/strong\u003e. (\u003cstrong\u003ea\u003c/strong\u003e) V-PIP\u003csub\u003e2\u003c/sub\u003e binding pocket with key residues displayed for KCNQ1-E1R/R2E bent conformation and KCNQ1-KCNE1 straight conformation, respectively. KCNQ1, CaM, KCNE1 and V-PIP\u003csub\u003e2\u003c/sub\u003e are colored in blue, orange, magenta, and black, respectively. (\u003cstrong\u003eb\u003c/strong\u003e) \u003cem\u003eIn silico\u003c/em\u003e screening of CA1 to the straight conformation of KCNQ1-KCNE1 (PDB: 9VEI) and the bent conformation of KCNQ1 (PDB: 6uzz). KCNQ1, KCNE1, and CA1 are colored in tan, sky blue and red, respectively. The CA1 binding pocket is represented in orange. (\u003cstrong\u003ec\u003c/strong\u003e) CA1 effects on I\u003csub\u003eKs\u003c/sub\u003e channels. Current traces were recorded before (black) and after (red) adding 10 μM CA1. Average G-V relationship (lower) of I\u003csub\u003eKs\u003c/sub\u003e channels before (black) and after (red) adding 10 μM CA1. (\u003cstrong\u003ed\u003c/strong\u003e) Current amplitude comparative effects of CA1 on KCNQ1-WT (yellow), KCNQ1-WT + KCNE3-WT (green), I\u003csub\u003eKs\u003c/sub\u003e and putative binding residues (rose pink) from molecular docking. Changes in current amplitudes were evaluated at +40 mV without KCNE1 and at +80 mV with KCNE1. (\u003cstrong\u003ee\u003c/strong\u003e) Representative current recordings of CA1 effects on KCNQ1-WT (upper) and KCNQ1-WT + KCNE3-WT (lower). Current traces were recorded before (black) and after (red) adding 10 μM CA1. (\u003cstrong\u003ef\u003c/strong\u003e) Representative structures from the Metadynamics MD simulation illustrating the conformational transition from the bent conformation to the straight conformation (left to right). CaM is shown in cyan, V-PIP\u003csub\u003e2\u003c/sub\u003e in green, S2S3L in pink, S4 in orange, and S6 in yellow.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/0c3a10cf50a218ec89ef767d.png"},{"id":94010673,"identity":"6223ecce-91b2-4181-92a6-4fbfcc3f07a5","added_by":"auto","created_at":"2025-10-21 10:16:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1263170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA model depicting the activation of KCNQ1 by VSD, C-PIP\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e, V-PIP\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and CaM\u003c/strong\u003e. In this model, KCNQ1-RC, IC and IO states adopt a bent conformation, while the AC and AO states adopt a straight conformation. V-PIP\u003csub\u003e2\u003c/sub\u003e associates with the channel during activation of the VSD to the I state, including IC and IO, through a voltage-dependent mechanism that triggers the IO-AO transition. CaM and V-PIP\u003csub\u003e2\u003c/sub\u003e compete for binding to the S2S3L; when CaM binds, the channel adopts a bent conformation, whereas binding of V-PIP\u003csub\u003e2\u003c/sub\u003e results in a straight conformation. C-PIP\u003csub\u003e2\u003c/sub\u003e interacts with the channel in both the I and A states, facilitating the coupling of VSD and pore during the IC-IO and AC-AO transitions.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/62b214df62005af148282a67.png"},{"id":94012948,"identity":"02e5d0cb-54c1-449b-9cfb-12fd7adf1f78","added_by":"auto","created_at":"2025-10-21 10:27:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17784770,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/ba2d912f-5f1e-4cb8-b46f-732adf589170.pdf"},{"id":94010617,"identity":"235bb200-75ed-4098-86a8-28cc397dbf86","added_by":"auto","created_at":"2025-10-21 10:16:41","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":27212034,"visible":true,"origin":"","legend":"Figure S13-The effects of CA1 on KCNQ1-mutation + KCNE1-WT and mutation channels","description":"","filename":"FigureS13.tif","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/04f90bd99b2cdd173ed0350b.tif"},{"id":94010603,"identity":"5adbc0cf-020c-4d10-a1a9-05c48d290130","added_by":"auto","created_at":"2025-10-21 10:16:40","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20747470,"visible":true,"origin":"","legend":"Figure S8-Electrophysiological recordings of additional C-PIP2 binding residues in KCNQ1","description":"","filename":"FigureS8.tif","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/35abcd93cc0e249f9ea55f60.tif"},{"id":94010616,"identity":"727ee9b3-a379-4e67-b7d0-b2abecb2c38f","added_by":"auto","created_at":"2025-10-21 10:16:41","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2076864,"visible":true,"origin":"","legend":"Figure S10-Electrophysiological recordings of C-PIP2 binding residues co-expressed with KCNE3","description":"","filename":"FigureS10.tif","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/f688a1bd2675d4d7ffedff8d.tif"},{"id":94010786,"identity":"2c466a49-3a3d-4338-9ba7-1f6dba731b4c","added_by":"auto","created_at":"2025-10-21 10:16:51","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":25665880,"visible":true,"origin":"","legend":"Figure S5-Mutation scanning of KCNQ1-S2S3L (residues: C180 to K183) with CaM-D96","description":"","filename":"FigureS5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/132721f01a6ba7adfa732014.tif"},{"id":94010728,"identity":"c22bb21b-b9ea-4463-a35b-a7697d206f13","added_by":"auto","created_at":"2025-10-21 10:16:46","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":20068399,"visible":true,"origin":"","legend":"Supplementary Movie 1","description":"","filename":"MovieS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/f1d8ea07a3e75c64ecca24dd.mp4"},{"id":94010790,"identity":"a416379e-c5eb-4cfe-803f-3c196e54c859","added_by":"auto","created_at":"2025-10-21 10:16:52","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":17024740,"visible":true,"origin":"","legend":"Figure S14-Conformation transition from the bent conformation to the straight conformation in the presence of V-PIP2","description":"","filename":"FigureS14.tif","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/4a55cb44f29b57313808b80c.tif"},{"id":94010765,"identity":"3f0ac5c0-efa7-42ef-9bff-bd9cfd3b3c3c","added_by":"auto","created_at":"2025-10-21 10:16:51","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":17561118,"visible":true,"origin":"","legend":"Figure S11-Electrophysiological recordings of V-PIP2 binding residues in the KCNQ1S338F and KCNQ1F351A backgrounds","description":"","filename":"FigureS11.tif","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/229a1d67e66b283412fbb98d.tif"},{"id":94010677,"identity":"d2190945-59eb-425b-84d0-352e2955b88f","added_by":"auto","created_at":"2025-10-21 10:16:43","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":6925330,"visible":true,"origin":"","legend":"Figure S1-KCNQ1 gating and structure models","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/ee26136d608df3e3032600b1.tif"},{"id":94010555,"identity":"77463a60-990e-40db-b931-5f6a91039024","added_by":"auto","created_at":"2025-10-21 10:16:38","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":14672050,"visible":true,"origin":"","legend":"Figure S4-Mutation scanning of KCNQ1-S2S3L and S3 (residues: R190 to K196) with CaM-D94","description":"","filename":"FigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/fa1120ddaee41ece97b06da8.tif"},{"id":94010513,"identity":"f673061e-b69b-42b7-b235-d8dddb383cdd","added_by":"auto","created_at":"2025-10-21 10:16:35","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":32159380,"visible":true,"origin":"","legend":"Figure S6-Fig. S6 Mutation scanning of KCNQ1-S2S3L and S3 (residues: R190 to K196) with CaM-D96","description":"","filename":"FigureS6.tif","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/604c27851266591155e4a516.tif"},{"id":94010675,"identity":"3d4e96e0-3f49-4fd5-be46-9fd889bbe126","added_by":"auto","created_at":"2025-10-21 10:16:43","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":17299374,"visible":true,"origin":"","legend":"Figure S3-Mutation scanning of KCNQ1-S2S3L (residues: C180 to Y184) with CaM-D94","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/f21a24aa1e31b14cc45d8690.tif"},{"id":94010559,"identity":"ecb6a0df-a778-420e-b9a8-d64527768bc4","added_by":"auto","created_at":"2025-10-21 