KChIP1 splice variants modulate Kv4 channels by promoting P/C-type inactivation features

KChIP1 splice variants modulate Kv4 channels by promoting P/C-type inactivation features | 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 KChIP1 splice variants modulate Kv4 channels by promoting P/C-type inactivation features Wuyou Cao, Georgios Tachtsidis, Robert Bähring This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7356877/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 14 You are reading this latest preprint version Abstract Kv4 channels mediate a somatodendritic A-type (i.e., rapidly inactivating) potassium current, which controls neuronal excitability and firing frequency. Kv4 channels form complexes with auxiliary DPPs and KChIPs, which modify channel gating, including an acceleration of recovery from inactivation. Although ternary Kv4 + DPP + KChIP complexes represent a likely native channel configuration, little is known about the concerted Kv4 channel modulation by DPPs and KChIPs. Here, we studied the modulatory effects of two functionally distinct KChIP1 splice variants (1a and 1b), utilizing two-electrode voltage-clamp in Xenopus oocytes. We tested Kv4.1, Kv4.2, Kv4.3S, and Kv4.3L, co-expressed with either KChIP1 splice variant in binary Kv4 + KChIP1 and ternary Kv4 + DPP + KChIP1 channel configurations. For all Kv4.x channels, we observed an extremely slow component of recovery from inactivation upon co-expression of either KChIP1 splice variant, which persisted in a ternary configuration with DPP. Our results suggest a special functional role of KChIP1b, limiting the time-dependent availability of the somatodendritic A-type current. Our mechanistic investigations of ternary Kv4.2 + DPP + KChIP1b channels revealed a strong enhancement of P/C-type inactivation features, which are normally vestigial in Kv4 channels, but may co-exist with preferential closed-state inactivation in the presence of KChIP1b. Biological sciences/Biophysics Biological sciences/Neuroscience A-type current repetitive firing recovery from inactivation exponential fitting Xenopus oocytes two-electrode voltage-clamp Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Voltage-gated potassium (Kv) channels control action potential generation, shape and frequency 1 . The Kv4 channel subfamily underlies a somatodendritic A-type (i.e., rapidly inactivating) current ( I SA ) 2 , 3 , involved in the control of low frequency neuronal firing, dendritic excitation and synaptic plasticity 4 – 7 . Like all Kv channels, Kv4 channels are formed by the assembly of four α-subunits, surrounding a central ion conduction pathway (Fig. S1 ). Each α-subunit consists of six transmembrane segments (S1 - S6) with intracellular N- and C-termini and an N-terminal tetramerization (T) domain, which controls subfamily-specific assembly 8 . The transmembrane portions of the α-subunit can be divided into a voltage sensing (S1-S4) and a pore module (S5-S6) with a selectivity filter sequence located between S5 and S6 (Fig. S1 ). The distal S6 segments constitute the gate of the tetrameric channel by forming an aperture, able to either constrict or splay apart in a voltage-dependent manner 9 . Blocking of the open channel from the cytoplasmic side by an intrinsic N-terminal inactivation domain (N-type inactivation) 10 , and conformational rearrangements of the external pore mouth near the selectivity filter (P/C-type inactivation) 11 , two well-investigated mechanisms of Kv channel autoinhibition, are vestigial in Kv4 channels, which undergo preferential closed-state inactivation (CSI) based on dynamic S6 rearrangements 12 – 14 . Kv4 channels form complexes with two types of auxiliary β-subunits, membrane-spanning dipeptidyl amino peptidase-related proteins (DPPs) 15 and cytoplasmic Kv channel interacting proteins (KChIPs 16 ; Fig. S1 ). Association of either type of β-subunit enhances Kv4 channel surface expression and modulates Kv4 channel inactivation gating in a specific manner. Typically, macroscopic inactivation of Kv4 channels is accelerated by DPPs and slowed by KChIPs, whereas recovery from inactivation is accelerated by both 15 – 21 . Structures of ternary Kv4 + DPP + KChIP complexes 14 , 22 , 23 , a likely native channel configuration 24 , indicate that subunit assembly occurs in a 4:4:4 stoichiometry (Fig. S1 ). There are four human KChIP subtypes (KChIP1–4; Fig. S2), encoded by four different genes ( KCNIP1–4 ) 25 . Utilizing alternative transcription start sites and alternative splicing, KCNIP1 yields different KChIP1 isoforms. The KChIP1a and KChIP1b splice variants differ by an N-terminal 11-amino-acid stretch, rich in aromatic side chains, only found in KChIP1b 26 , 27 (Fig. S2). This N-terminal aromatic cluster has been reported to be responsible for the differential channel modulation by KChIP1b when co-expressed by transient cDNA transfection of a stable Kv4.2-expressing cell line. In particular, KChIP1b was found to induce an extremely slow component of recovery from inactivation 27 , 28 , when other KChIPs, including KChIP1a, usually accelerate the recovery of Kv4 channels from inactivation 16 – 18 , 27 . Here, we asked whether the peculiar KChIP1b feature of slowing recovery from inactivation is also observed in a ternary configuration with DPP and for other Kv4.x channels. Intriguingly, our data reveal the induction of a slow recovery component for both KChIP1a and KChIP1b (referred to as 1a and 1b for simplicity below) co-expression. The effect is consistently observed for all Kv4.x channels, both in a binary configuration and in a ternary configuration with DPP. These results suggest a functional role for the KChIP1-mediated slow recovery from inactivation, which was therefore addressed mechanistically. Results Modulation of Kv4 channel-mediated A-type currents by the KChIP splice variants 1a and 1b To study different aspects of Kv4 channel modulation by the two KChIP splice variants 1a and 1b, we expressed Kv4.x channels (Kv4.1, Kv4.2, Kv4.3S or Kv4.3L) in the following configurations (see Fig. S1 ): 1. Kv4.x α-subunit alone; 2. Kv4.x α-subunit together with either 1a or 1b; 3. Kv4.x α-subunit together with DPP; 4. Kv4.x α-subunit together with DPP and either 1a or 1b. The effects of 1a or 1b co-expression on the following parameters were examined: 1. Macroscopic inactivation kinetics; 2. Kinetics of recovery from inactivation; 3. Voltage dependences of activation and steady-state inactivation (see Methods). The present study was primarily focussed on the effects of KChIP1 splice variant co-expression on Kv4.2 (Figs. 1 – 3 ), in order to complement previously published data obtained with a Kv4.2 stable Human Embryonic Kidney (HEK) 293 cell line, transiently transfected with 1a or 1b cDNA in the absence of DPP 27 , 28 . In the Supplements our results for Kv4.1, Kv4.3S and Kv4.3L are illustrated (Figs. S3 - S5), and the data for all Kv4.x channels, including statistical analysis results, are summarized (Tables S1 - S4). We initially analysed the kinetics of macroscopic inactivation during prolonged depolarizing voltage pulses (Fig. 1 A, B; see Methods). We obtained time constants of ∼ 16, 68, 809 ms (81, 15, 4%) for Kv4.2 alone and ∼ 9, 50, 1136 ms (86, 11, 3%) for Kv4.2 + DPP (Fig. 1 C, D; Table S2). Both in a binary and in a ternary configuration (see Fig. S1 ), the co-expression of the KChIP splice variants 1a and 1b caused the typical crossover of normalized current traces when overlayed with control traces obtained in the absence of KChIP1 18 (Fig. 1 A, B). This was due to a slowing of the initial and intermediate decay components, while the final decay component was unaffected or slightly accelerated by KChIP1 co-expression. In the binary configuration we obtained time constants of ∼ 47, 113, 697 ms (18, 80, 2%) for Kv4.2 + 1a, and ∼ 34, 118, 615 ms (42, 48, 10%) for Kv4.2 + 1b (data for 10 ng KChIP1 cRNA per oocyte, but the differences between 1a and 1b co-expression in a binary configuration did not depend on the amount of KChIP1 cRNA; Fig. 1 C). In the ternary configuration the time constants were ∼ 32, 65, 531 ms (41, 57, 2%) for Kv4.2 + DPP + 1a, and ∼ 33, 85, 483 ms (48, 48, 4%) for Kv4.2 + DPP + 1b (Fig. 1 D; Table S2). The effects of auxiliary subunit co-expression on Kv4.1, Kv4.3S and Kv4.3L-mediated current decay kinetics showed a considerable variability. However, the typical acceleration of the final cumulative component of macroscopic