10:16:38","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":11731794,"visible":true,"origin":"","legend":"Figure 9-Electrophysiological recordings of V-PIP2 binding residues in KCNQ1 and KCNE1","description":"","filename":"FigureS9.tif","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/d8c76d24b6141c16b6adad5e.tif"},{"id":94010678,"identity":"b07898ac-e4ec-4fd7-aa16-12745a858b89","added_by":"auto","created_at":"2025-10-21 10:16:43","extension":"tif","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":4395504,"visible":true,"origin":"","legend":"Figure S2-Putative interactions between KCNQ1-VSD and CaM","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/31a0caac5008b7cc25999f20.tif"},{"id":94010528,"identity":"6831b512-2c5f-453e-bedb-06f375282990","added_by":"auto","created_at":"2025-10-21 10:16:38","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":25979634,"visible":true,"origin":"","legend":"Figure S7-Electrophysiological recordings of C-PIP2 binding residues in both KCNQ1 and KCNE1","description":"","filename":"FigureS7.tif","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/e39404829f4ce0d16d9dae51.tif"},{"id":94010527,"identity":"2d00abb4-a765-4126-943f-97c99cb3eff7","added_by":"auto","created_at":"2025-10-21 10:16:37","extension":"tif","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":5389854,"visible":true,"origin":"","legend":"Figure S12-Structural comparison of the V-PIP2 binding site on KCNQ1 between the straight and bent conformations","description":"","filename":"FigureS12.tif","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/0c31db8b927d36fa31977d03.tif"},{"id":94010733,"identity":"99b6dfe6-78ee-4985-a16e-7014cd451d32","added_by":"auto","created_at":"2025-10-21 10:16:47","extension":"docx","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":9131500,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7609003/v1/1dc43d3480c40317254a09a0.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003e\u003cstrong\u003ePIP\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e activation of the cardiac I\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eKs\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e potassium channel\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Main","content":"\u003cp\u003ePhosphatidylinositol 4,5-bisphosphate (PIP\u003csub\u003e2\u003c/sub\u003e) is an important signaling molecule that regulates the function of a variety of membrane proteins \u003csup\u003e1-3\u003c/sup\u003e including many of ion channels \u003csup\u003e4,5\u003c/sup\u003e. Among PIP\u003csub\u003e2\u003c/sub\u003e-regulated ion channels, the family of KCNQ1-5 potassium (K\u003csup\u003e+\u003c/sup\u003e) channels plays a crucial role in regulating membrane excitability in the brain, heart and epithelium \u003csup\u003e6-9\u003c/sup\u003e. PIP\u003csub\u003e2\u003c/sub\u003e is required for KCNQ channel function, and channel activity diminishes when membrane PIP\u003csub\u003e2\u003c/sub\u003e levels are reduced \u003csup\u003e7,10,11\u003c/sup\u003e. Thus, investigating PIP\u003csub\u003e2\u003c/sub\u003e-dependent activation of KCNQ channels could yield valuable insights into the physiological effects and molecular mechanisms of PIP\u003csub\u003e2\u003c/sub\u003e regulation in ion channels. We recently solved the structure of KCNQ1 in association with its regulatory subunit, KCNE1, and PIP\u003csub\u003e2\u003c/sub\u003e molecules (\u003cstrong\u003eFig. S1b\u003c/strong\u003e) \u003csup\u003e11\u003c/sup\u003e, providing a structural foundations for understanding mechanisms of the PIP\u003csub\u003e2\u003c/sub\u003e-dependent activation of KCNQ1 channels.\u003c/p\u003e\n\u003cp\u003eKCNQ1 channels are voltage-gated K\u003csup\u003e+\u003c/sup\u003e (Kv) channels \u003csup\u003e12-14\u003c/sup\u003e. KCNQ1, together with the regulatory subunit KCNE1, forms the slow-delayed rectifier potassium (I\u003csub\u003eKs\u003c/sub\u003e) channels in the heart and inner ear, which are crucial for regulating heart rhythm and supporting K\u003csup\u003e+\u003c/sup\u003e recycling in the endolymph \u003csup\u003e15-17\u003c/sup\u003e. When associated with the regulatory subunit KCNE3, KCNQ1 is constitutively active at physiological voltages, functioning as a K\u003csup\u003e+\u003c/sup\u003e transporter to maintain ionic homeostasis in epithelial and endothelial cells \u003csup\u003e18,19\u003c/sup\u003e. Malfunctions of KCNQ1 caused by drugs, mutations, and single nucleotide polymorphisms are linked to arrhythmia, deafness, atrial fibrillation and type-2 diabetes mellitus \u003csup\u003e12,20-22\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe voltage-dependent gating of K\u003csub\u003ev\u003c/sub\u003e channels involves three fundamental processes, voltage sensor domain (VSD) activation, VSD-pore coupling, and pore opening. The VSD of Kv channels, such as Shaker and KCNQ1, can exist in three resolvable states: resting (R), intermediate (I) and activated (A). Upon membrane depolarization the VSD of these channels transition from the R state to the A state via the I state \u003csup\u003e23-25\u003c/sup\u003e. However, in Shaker channels, the pore conducts only at the activated open (AO) state when the VSD is in the A state \u003csup\u003e23,26\u003c/sup\u003e. In contrast, in KCNQ1, the pore opens when the VSD is in either the I or the A state, resulting in the IO (intermediate open) and AO states \u003csup\u003e14,24,27,28\u003c/sup\u003e (\u003cstrong\u003eFig. S1a\u003c/strong\u003e). The IO and AO states exhibit drastically different properties in their voltage dependence, activation kinetics, current amplitude, ion permeability, and pharmacology \u003csup\u003e13,14,24\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe IO and AO states of KCNQ1 are differentially regulated by various KCNE subunits, which exhibit distinct tissue distributions \u003csup\u003e15,16,18,19\u003c/sup\u003e. This differential regulation enables KCNQ1 + KCNE channels to function across diverse tissues, supporting a wide range of physiological functions. For instance, both KCNE1 and KCNE3 selectively shift the VSD activation to the I state at more negative voltages \u003csup\u003e18\u003c/sup\u003e. However, KCNE1 suppresses IO but enhances AO of KCNQ1, making the channel open exclusively in the AO state, with a more positively shifted voltage dependence, slower activation kinetics, and larger current amplitudes \u003csup\u003e24,28\u003c/sup\u003e. These features are essential for terminating cardiac action potential. In contrast, KCNE3 does not suppress IO, allowing KCNQ1 + KCNE3 channels open at more negative voltages and remain constitutively open at physiological voltages to maintain K\u003csup\u003e+\u003c/sup\u003e homeostasis in the epithelium \u003csup\u003e18,19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe structural basis underlying the IO and AO states in KCNQ1 remains unclear. KCNQ1 channels adopt a homo-tetrameric assembly, where each subunit contains six transmembrane α-helices S1-S6, with S1-S4 forming the VSD and S5 and S6 forming the central pore \u003csup\u003e29,30\u003c/sup\u003e. Each KCNQ1 subunit interacts with a calmodulin (CaM) molecule mainly through its cytosolic helix A (HA) and helix B (HB) (\u003cstrong\u003eFig. S1b\u003c/strong\u003e). Our previous structural studies revealed two distinct conformations of the cytosolic domain in KCNQ1 channels; straight and bent \u003csup\u003e29,30\u003c/sup\u003e. In the straight conformation, HA forms a continuous helix with the transmembrane helix S6, positioning CaM deep in the cytosol, away from the membrane-spanning domain of KCNQ1. The bent conformation is facilitated by a structural kink at the “RQKH” motif, located between helix S6 and HA. This kink allows HA to adopt an upward-bent orientation, enabling CaM to undergo rotational displacement to interact with the cytosolic S2-S3 linker (S2S3L). Our previous functional studies demonstrated that mutations disrupting the S2S3L-CaM interface shift KCNQ1 channel properties from predominantly IO to characteristics resembling the AO state. Based on