inactivation was consistently observed for both 1a and 1b co-expression and in either channel configuration (Fig. S3; Tables S1, S3 and S4). - Fig. 1 - Unusual modulation of Kv4 channel recovery kinetics by the KChIP splice variants 1a and 1b It has been reported previously that, unlike 1a, 1b causes biphasic kinetics of recovery from inactivation, resulting in a slowing of the overall recovery process in Kv4.2 channels 27 , 28 . We found that in the absence of KChIP1 the recovery of Kv4.2 channels from inactivation followed a single-exponential time course, and the recovery kinetics were accelerated by DPP co-expression 15 (Fig. 2 A - D). The recovery time constants were 563 ms for Kv4.2 alone and 117 ms for Kv4.2 + DPP (Fig. 2 E, F; Table S2). Surprisingly, we observed biphasic recovery kinetics for both 1a and 1b co-expression in either channel configuration (Fig. 3 C, D), albeit the obtained time constants and their relative amplitudes differed for the two KChIP1 splice variants, with a more pronounced slowing for 1b. In the binary configuration, we obtained recovery time constants of 126 ms and ∼1.5 s (87 and 13%) for Kv4.2 + 1a, and 218 ms and ∼5.4 s, (56 and 44%) for Kv4.2 + 1b (data for 10 ng KChIP1 cRNA per oocyte, but the distinct remodelling features seen for 1a and 1b co-expression in a binary configuration did not depend on the amount of KChIP1 cRNA; Fig. 2 E; Table S2). In a ternary configuration, the recovery time constants were ∼24 and 395 ms (79 and 21%) for Kv4.2 + DPP + 1a, and ∼ 41 ms and 1.3 s (57 and 43%) for Kv4.2 + DPP + 1b (Fig. 2 F; Table S2). The modulation of recovery kinetics caused by 1a and 1b co-expression observed for Kv4.1, Kv4.3S and Kv4.3L in a binary and a ternary configuration was very similar to the results obtained with Kv4.2 (Fig. S4; Tables S1, S3 and S4). - Fig. 2 - Voltage dependence of Kv4 channel steady-state inactivation is differentially modulated by KChIP splice variants 1a and 1b Finally, we examined the effects of 1a and 1b co-expression on the voltage dependences of activation and steady-state inactivation (Fig. 3 ; see Methods). For all channel configurations tested, the Kv4.2 activation and inactivation curves showed little overlap, characteristic for preferential CSI 12 , and inactivation curves were steeper than activation curves (Fig. 3 A, C). For Kv4.2 alone, halfmaximal activation occurred at + 5.5 mV with a slope factor of 33.8 mV (Fig. 3 B; Table S2). DPP co-expression resulted in a negative shift of the activation curve 15 , with halfmaximal activation at -28.5 mV (slope factor 23.2 mV) for Kv4.2 + DPP (Fig. 3 D; Table S2). In a binary configuration, co-expression of KChIP1 splice variants caused a steepening and a negative shift of activation curves with halfmaximal activation at -9.6 mV (slope factor 25.6 mV) and − 5.7 mV (slope factor 30.8 mV) for Kv4.2 + 1a and Kv4.2 + 1b, respectively (data for 10 ng KChIP1 cRNA per oocyte, but the differences between 1a and 1b co-expression in a binary configuration were not dependent on the amount of KChIP1 cRNA; Fig. 3 A, B; Table S2). In a ternary configuration with DPP, KChIP1 co-expression caused a positive shift of activation curves, with halfmaximal activation at -19.2 mV (slope factor 26.1 mV) and − 12.1 mV (slope factor 33.7 mV) for Kv4.2 + DPP + 1a and Kv4.2 + DPP + 1b, respectively (Fig. 3 C, D; Table S2). - Fig. 3 - The analysis of steady-state inactivation revealed that halfmaximal inactivation occurred at -68.3 mV (slope factor 6.4 mV) for Kv4.2 alone (Fig. 3 B). Similar to the voltage dependence of activation, the inactivation curve was shifted negative by DPP co-expression 15 , with halfmaximal inactivation at -73.5 mV (slope factor 4.7 mV) for Kv4.2 + DPP (Fig. 3 D; Table S2). The modulation of the voltage dependence of steady-state inactivation differed between 1a and 1b co-expression. While 1a caused virtually no effect, 1b shifted inactivation curves in the negative direction (Fig. 3 A, C; Table S2). In a binary configuration, halfmaximal inactivation occurred at -66.3 mV (slope factor 5.2 mV) for Kv4.2 + 1a and at -76.0 mV (slope factor 6.1 mV) for Kv4.2 + 1b (data for 10 ng KChIP1 cRNA per oocyte, but the differences between 1a and 1b co-expression