these findings, we proposed that the IO state likely adopts the bent conformation, while the AO state corresponds to the straight conformation, and that disrupting S2S3L-CaM interactions promotes the transition from the IO to the AO state \u003csup\u003e31\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur prior studies demonstrated that PIP\u003csub\u003e2\u003c/sub\u003e is required for VSD-pore coupling in KCNQ1 in both the IO and AO states \u003csup\u003e7,10\u003c/sup\u003e. Without PIP\u003csub\u003e2\u003c/sub\u003e, the VSD activates but fails to induce pore opening. Functional and mutagenesis studies indicated a PIP\u003csub\u003e2\u003c/sub\u003e binding site at the interface between the VSD and the pore, essential for KCNQ1 channel opening \u003csup\u003e7\u003c/sup\u003e. However, our previous structural analyses of KCNQ1 and KCNQ1 + KCNE3 did not clearly show PIP\u003csub\u003e2\u003c/sub\u003e binding at the VSD-pore interface but instead revealed a PIP\u003csub\u003e2\u003c/sub\u003e-binding site adjacent to the VSD \u003csup\u003e30\u003c/sup\u003e. We recently solved the structure of the KCNQ1-KCNE1 complex, which revealed not only the PIP\u003csub\u003e2\u003c/sub\u003e-binding site near the VSD, but also an additional PIP\u003csub\u003e2\u003c/sub\u003e site at the VSD-pore interface, yielding a total of eight PIP\u003csub\u003e2\u003c/sub\u003e molecules in the channel complex \u003csup\u003e11\u003c/sup\u003e (\u003cstrong\u003eFig. S1b\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eIn this study we solved the structure of KCNQ1 in an I state. We examined the structural and functional properties of the two PIP\u003csub\u003e2\u003c/sub\u003e-binding sites in KCNQ1. These results, together with the identification of a compound, CA1, that selectively binds to one of PIP\u003csub\u003e2\u003c/sub\u003e sites and specifically enhances the activation of KCNQ1 only in the presence of KCNE1, support that the IO and AO states adopt the bent and straight conformations, respectively. Through electrophysiological experiments and structural characterization of KCNQ1 channel at the I state, we found that the first PIP\u003csub\u003e2\u003c/sub\u003e molecule located near the VSD and termed V-PIP\u003csub\u003e2\u003c/sub\u003e for its role in VSD function (\u003cstrong\u003eFig. S1b\u003c/strong\u003e), drives the conformational shift from IO to AO during VSD activation. VSD activation to the A state alters the conformation of V-PIP\u003csub\u003e2\u003c/sub\u003e and its binding site (\u003cstrong\u003eFig. 4a\u003c/strong\u003e), detaching CaM from the VSD and promoting the transition from bent to straight. The second PIP\u003csub\u003e2\u003c/sub\u003e molecule, designated C-PIP\u003csub\u003e2\u003c/sub\u003e, was revealed in our recently determined KCNQ1-KCNE1 structure at the VSD-pore interface, promoting VSD-pore coupling to enable channel opening in both IO and AO states while stabilizing the straight conformation (\u003cstrong\u003eFigs. 4g, S1b\u003c/strong\u003e) \u003csup\u003e11\u003c/sup\u003e. VSD-pore coupling determines the channel’s ability to open, whereas the IO-to-AO transition is critical for the function of I\u003csub\u003eKs\u003c/sub\u003e channels in the heart. Therefore, both PIP\u003csub\u003e2\u003c/sub\u003e molecules are vital for the physiological functions of KCNQ1 channels.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConformations of KCNQ1 in various functional states\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVoltage dependent gating of KCNQ1 involves two measurable steps of VSD activation to the I and A states, both of which trigger pore opening to the IO and AO (\u003cstrong\u003eFig. S1a\u003c/strong\u003e). Previous structural studies indicate that KCNQ1 may adopt a bent conformation in the resting-closed (RC) state \u003csup\u003e32\u003c/sup\u003e and a straight conformation in the AO state \u003csup\u003e11,30\u003c/sup\u003e. Then, what is the conformation, bent or straight, in the IO state? To address this question, we trapped the VSD of KCNQ1 in I state by introducing E160R/R231E (E1R/R2E) mutations \u003csup\u003e24,33\u003c/sup\u003e (\u003cstrong\u003eFig. 1a\u003c/strong\u003e). The structure of KCNQ1-E1R/R2E in I state shares high similarity with KCNQ1-PIP\u003csub\u003e2\u003c/sub\u003e structure (PDB: 9VEN), and both adopt a bent conformation: the S6 and HA helices form a helix-loop-helix structure. The R237 (R4) residue of the S4 helix points to the gate charge transfer center formed by F167, E170 and D202, while R231 (R2) and H240 (H5) of S4 helix sit within the gate charge transfer center in the resting state (PDB: 8SIN) and activated state (PDB: 9VE1), respectively. This supports that the structure is captured in the I state, with the S4 is ~5 Å lower than in the A state \u003csup\u003e34\u003c/sup\u003e. The ion conductance pathway plotted using HOLE program shows that the pore radius (~1 Å at the narrowest point) is too small for hydrated potassium to pass, suggesting that the structure may represent an intermediate-closed (IC) state. In the structure, one PIP\u003csub\u003e2\u003c/sub\u003e molecule is observed at the V-PIP\u003csub\u003e2\u003c/sub\u003e site, which is mainly formed by the S2S3L, S3 and S4-S5 linker (S4S5L) (\u003cstrong\u003eFigs. 1a-c\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eIn functional studies, while the KCNQ1-E1R/R2E channels showed constitutive macroscopic currents independent of voltage \u003csup\u003e24,33\u003c/sup\u003e, single-channel recordings exhibited flickering openings with low open probability \u003csup\u003e28\u003c/sup\u003e. These results are consistent with the KCNQ1-E1R/R2E structure being in the IC conformation. Then, does the IO state adopt the bent conformation? In KCNQ1 channel structures, each subunit binds a CaM molecule at the cytosolic domain\u003csup\u003e11,29,30,32,34\u003c/sup\u003e. In the bent conformation, CaM interacts with the S2S3L on the cytosolic side of the VSD (\u003cstrong\u003eFig. 1a\u003c/strong\u003e). During VSD activation (R →I →A), if the channel remains bent, with S2S3L and CaM likely interacting differently before reaching the straight A state, these S2S3L-CaM interactions would influence activation of the IO state more than to the AO state.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo test this hypothesis, we performed mutational scanning in both CaM (D94 and D96) and S2S3L (C180, R181, S182, K183, Y184, L191 and R192) as well as in S3 (R195 and K196) to evaluate the function of individual mutations and their pairs in the activation of KCNQ1 and I\u003csub\u003eKs\u003c/sub\u003e, respectively (\u003cstrong\u003eFigs. S2-6\u003c/strong\u003e). Four pairs of these mutations are noteworthy: KCNQ1-C180D in S2S3L paired with CaM-D94K and R195Q in S3 paired with CaM-D94K (\u003cstrong\u003eFigs. 1e, S3a, S4d\u003c/strong\u003e), and KCNQ1-Y184W and L191D in S2S3L, each paired with CaM-D96R (\u003cstrong\u003eFigs. 1d, e, S6b\u003c/strong\u003e). Among these four pairs of mutations, three double mutations (KCNQ1-C180D and CaM-D94K, KCNQ1-R195Q and CaM-D94K, KCNQ1-Y184W and CaM-D96R) caused near complete loss of current compared to single mutations in KCNQ1 or CaM alone, whereas the KCNQ1-L191D and CaM-D96R double mutation pair significantly recued the current of KCNQ1-L191D mutation (\u003cstrong\u003eFigs. 1e, S6b\u003c/strong\u003e). Such drastic changes in current amplitude by the double mutations suggests that these paired residues may interact during channel opening. The four mutation pairs specifically affect KCNQ1, as their double mutations in I\u003csub\u003eKs\u003c/sub\u003e produced current amplitudes similar to those of single KCNQ1 or CaM mutations (\u003cstrong\u003eFigs. 1d, e, S3a, S4d, S6b\u003c/strong\u003e). It is unlikely that these double mutations reduce surface expression, as evidenced by the robust currents exhibited by the mutant I\u003csub\u003eKs\u003c/sub\u003e complexes, although it is known that KCNQ1 and KCNE1 traffic to the plasma membrane via independent pathways in both cardiac myocytes and \u003cem\u003eXenopus\u003c/em\u003e oocytes \u003csup\u003e35\u003c/sup\u003e. Since KCNQ1 opens predominantly to the IO state and I\u003csub\u003eKs\u003c/sub\u003e opens exclusively to the AO state, these results support that the channel adopts a bent conformation in the IO state and a straight conformation in the AO state. Further support for the correlation between the conformation and functional state is shown below.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC-PIP\u003csub\u003e2\u003c/sub\u003e binding is essential for channel activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the KCNQ1-E1R/R2E structure, no obvious C-PIP\u003csub\u003e2\u003c/sub\u003e was observed. Is C-PIP\u003csub\u003e2\u003c/sub\u003e important for both the IO and AO states, and what role does C-PIP\u003csub\u003e2\u003c/sub\u003e play? The KCNQ1-KCNE1 structure reveals that C-PIP\u003csub\u003e2\u003c/sub\u003e interacts with residues in the S4S5 linker (V255 and F256), S5 (R259, Q260, L262 and L263) of one KCNQ1 subunit, and S6-HA (Q359, K362, and R366) of a neighboring KCNQ1 subunit, and the KCNE1 (F57, I61, L63, S64, R67, S68, K69, and K70) subunit (\u003cstrong\u003eFig. 2a\u003c/strong\u003e). First, we mutated each C-PIP\u003csub\u003e2\u003c/sub\u003e-interacting residue in both KCNQ1 and KCNE1 to alanine (Ala) or neutralized charged amino acids (glutamine Q or asparagine N) and assessed the function of the mutant channels. All mutations, except KCNE1-F57A, significantly reduced I\u003csub\u003eKs\u003c/sub\u003e current amplitudes (\u003cstrong\u003eFigs. 2b, c, S7\u003c/strong\u003e). The similar effect of KCNE1-K70N was reported previously \u003csup\u003e10\u003c/sup\u003e. The C-PIP\u003csub\u003e2\u003c/sub\u003e site mutations also reduced KCNQ1 current amplitude in the absence of KCNE1 (\u003cstrong\u003eFigs. 2b, c, S7\u003c/strong\u003e). \u0026nbsp;These results are consistent with observations made when membrane PIP\u003csub\u003e2\u003c/sub\u003e levels were reduced \u003csup\u003e10,11\u003c/sup\u003e, indicating that these mutations diminish PIP\u003csub\u003e2\u003c/sub\u003e binding to the KCNQ1 channel and that C-PIP\u003csub\u003e2\u003c/sub\u003e is critical for the conductance of KCNQ1 and I\u003csub\u003eKs\u003c/sub\u003e. Importantly, all these mutations caused only minor shifts of the voltage-dependent activation, with the conductance-voltage (G-V) relationship deviating from the wild-type (WT) less than 25 mV (\u003cstrong\u003eFigs. 2d, S7\u003c/strong\u003e). This is consistent with the mechanism whereby C-PIP\u003csub\u003e2\u003c/sub\u003e binding is crucial for coupling of VSD movements and pore opening but not for VSD activation \u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAmong the exanimated mutations, KCNQ1-I263A, Q359A, K362A and R366A produced less pronounced reductions in KCNQ1 current amplitude than the reduction observed in the I\u003csub\u003eKs\u003c/sub\u003e current (\u003cstrong\u003eFigs. 2c, S8a-d\u003c/strong\u003e). These results indicate that the C-PIP\u003csub\u003e2\u003c/sub\u003e binding sites in KCNQ1 may alter in the absence of KCNE1 \u003csup\u003e32\u003c/sup\u003e. Notably, residues Q359, K362 and R366 within or around the “RQKH” motif, which is the hinge for the bent and straight conformation switch (\u003cstrong\u003eFig. 2e\u003c/strong\u003e), interact with C-PIP\u003csub\u003e2\u003c/sub\u003e primarily in the presence of KCNE1 (\u003cstrong\u003eFigs. 2c, S8b-d\u003c/strong\u003e). Thus, the binding of C-PIP\u003csub\u003e2\u003c/sub\u003e to these residues may help KCNE1 stabilize the straight conformation \u003csup\u003e7,10\u003c/sup\u003e, while the disruption of the “RQKH” motif from it α-helical structure into a loop may alter C-PIP\u003csub\u003e2\u003c/sub\u003e binding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo verify this notion, we evaluated the structure of the C-PIP\u003csub\u003e2\u003c/sub\u003e binding sites in the bent conformation. Since C-PIP\u003csub\u003e2\u003c/sub\u003e binding was not observed in any available structures of KCNQ1 in the bent conformation, we performed molecular docking of C-PIP\u003csub\u003e2\u003c/sub\u003e to the bent conformation of KCNQ1 (\u003cstrong\u003eFig. 2f\u003c/strong\u003e). The results revealed that the residues I263, Q359, K362, and R366 no longer interact with C-PIP\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e(\u003cstrong\u003eFigs. 2f, g\u003c/strong\u003e). These findings align with the functional data showing that mutations of these residues reduce KCNQ1 current amplitudes to a lesser extent than I\u003csub\u003eKs\u0026nbsp;\u003c/sub\u003e(\u003cstrong\u003eFigs. 2c, S8a-d\u003c/strong\u003e). Molecular docking further revealed that, in the bent conformation, residues T264, K358 and Q361 interacted with the C-PIP\u003csub\u003e2\u003c/sub\u003e, which are not part of the C-PIP\u003csub\u003e2\u003c/sub\u003e site in the I\u003csub\u003eKs\u003c/sub\u003e structure. Mutation of these residues reduced KCNQ1 current amplitudes to a greater extent than I\u003csub\u003eKs\u0026nbsp;\u003c/sub\u003e(\u003cstrong\u003eFigs. 2g-i, S8e, f\u003c/strong\u003e). These results indicate that the opening of KCNQ1 channel is associated with the bent conformation, whereas the opening of I\u003csub\u003eKs\u003c/sub\u003e channel is associated with the straight conformation, and the C-PIP\u003csub\u003e2\u003c/sub\u003e site alters between the two conformations. Our previous findings indicated that both KCNQ1 and I\u003csub\u003eKs\u003c/sub\u003e require PIP\u003csub\u003e2\u003c/sub\u003e for channel function, but the EC\u003csub\u003e50\u003c/sub\u003e of PIP\u003csub\u003e2\u003c/sub\u003e dose-response for KCNQ1 was over 100-fold greater than that for I\u003csub\u003eKs\u003c/sub\u003e, suggesting a higher PIP\u003csub\u003e2\u003c/sub\u003e affinity for I\u003csub\u003eKs\u003c/sub\u003e \u003csup\u003e10\u003c/sup\u003e. The differences in C-PIP\u003csub\u003e2\u003c/sub\u003e binding sites within KCNQ1 across different states, combined with the role of KCNE1 in facilitating C-PIP\u003csub\u003e2\u003c/sub\u003e binding, explain the\u0026nbsp;﹥100-fold increase in PIP\u003csub\u003e2\u003c/sub\u003e sensitivity observed in I\u003csub\u003eKs\u003c/sub\u003e channels compared to KCNQ1 alone. This mechanism also accounts for the observation that no PIP\u003csub\u003e2\u003c/sub\u003e is resolved in the C-site in the bent conformation due to its low affinity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eV-PIP\u003csub\u003e2\u003c/sub\u003e binding is essential for channel activation to AO but not to IO\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the structure of KCNQ1-E1R/R2E, V-PIP\u003csub\u003e2\u003c/sub\u003e interacts with residues in the S2S3L (R181, K183, and Y184), the S3 (K196 and I198) and S4S5L (Q244, W248, and R249) (\u003cstrong\u003eFigs. 1a, 4a\u003c/strong\u003e). In the structure of KCNQ1-KCNE1 (PDB: 9VEI), V-PIP\u003csub\u003e2\u003c/sub\u003e additionally interacts with the N-terminus S0 (Y111 and R116) of KCNQ1, as well as with KCNE1 (E72 and H73) (\u003cstrong\u003eFig. 3a\u003c/strong\u003e). We mutated these V-PIP\u003csub\u003e2\u003c/sub\u003e interacting residues to A or N/Q using a mutation scanning. Most mutations in KCNQ1 (R181, K183, Y184, K196, and R249) did not reduce but rather increased KCNQ1 current amplitudes (\u003cstrong\u003eFigs. 3c, S9c-f, i\u003c/strong\u003e), with the exceptions of Q244N and W248A (\u003cstrong\u003eFigs. 3c, S9g, h\u003c/strong\u003e), which are critical for VSD-pore coupling \u003csup\u003e25\u003c/sup\u003e. Y111A and R116A reduced KCNQ1 current amplitudes (\u003cstrong\u003eFigs. 3c, S9a, b\u003c/strong\u003e); however, these residues did not interact with V-PIP\u003csub\u003e2\u003c/sub\u003e in the bent conformation and the IO state, indicating that the effects of Y111A and R116A on KCNQ1 current amplitudes may not directly disrupt V-PIP\u003csub\u003e2\u003c/sub\u003e binding. On the other hand, most of the mutations significantly reduced the current amplitudes of I\u003csub\u003eKs\u003c/sub\u003e except for the KCNQ1-R181Q + KCNE1 and KCNQ1-K183N + KCNE1 (\u003cstrong\u003eFigs. 