in a binary configuration were not dependent on the amount of KChIP1 cRNA; Fig. 3 B; Table S2). In a ternary configuration with DPP, halfmaximal inactivation occurred at -74.4 mV (slope factor 4.3 mV) for Kv4.2 + DPP + 1a and at -81.3 mV (slope factor 7.4 mV) for Kv4.2 + DPP + 1b (Fig. 3 D; Table S2). The modulation of the voltage dependences of Kv4.1, Kv4.3S and Kv4.3L gating caused by 1a and 1b co-expression in a binary and a ternary configuration was very similar to the results described for Kv4.2. Although the results showed some variability among the different Kv4.x channels, binary and ternary channels containing 1b had the most negative inactivation curves (Fig. S5; Tables S1, S3 and S4). High external K + identifies P/C-type inactivation features of the 1b-mediated slow recovery component Our kinetic analyses of recovery from inactivation have corroborated the view that the biphasic nature of the recovery process represents an intrinsic feature of KChIP1-containing Kv4 channels. Moreover, our data suggest that, unlike 1a, 1b co-expression induces only negligible acceleration of recovery, but more or less exclusively adds a slow recovery component (see Fig. 2 C, D). Therefore, we chose Kv4.2 + DPP + 1b ternary channels (1.7 + 5 + 10 ng cRNA per oocyte) to study the kinetics of recovery from inactivation in more mechanistic detail. To this end, we intended to experimentally interfere with different mechanisms of inactivation, normally vestigial in Kv4 channels 12 , but possibly promoted by 1b co-expression, such as N-type inactivation 29 or P/C-type inactivation 30 . Vestigial N-type inactivation features of Kv4 channels are thought to be largely suppressed by KChIP binding 29 . Nevertheless, in order to abolish putative residual or newly generated N-type inactivation features in Kv4.2 + DPP + 1b channels, we used an N-terminally truncated version of Kv4.2, which lacks the first 10 amino acids (Δ10; Fig. S1 ). This deletion removes the Kv4.2 N-terminal inactivation domain, but leaves KChIP binding intact 17 , 29 , 31 . We found that macroscopic inactivation kinetics of the truncated channels were almost identical to wild-type (Fig. 4 A). Also, the kinetics of recovery from inactivation were still biphasic and very similar to wild type (Fig. 4 B; Table S5). Based on these results, residual or newly generated N-type inactivation as a possible mechanism underlying the slow recovery kinetics of 1b containing Kv4.2 channel complexes was excluded. - Fig. 4 - Next, we intended to interfere with putative residual or newly generated P/C-type inactivation in Kv4.2 + DPP + 1b channels by applying a high K + solution (see Methods). Elevated external K + has been shown previously to slow current decay and to accelerate recovery kinetics by interfering with the classical P/C-type inactivation of Shaker -related (Kv1) channels 32 – 35 . Under these conditions, the macroscopic inactivation of Kv4.2 + DPP + 1b channels was accelerated, in accordance with previous reports 30 , 36 – 39 (Fig. 4 C; see Discussion). Intriguingly, high K + solution also affected the kinetics of recovery from inactivation by specifically accelerating the slow component (2.4-fold; Fig. 4 D; Table S5). High K + solution also influenced Kv4.2 Δ10 + DPP + 1b channels, in the same manner as wild-type ternary channels, albeit with a somewhat weaker effect on the slow recovery component (2.1-fold acceleration; Fig. 4 E, F; Table S5). Notably, superfusion with TEA solution (see Methods) had no obvious effects, except for an acceleration of current decay kinetics, which was more pronounced for Kv4.2 Δ10 than for Kv4.2 wild-type ternary channels (Fig. S6; Table S5). From our experimental results with high K + solution we concluded that Kv4.2 + DPP + 1b channels may not only undergo CSI related to dynamic S6 rearrangements 13 , 14 , from which recovery is thought to be fast. Rather, promoted by 1b co-expression, a fraction of channels may also undergo strong Shaker -like P/C type inactivation with slow recovery kinetics. Therefore, we finally intended to study Kv4.2 channel constructs in which S6-related CSI was specifically modified. For this purpose, we chose two previously characterized S6 mutants (Fig. S1 ), in which recovery from inactivation had been found to be either drastically slowed (Kv4.2 L400A) or drastically accelerated (Kv4.2 N408A) 13 . We expected the putative newly generated P/C-type inactivation with slow recovery kinetics in the presence of 1b to be further augmented in Kv4.2 N408A, where the affinity towards S6-related CSI states is lowered 13 (see Discussion), but not in Kv4.2 L400A. The S6 mutations influenced both macroscopic inactivation and recovery from inactivation in Kv4.2 + DPP + 1b channels in a characteristic manner. While L400A caused an overall slowing, N408A caused a strong acceleration of current decay kinetics (Fig. 5 A; Table S5). Kv4.2 L400A + DPP + 1b recovery kinetics were very similar to wild-type ternary, whereas Kv4.2 N408A + DPP + 1b recovery kinetics differed substantially: They followed a single exponential time course, apparently corresponding to the slow recovery components of wild-type and L400A ternary channels (Fig. 5 B; Table S5). Finally, we tested the two mutants in high K + solution. For both mutants, macroscopic inactivation was accelerated under these conditions, similar to wild type (Fig. 5 C, E; see also Fig. 4 C). Also, the slow recovery component was accelerated in L400A ternary channels, as seen for wild-type, albeit only 1.4-fold (Fig. 5 D; Table S5). Remarkably, in N408A ternary channels the recovery kinetics remained single-exponential, and were accelerated 2.2-fold in high K + solution (Fig. 5 F; Table S5), suggesting that Kv4.2 N408A + DPP + 1b recovery kinetics fully reflect the recovery from a putative Shaker -like P/C type inactivation. Taken together, our findings support the notion that otherwise vestigial P/C-type inactivation features of Kv4.2 channels are strongly promoted by 1b and may co-exist with S6-related CSI. - Fig. 5 - Discussion In the present paper we set out to critically revise a previous report on the distinctive features of the KChIP splice variants 1a and 1b. The previously reported induction of a novel extremely slow component of recovery from inactivation by 1b is confirmed by our results, but they reveal that 1a co-expression can also cause biphasic recovery kinetics. KChIP1 co-expression effects in heterologous systems and the putative mechanism, including possible structure-function relationships, underlying the slow recovery component, will be discussed in a physiological context. Co-expression of an initially identified KChIP1, which is identical to the 1a splice variant used herein, with Kv4.2 in tissue culture cells and Xenopus oocytes defined the hallmarks of Kv4 channel modulation by KChIPs. These included an increase in current density, a slowing of macroscopic inactivation and an acceleration of recovery from inactivation 16 . The initially identified KChIP1 (= 1a) was used for a detailed analysis of Kv4 channel assembly and trafficking, to demonstrate the stabilizing effect of KChIP1 on Kv4.3 tetramers, as well as the KChIP1-mediated release of Kv4.2 ER retention, and the role of KChIP1-specific N-terminal myristoylation in subcellular targeting of the Kv4.2 + KChIP1 complex to post-ER transport vesicles 20 , 40 – 42 . These results provided an explanation for the observed increase in current density upon KChIP1 co-expression. The initially identified KChIP1 (= 1a) was also used for a detailed biophysical analysis of Kv4 channel gating modulation, showing similar remodelling of Kv4.1 and Kv4.3 channel inactivation 18 . The remodelling included the typical streamlining effect on macroscopic currents, with a crossover of normalized current traces obtained in the absence and presence of KChIP1, respectively (see also Fig. 1 and Fig. S3), as well as a shift of inactivation curves to more positive voltages, most likely reflecting the accelerated recovery from inactivation, upon KChIP1 co-expression 18 . Our experimental results obtained with Kv4.x + 1a co-expression largely confirm these initial observations. The alternatively spliced KChIP variant 1b differs from 1a by an 11-amino-acid N-terminal insertion (residues 22–33 in 1b; Fig. S2), rich in aromatic side chains 25 – 27 . The 1b splice variant has been reported previously to differ from 1a quite substantially, by inducing