3c, S9c, d\u003c/strong\u003e). Although these two mutations enhanced I\u003csub\u003eKs\u003c/sub\u003e current amplitudes, the enhancement was significantly smaller compared to the enhanced KCNQ1 current amplitudes of the same mutant. All mutations induce small GV shifts in both KCNQ1 (less than\u0026nbsp;15 mV) and I\u003csub\u003eKs\u003c/sub\u003e (less than\u0026nbsp;25 mV) (\u003cstrong\u003eFigs. 3d, S9\u003c/strong\u003e), indicating that the mutations did not modify current amplitudes by altering voltage dependence. Additionally, we observed that mutations at the V-PIP\u003csub\u003e2\u003c/sub\u003e sites did not significantly affect the current amplitudes of KCNQ1 + KCNE3 (\u003cstrong\u003eFigs. 3e, f\u003c/strong\u003e), whereas mutations at the C-PIP\u003csub\u003e2\u003c/sub\u003e sites drastically reduced the current amplitudes of KCNQ1 + KCNE3 (\u003cstrong\u003eFig. S10\u003c/strong\u003e). Thus, these results suggest that V-PIP\u003csub\u003e2\u003c/sub\u003e is essential for the AO state but not the IO state, as both KCNQ1 and KCNQ1 + KCNE3 primarily open to the IO state, whereas I\u003csub\u003eKs\u003c/sub\u003e exclusively opens to the AO state\u0026nbsp;\u003csup\u003e24,25,28,34\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo validate this mechanism, we introduced some of the V-PIP\u003csub\u003e2\u003c/sub\u003e site mutations into the mutant KCNQ1-S338F and KCNQ1-F351A, which were previously established as opening only in the IO and AO state, respectively \u003csup\u003e24,28\u003c/sup\u003e. Notably, these mutations either had no impact (Y184, K196, I198, Q244, and R249) or enhanced KCNQ1-S338F current amplitudes (K183), while significantly reducing KCNQ1-F351A current amplitudes (\u003cstrong\u003eFigs. 3g, h, S11a-d\u003c/strong\u003e), supporting the notion that V-PIP\u003csub\u003e2\u003c/sub\u003e is specifically required for the AO state, but not the IO state. By contrast, KCNQ1-R259Q, which affects C-PIP\u003csub\u003e2\u003c/sub\u003e binding, reduced the current amplitudes of both KCNQ1-S338F and KCNQ1-F351A (\u003cstrong\u003eFig. S11e\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eV-PIP\u003csub\u003e2\u003c/sub\u003e modulates IO to AO transition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComparing V-PIP\u003csub\u003e2\u003c/sub\u003e and its binding site in the structures of KCNQ1-E1R/R2E and KCNQ1-KCNE1 (\u003cstrong\u003eFig. 4a\u003c/strong\u003e), notable differences were observed in the interaction network of PIP\u003csub\u003e2\u003c/sub\u003e. In the I state, the head group of PIP\u003csub\u003e2\u003c/sub\u003e contacts S4, S4S5L, S2S3L and CaM. Upon further activation of the VSD, S4 moves upward, followed by S4S5L and S2S3L, a motion that appears to elevate the center of the PIP\u003csub\u003e2\u003c/sub\u003e-binding pocket. This motion weakens interactions between CaM and the rest of the V-PIP\u003csub\u003e2\u003c/sub\u003e site, facilitating the alteration of the V-PIP\u003csub\u003e2\u003c/sub\u003e site and repositioning of V-PIP\u003csub\u003e2\u003c/sub\u003e to form closer interactions with S2S3L (\u003cstrong\u003eFigs. 4a, S12\u003c/strong\u003e). The upward movement of the PIP\u003csub\u003e2\u003c/sub\u003e-interaction network, the detachment of CaM from the V-PIP\u003csub\u003e2\u003c/sub\u003e site, and the alteration of V-PIP\u003csub\u003e2\u003c/sub\u003e binding are associated with the “bent-to-straight” conformational transition of the KCNQ1 channel.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo validate the observed structural difference in V-PIP\u003csub\u003e2\u003c/sub\u003e binding between the I and A states, we employed small-molecule screening approaches \u003csup\u003e36\u003c/sup\u003e. We reasoned that a compound selectively binding to the V-PIP\u003csub\u003e2\u003c/sub\u003e site in the straight conformation would modulate the I\u003csub\u003eKs\u003c/sub\u003e channel without affecting KCNQ1 alone in the I state. We screened the Available Chemical Database (ACD, Molecular Design Ltd.) by docking its compound to the V-PIP\u003csub\u003e2\u003c/sub\u003e binding sites of KCNQ1-KCNE1in the straight conformation. Docking results revealed that the compound CA1 (\u003cstrong\u003eFig. S13a\u003c/strong\u003e), selectively binds to the straight conformation of the channel, with the binding site nearly overlapping with the V-PIP\u003csub\u003e2\u003c/sub\u003e site. In contrast, the binding pocket undergoes structural rearrangement in the bent conformation, preventing CA1 from binding (\u003cstrong\u003eFig. 4b\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur functional data showed that CA1 significantly enhanced the current amplitudes of I\u003csub\u003eKs\u003c/sub\u003e and shifted its GV to more negative voltages (\u003cstrong\u003eFig. 4c\u003c/strong\u003e). Mutations in the putative CA1-binding residues (\u003cstrong\u003eFig. S13b\u003c/strong\u003e) attenuated the effects of CA1 (\u003cstrong\u003eFigs. 4d, S13d-o\u003c/strong\u003e), supporting our docking results for the CA1 binding site in the straight conformation of the I\u003csub\u003eKs\u003c/sub\u003e channel, which opens exclusively to the AO state. Conversely, CA1 exhibited no enhancing effect on channels predominantly open to the IO state in the bent conformation, including KCNQ1 alone and the KCNQ1-KCNE3 complex (\u003cstrong\u003eFigs. 4d, e\u003c/strong\u003e). Notably, KCNQ1-KCNE3 could adopt a straight conformation and open in the AO state, albeit with a low probability \u003csup\u003e30\u003c/sup\u003e. Docking of CA1 onto the straight conformation of KCNQ1-KCNE3 showed that CA1 could bind to the same pocket (\u003cstrong\u003eFig. S13c\u003c/strong\u003e). Thus, the absence of CA1-induced current augmentation in KCNQ1 + KCNE3 channels is consistent with the complex primarily conducting in the IO state via the bent conformation, which precludes CA1 binding.\u003c/p\u003e\n\u003cp\u003eTo support the above mechanism that the VSD movement to the A state and the change in V-PIP\u003csub\u003e2\u003c/sub\u003e trigger the bent to straight conformational switch, we performed molecular dynamics (MD) simulations by first constructing a hybrid starting model. In this model, the VSD and pore domains (residues 1–236) preceding the bending point (RQKH, residues 251–254), including the bound PIP\u003csub\u003e2\u003c/sub\u003e, were taken from the straight conformation, whereas the remaining regions were retained from the bent conformation. We then performed metadynamics simulations to accelerate sampling along the “bent-to-straight” transition. Representative snapshots are shown in \u003cstrong\u003eFig. 4f\u003c/strong\u003e. Along the biased trajectory, CaM progressively dissociated from the VSD and rotated while KCNQ1 spontaneously adopted a straight configuration. These conformational changes align with our structures of the I and A states and support the mechanism in which upward S4 movement facilitates VSD activation and conformational changes in the V-PIP\u003csub\u003e2\u003c/sub\u003e binding site, displacing CaM and stabilizing a straight cytosolic conformation, thus promoting the AO state.\u003c/p\u003e\n\u003cp\u003eTo visualize the conformational transition from the bent conformation to the straight conformation, we also performed targeted MD simulations. Snapshots from the simulation are presented in \u003cstrong\u003eFig. S14 and Movie. S1\u003c/strong\u003e. During the transition, the S4 helix exhibits an upward movement compared to its position in the bent conformation (\u003cstrong\u003eFig. S14a\u003c/strong\u003e). The phosphatidylinositol head group of V-PIP\u003csub\u003e2\u003c/sub\u003e undergoes a reorientation toward S2S3L, establishing contacts with this linker (\u003cstrong\u003eFig S14b\u003c/strong\u003e). Concurrently, CaM gradually dissociates from the VSD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA model of the activation of KCNQ1 and I\u003csub\u003eKs\u003c/sub\u003e by VSD, PIP\u003csub\u003e2\u003c/sub\u003e and CaM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on our findings and prior studies, we propose a model for KCNQ1 and I\u003csub\u003eKs\u003c/sub\u003e activation (\u003cstrong\u003eFig. 5\u003c/strong\u003e). In this model, V-PIP\u003csub\u003e2\u003c/sub\u003e interacts with the channel via a voltage-dependent mechanism to trigger the IO to AO transition, while C-PIP\u003csub\u003e2\u003c/sub\u003e is associated with the channel to mediate the VSD-pore coupling and stabilize the straight conformation. The recently published structure of KCNQ1 with the VSD in the RC state indicated that the channel adopts a bent conformation, which obstructs the V-PIP\u003csub\u003e2\u003c/sub\u003e site and prevents its binding \u003csup\u003e32\u003c/sup\u003e. Therefore, V-PIP\u003csub\u003e2\u003c/sub\u003e binds to the channel upon VSD activation to the I state, in both IC and IO, which adopt the bent conformation (\u003cstrong\u003eFig. 5\u003c/strong\u003e). V-PIP\u003csub\u003e2\u003c/sub\u003e is subsequently reoriented upon VSD further activation to the A state, triggering the switch from bent to straight, which culminates in the activating-closed (AC) and AO states (\u003cstrong\u003eFigs. 3, 4f\u003c/strong\u003e). C-PIP\u003csub\u003e2\u003c/sub\u003e binds to the channel in both the I and A states to facilitate the VSD-pore coupling during the IC-IO and AC-AO transitions, but whether C-PIP\u003csub\u003e2\u003c/sub\u003e binds to the RC state is not clear. In the I state, C-PIP\u003csub\u003e2\u003c/sub\u003e binds to the channel with a lower affinity than in the A state owing to the alteration of the C-PIP\u003csub\u003e2\u003c/sub\u003e binding sites during the “bent-to-straight” transition (\u003cstrong\u003eFig. 2\u003c/strong\u003e). The KCNE1 association inhibits the IO state and enhances the AO state (\u003cstrong\u003eFig. S1a\u003c/strong\u003e) \u003csup\u003e24,25,28\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe propose that, even in association with KCNE1, the KCNQ1 subunit undergoes the same bent and straight transition as the VSD moves from the I state to the A state, based on the following observations. First, V-PIP\u003csub\u003e2\u003c/sub\u003e is essential for the activation of the KCNQ1 + KCNE1 channel (\u003cstrong\u003eFig. 3\u003c/strong\u003e), potentially by stabilizing the straight conformation. Second, C-PIP\u003csub\u003e2\u003c/sub\u003e in I\u003csub\u003eKs\u003c/sub\u003e exhibits a state-dependent change in binding affinity; in the open state (AO, straight), the affinity is enhanced, and C-PIP\u003csub\u003e2\u003c/sub\u003e is resistant to digestion by the lipid phosphatase CiVSP. By contrast, the C-PIP\u003csub\u003e2\u003c/sub\u003e affinity decreases following channel deactivation at hyperpolarized voltages (IC and RC, bent), allowing for digestion by CiVSP \u003csup\u003e37,38\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe correspondence between RC, IC, and IO (all adopting bent conformation) and AO (adopting the straight conformation) is supported by structural and functional evidence from previous studies \u003csup\u003e24,25,28-31,34\u003c/sup\u003e and from this study. However, whether the AC state corresponds to the straight conformation is not supported by available data and remains an assumption in the model. This model accounts for many aspects of KCNQ1/ I\u003csub\u003eKs\u003c/sub\u003e channel function and underscores the pivotal roles of PIP\u003csub\u003e2\u003c/sub\u003e in their gating mechanism.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompounds, such as CA1, which target the V-PIP\u003csub\u003e2\u003c/sub\u003e binding site modulate the activity of I\u003csub\u003eKs\u003c/sub\u003e channel (\u003cstrong\u003eFig. 4b-e\u003c/strong\u003e) and may specifically modify cardiac physiology without affecting other tissues, such as epithelium, due to the tissue-specific distribution of KCNE1. This feature potentially aids in the development of safe and effective antiarrhythmic therapies.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eConstructs and mutagenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePoint mutations were introduced in KCNQ1, KCNE1, and CaM via overlap extension and high-fidelity polymerase chain reaction. DNA sequencing verified the existence of all introduced mutations. Mutant complementary RNA (cRNA) was synthesized using the mMessage T7 polymerase kit (Applied Biosystems-Thermo Fisher Scientific). cRNA stocks were stored at -80 ℃.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM sample preparation and data collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConstruct design, protein expression and purification, nanodisc reconstitution for KCNQ1-KCNE1 followed the same protocol as the recent study \u003csup\u003e11\u003c/sup\u003e. Briefly, N- and C-terminus loop of KCNQ1 are truncated for stability, resulting in a construct including residues 76-620. I145C and K41C mutations were introduced to KCNQ1 and KCNE1, respectively, for KCNQ1-KCNE1 stabilization and purification. To trap KCNQ1-VSD into I state, E160R (E1R) and R231E (R2E) were introduced to KCNQ1. Viruses of KCNQ1-I145C-E1R-R2E and KCNE1-K41C were added with volume ratio 1:1. Resolved structure lacks KCNE1, but CaM binds to KCNQ1 at a 1:1 stoichiometry, as an essential structural component.\u003c/p\u003e\n\u003cp\u003eQuantifoil R1.2/1.3 (400 mesh) holey carbon gold grids were glow-discharged for 30-s. The concentrated protein sample was mixed with 120 mM Fos-Choline-8 at a volume ratio of 1:50 immediately before applying to the grid. 3.5 \u0026mu;L of ~2.5 mg/mL protein sample was applied to each grid, after 20-s waiting, which were double-blotted for 5-s under blot force -3 at 100% humidity and 16 ℃, then vitrified by plunging into liquid ethane cooled by liquid nitrogen using Vitrobot Mark IV (FEI).\u003c/p\u003e\n\u003cp\u003eDataset was acquired on 300 keV Titan Krios microscope (FEI) equipped with a K3 direct electron detector (Gatan) using EPU software with magnification of 130,000. Data collection was conducted in super-resolution mode with pixel size of 0.6485 \u0026Aring;. Images were recorded with a defocus range of \u0026minus;1.0 to \u0026minus;2.0\u0026thinsp;\u0026mu;m at a dose rate of 14.7 e/frame/s with images captured over 50 frames.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM image processing and 3D reconstruction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImage stacks were gain-normalized and corrected for beam-induced motion using MotionCor2 \u003csup\u003e39\u003c/sup\u003e. The contrast transfer function parameters were estimated from motion-corrected summed images without dose-weighting using CTFFIND4 \u003csup\u003e40\u003c/sup\u003e. All subsequent processing steps were performed on motion-corrected, dose-weighted summed images. Data processing was performed in CryoSPARC \u003csup\u003e41\u003c/sup\u003e. 2D classification was conducted to remove junk particles. Good particles were subjected to Ab-Initio Reconstruction and Heterogeneous Refinement with C1 symmetry. Correct conformational particles were sorted out and input to Non-Uniform Refinement to generate a final reconstruction map with C4 symmetry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM structural refinement and model building\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInitially, KCNQ1 bent (PDB 6UZZ) was docked into the cryo-EM map KCNQ1-I145C-E160R-R231E. Model was manually built in Coot \u003csup\u003e42\u003c/sup\u003e. PIP\u003csub\u003e2\u003c/sub\u003e molecule was generated as CIF file by the phenix.eLBOW \u003csup\u003e43\u003c/sup\u003e and imported as PT5. The structural model was iteratively refined using phenix.real_space_refine \u003csup\u003e44\u003c/sup\u003e with secondary structure restraints and checked in Coot. The quality of the structures was assessed using the MolProbity server \u003csup\u003e45\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe pore radii were calculated using HOLE \u003csup\u003e46\u003c/sup\u003e. Figures were created using PyMOL (The PyMOL Molecular Graphics System, Version 2.6.0 and 3.1.1, Schr\u0026ouml;dinger, LLC) and UCSF Chimera \u003csup\u003e47\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe interaction between C-PIP\u003csub\u003e2\u003c/sub\u003e and KCNQ1 in the bent conformation (\u003cstrong\u003eFigs. 2f, g\u003c/strong\u003e) was modeled using \u0026nbsp;AutoDock Vina \u003csup\u003e48\u003c/sup\u003e. The docking box was set to include residues surrounding the \u0026ldquo;RQKH\u0026rdquo; motif, and the side chains of residues Q360, K354, K358, and Q361 at the C-PIP\u003csub\u003e2\u003c/sub\u003e binding site were treated as flexible during docking. It is noteworthy that PIP\u003csub\u003e2\u003c/sub\u003e has two long and highly flexible fatty acid chains, which pose challenges for molecular docking. To address this, we used a PIP\u003csub\u003e2\u003c/sub\u003e structure with shortened tails (each containing four carbon atoms) for docking. The PIP\u003csub\u003e2\u003c/sub\u003e structure was treated as flexible. The exhaustiveness parameter was increased from the default value of 8 to 64. A predicted model of the C-PIP\u003csub\u003e2\u003c/sub\u003e-KCNQ1 complex in the bent conformation is consistent with mutagenesis results (\u003cstrong\u003eFigs. 2g-i, S8e, f\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn silico\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;compound screening\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ein silico\u003c/em\u003e screening strategy employed our recently developed template-guided docking method \u003csup\u003e36\u003c/sup\u003e as the search engine to screen a subset of the Available Chemical Database (ACD, Molecular Design Ltd.), consisting of approximately 10,000 compounds. Each compound in this subset carries two formal charges and was screened against the V-PIP\u003csub\u003e2\u003c/sub\u003e binding site in the KCNQ1-KCNE1 complex structure.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor each compound in the chemical library, OMEGA2 (Version 3.0.1.2, OpenEye, Cadence Molecular Sciences, Santa Fe, NM, USA, http://www.eyesopen.com/) \u003csup\u003e49,50\u003c/sup\u003e was used to generate up to 200 conformers. These 3D conformers were then superimposed onto the co-bound V-PIP\u003csub\u003e2\u003c/sub\u003e (focused on the head group) using the 3D similarity calculation program SHAFTs \u003csup\u003e51\u003c/sup\u003e. Subsequently, the molecular docking program AutoDock Vina \u003csup\u003e48\u003c/sup\u003e was employed to refine the complex structures generated during the superposition step. Compounds were ranked using a hybrid scoring function implemented in our method. Briefly, the ranking score combines the 3D similarity between each compound and the template PIP\u003csub\u003e2\u003c/sub\u003e with the binding score of the compound at the V-PIP\u003csub\u003e2\u003c/sub\u003e site. Additional details of our template-guided docking method are provided in Ref. \u003csup\u003e36\u003c/sup\u003e. The predicted binding modes of the top-ranked compounds were further evaluated by visual inspection. Given that the V-PIP\u003csub\u003e2\u003c/sub\u003e binding site contains several positively charged residues (e.g., R181, K183, and K196), we prioritized compounds with negatively charged groups capable of forming salt bridges with these residues.\u003c/p\u003e\n\u003cp\u003eAs a result, compound CA1 was identified as an active binder of KCNQ1 + KCNE1 at the V-PIP\u003csub\u003e2\u003c/sub\u003e site in experimental assays (\u003cstrong\u003eFig. 4c\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetadynamics simulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe examined the conformational transition of the KCNQ1 channel from the bent to straight conformation without applying external steering forces, as is done in targeted MD (see the next subsection). Well-tempered metadynamics simulations were performed to accelerate this transition using Amber22 \u003csup\u003e52\u003c/sup\u003e patched with PLUMED version 2.9.3 \u003csup\u003e53\u003c/sup\u003e. Simulations were carried out using a single subunit of KCNQ1 in complex with CaM. The initial hybrid structure was constructed by combining the VSD and pore domains (residues 1\u0026ndash;236) preceding the bending point (\u0026ldquo;RQKH\u0026rdquo;, residues 251\u0026ndash;254), including the bound PIP₂, from the straight conformation, with the remaining regions taken from the bent conformation. The system was embedded in a POPC bilayer and solvated with TIP3P water and 0.15 M NaCl using the CHARMM-GUI server \u003csup\u003e54\u003c/sup\u003e. PIP\u003csub\u003e2\u003c/sub\u003e structures were optimized at the HF/6-31G** level using the Gaussian 16 package \u003csup\u003e55\u003c/sup\u003e, followed by single-point energy calculations at the B3LYP/cc-pVTZ level to obtain the electrostatic potential (ESP). Restrained ESP (RESP) charges were derived from these calculations for use in force field parameterization. Protein parameters were assigned using the AMBER ff14SB force field \u003csup\u003e56\u003c/sup\u003e, and ligand parameters were generated using the general AMBER force field 2 (GAFF2) \u003csup\u003e57\u003c/sup\u003e. The collective variable (CV) was defined as the RMSD of KCNQ1 relative to the straight conformation, and well-tempered metadynamics biasing was applied along this CV to enhance sampling of the \u0026ldquo;bent-to-straight\u0026rdquo; transition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTargeted molecular dynamics (TMD) simulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTMD simulations were performed to visualize the conformational transition of the KCNQ1 channel from the IO state (initial structure) to the AO state (target structure). TMD applies a biasing potential that drives the system from an initial to a target conformation by minimizing the root-mean-square deviation (RMSD) between selected atoms of the current and target structures over the course of the simulation. To reduce system complexity and computational cost, we performed the simulations using a single subunit of KCNQ1 in complex with CaM in an implicit solvent system, rather than the full tetrameric complex embedded in a lipid bilayer. This simplification is justified because both the IO and AO conformations have been experimentally determined, and the major structural differences occur at the single-chain level. Our TMD simulation goal was to visualize the conformational changes within one KCNQ1-CaM unit. In our setup, the C\u0026alpha; atoms of the AO state conformation were defined as the target reference. A force constant of 100 kJ\u0026middot;mol⁻\u0026sup1;\u0026middot;nm⁻\u0026sup2; was applied to drive the transition. The initial and target structures were pre-aligned prior to simulation to remove overall translational and rotational differences. Artificial rotational motion was further removed every 10 simulation steps to ensure smooth convergence toward the target. TMD simulations were carried out using the Amber22 software suite patched with PLUMED version 2.9.3.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIon channel expression in \u003cem\u003eXenopus\u003c/em\u003e oocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStage\u0026nbsp;\u003cem\u003eⅤ\u003c/em\u003e or\u0026nbsp;\u003cem\u003eⅥ\u003c/em\u003e oocytes were procured from \u003cem\u003eXenopus\u003c/em\u003e laevis via laparotomy, in accordance with the protocol approved by the Washington University Animal Studies Committee (protocol #24-0405). Oocytes were subjected to digestion with collagenase (0.5 mg/ml, sigma-Aldrich, St. Louis) and subsequently injected with channel cRNAs using a Nanoject (Drummond, Broomall). Each oocyte was injected an identical amount of cRNAs corresponding to either WT or mutant KCNQ1. In tests using KCNE1 or CaM co-expression, KCNE1 and CaM cRNAs were co-injected at mass ratios of 4:1 (KCNQ1: KCNE1) and 1:1 (KCNQ1: CaM), respectively. Injected oocytes were incubated in ND96 solution [96 mM NaCl, 2 mM KCl, 1.8 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 5 mM Hepes, 2.5 mM CH\u003csub\u003e3\u003c/sub\u003eCOCO\u003csub\u003e2\u003c/sub\u003eNa, and 1: 100 penicilin-streptomycin (pH 7.6)] at 18 ℃ for 3-5 days before recording.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTwo-electrode voltage clamp (TEVC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicroelectrodes were fabricated using thin wall borosilicate glass (B150-117-10) and a micropipette puller (P-1000, Sutter Instrument, Novato, CA). The pipette resistance ranged from 0.5 to 3 M\u0026Omega; when filled with a 3 M KCl solution and immerged in ND96 solution, which comprises 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 5 mM Hepes, and 2.5 mM CH\u003csub\u003e3\u003c/sub\u003eCOCO\u003csub\u003e2\u003c/sub\u003eNa at pH 7.6. The experiments were recorded in ND96 solutions at room temperature. Whole-cell currents were recorded using a GeneClamp 500B amplifier (Axon Instruments, CA) driven by Patchmaster (HEKA, Holliston, MA). To prevent aliasing, the device applied a low-pass filter to the measured currents at 2 kHz.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe testing voltages were from -100 mV to +60 mV (without KCNE1) or +80 mV (with KCNE1 or KCNQ1-F351A) with 10 mV increments and then returned to -40 mV for measuring the tail currents. The holding potential is -80 mV for all electrophysiology recordings.\u003c/p\u003e\n\u003cp\u003eIn compound CA1 application experiments, CA1 was initially dissolved in Dimethyl Sulfoxide (DMSO) to make a 10 mM stock solution, and 1 \u0026mu;L of this stock was added to 1000 \u0026mu;L bath solution (ND96) to achieve a final concentration of 10 \u0026mu;M using a manual pipette. The recording chamber was thoroughly rinsed with 70% ethanol and the deionized water following each experiment involving CA1 administrations.\u003c/p\u003e\n\u003cp\u003eAll the electrophysiological recordings were repeated with at least two different batches of oocyte.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectrophysiological data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were analyzed using MATLAB (MathWorks, MA) and Sigmaplot (SPSS) software. The G-V relationship calculation involved normalizing the instantaneous tail currents subsequent to test pulses to the maximum current. The G-V relationship was fitted using a single Boltzmann equation in the form of\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eG(V)\u003c/em\u003e = (1 + exp (-\u003cem\u003eV\u003csub\u003es\u003c/sub\u003e\u003c/em\u003e(\u003cem\u003eV\u003c/em\u003e-\u003cem\u003eV\u003csub\u003e1/2\u003c/sub\u003e\u003c/em\u003e))\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003ewhere \u003cem\u003eV\u003c/em\u003e represents the test pulse voltage, \u003cem\u003eV\u003csub\u003e1/2\u003c/sub\u003e\u003c/em\u003e represents the half-activation voltage, and \u003cem\u003eV\u003csub\u003es\u003c/sub\u003e\u003c/em\u003e regulates the steepness of the Boltzmann equation. \u003cem\u003eV\u003csub\u003es\u003c/sub\u003e\u003c/em\u003e is related to \u003cem\u003eRT/zF\u003c/em\u003e, where \u003cem\u003eR\u003c/em\u003e denotes the gas constant, \u003cem\u003eT\u003c/em\u003e represents the temperature, \u003cem\u003ez\u003c/em\u003e signfies the equivalent valence, and \u003cem\u003eF\u003c/em\u003e indicates the Faraday constant. The current amplitude comparison was determined using the steady-state current amplitude at the end of the four-seconds test pulse.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData was presented as mean \u0026plusmn; standard error of mean (SEM), with n specifying the number of independent experiments. Statistical analysis, including t-test and one-way ANOVA, were performed using SPSS. Statistical significance was designated as \u0026ldquo;*\u0026rdquo; for p ≦ 0.05, \u0026ldquo;**\u0026rdquo; for p ≦ 0.005, and \u0026ldquo;***\u0026rdquo; for p ≦0.0005; \u0026ldquo;NS.\u0026rdquo; represents no statistical difference.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Institute of Health (NIH) grant R01HL155398 and R01HL166628 for Dr. Jianmin Cui and Dr. Xiaoqin Zou. NIH 1R35GM136409 and 2R35GM136409 for Dr. Xiaoqin Zou. American Lebanese Syrian Associated Charities (ALSAC), President Young Professional (PYP) from National University of Singapore, MOE Tier 1 grant A-8002958-00-00 and NIH R00HL143037 to Dr. Ji Sun. We are grateful to Cadence Molecular Science (Santa Fe, NM, USA) for providing the OMEGA2 program and the OEChem Python toolkit (http://www.eyeopen.com/)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLZ, JShi, LH, and JC performed mutagenesis and voltage clamp studies. XX, DR, and XZ performed MD simulation and \u003cem\u003ein silico\u003c/em\u003e docking, CC, AK and JSun performed cryo-EM data process and analyzed the structure. LZ, XX, CC, RD, XZ, JSun, and JC wrote the manuscript with the input from all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary Information is available for this paper. Correspondence and requests for materials should be addressed to Lu Zhao ([email protected]), Jianmin Cui ([email protected]), Xiaoqin Zou ([email protected]), and Ji Sun ([email protected]).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCzech, M. P. PIP2 and PIP3: complex roles at the cell surface. \u003cem\u003eCell\u003c/em\u003e 100, 603-606 (2000). \u003c/li\u003e\n\u003cli\u003eMartin, T. F. 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The Journal of chemical physics 153 (2020). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"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":"V-PIP2, C-PIP2, KCNQ1, IKs, anti-arrhythmia therapy","lastPublishedDoi":"10.21203/rs.3.rs-7609003/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7609003/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe I\u003csub\u003eKs\u003c/sub\u003e channel complex, composed of the voltage-gated potassium channel KCNQ1 and its regulatory subunit KCNE1, is essential for the termination of cardiac action potentials. The function of KCNQ1 and I\u003csub\u003eKs\u003c/sub\u003e requires PIP\u003csub\u003e2\u003c/sub\u003e, and its depletion abolishes channel opening. Previous studies revealed that KCNQ1 adopts both bent and straight conformations and can bind two PIP\u003csub\u003e2\u003c/sub\u003e molecules: one adjacent to VSD (V-PIP\u003csub\u003e2\u003c/sub\u003e), and the other at the VSD-pore interface (C-PIP\u003csub\u003e2\u003c/sub\u003e). Here we show that the two PIP\u003csub\u003e2\u003c/sub\u003e perform essential yet distinct roles: V-PIP\u003csub\u003e2\u003c/sub\u003e enables the bent-to-straight transition, whereas C-PIP\u003csub\u003e2\u003c/sub\u003e mediates VSD-pore coupling and stabilizes the straight conformation. Structure-function analysis and molecular dynamic simulations show that VSD activation elevates the V-PIP\u003csub\u003e2\u003c/sub\u003e site and weakens the CaM-VSD interaction, permitting the conformational shift from the bent, intermediate open (IO) state associated with KCNQ1 to the straight, I\u003csub\u003eKs\u003c/sub\u003e-exclusive activated open (AO) state, which is further stabilized by C-PIP\u003csub\u003e2\u003c/sub\u003e. Leveraging this mechanism, we developed a compound CA1, which selectively targets the V-PIP\u003csub\u003e2\u003c/sub\u003e site and modulates I\u003csub\u003eKs\u003c/sub\u003e channel activity without affecting KCNQ1, offering a novel and promising conceptional path for specific and safe antiarrhythmic therapeutics.\u003c/p\u003e","manuscriptTitle":"PIP2 activation of the cardiac IKs potassium channel","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-21 09:56:50","doi":"10.21203/rs.3.rs-7609003/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":"94099d51-7fd7-468b-be6b-dbb2f3fa1e79","owner":[],"postedDate":"October 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56629930,"name":"Biological sciences/Biophysics/Membrane biophysics/Ion transport"},{"id":56629931,"name":"Physical sciences/Engineering/Biomedical engineering"}],"tags":[],"updatedAt":"2026-04-24T20:40:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-21 09:56:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7609003","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7609003","identity":"rs-7609003","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}