biphasic kinetics of recovery from inactivation with a newly generated extremely slow component 27 . Our critical revision of this previous report on the distinctive features of 1b was motivated by the fact that the authors had used transient transfection of a stable Kv4.2 cell-line with KChIP1 cDNA. We suspected that in this system, cell-to-cell variations in cDNA uptake and differences in the temporal expression profiles of α- (stable) and β-subunits (transient), may have caused two different Kv4.2 channel populations present at roughly equal amounts in the plasma membrane; i.e., Kv4.2 + 1b and Kv4.2 alone (stable), with fast and slow recovery kinetics, respectively. Therefore, we subjected the previous findings to a rigorous test by studying KChIP1 co-expression effects on Kv4.2 channel gating in a more quantitative manner in cRNA-injected Xenopus oocytes. With this approach the previously reported biphasic recovery kinetics were confirmed, irrespective of the amount of 1b cRNA injected (see Fig. 2 ). Thus, our initial concerns regarding the previously used expression system and transfection procedure were clearly unfounded. We further asked whether the 1b effect on recovery kinetics is also observed with other Kv4.x subtypes, and whether it is still visible in a ternary configuration with DPP, which itself strongly accelerates the recovery of Kv4 channels from inactivation 15 and may therefore be able to mask the 1b effect. The results of our experiments performed with all Kv4.x channel subtypes co-expressed with excess amounts of 1b, both in a binary configuration and in a ternary configuration with DPP, support the notion that the induction of a slow recovery component represents an intrinsic feature of the KChIP splice variant 1b with general applicability. Unexpectedly, we found that, similar to 1b, 1a is also capable of inducing a slow recovery component. It is remarkable that, for the most part, previous reports of KChIP1 (= 1a) co-expression effects on Kv4 channels, in both cDNA-transfected tissue culture cells 16 , 27 , 28 , 43 – 46 and cRNA-injected Xenopus oocytes 15 , 16 , 18 , 22 , 26 , 30 , 47 – 52 , used a single-exponential function to describe the kinetics of recovery from inactivation. Also, previous reports on the recovery kinetics of ternary Kv4.2 + DPP + 1a channels in different expression systems 15 , 43 used a single-exponential function for their analyses. Multi-exponential fitting of the 1a-induced recovery kinetics was not considered in these previous studies, despite the sometimes obvious inadequacy of the single-exponential fit. In some of the previously used experimental protocols the chosen interpulse durations may have been not long enough to reliably capture such a slow recovery component. Notably, in one study, performed in Xenopus oocytes with 2 mM standard external K + , like in the present study, the initially identified KChIP1 (= 1a) was reported to cause biphasic recovery kinetics for Kv4.1 and Kv4.2 53 . Double-exponential fitting in that study resulted in a numerical ratio of fast and slow recovery time constants and in fractional amplitudes very similar to our results. Van Hoorick and co-workers were able to convert biphasic into virtually monophasic recovery kinetics by mutating three of the five aromatic amino acid residues in 1b to alanine 28 . However, our kinetic analyses suggest that additional structural determinants in KChIP1 may contribute to the special remodelling features. We have chosen Kv4.2 + DPP + 1b ternary channels to study the newly generated slow recovery component mechanistically. In addition to CSI, which is most prominent in Kv4 channels 12 , Kv channels may undergo N-type inactivation and/or P/C-type inactivation, which are the major inactivation mechanisms of Shaker -related (Kv1) channels 10 , 11 . These classical Shaker inactivation mechanisms are also present in Kv4 channels, but in vestigial forms 12 , 29 , 30 . Commonly used approaches; i.e., N-terminal truncation and application of high external K + , respectively, were applied in the present study to test for a possible enhancement of the classical Shaker inactivation mechanisms in Kv4 channels, caused by the KChIP splice variant 1b. Since KChIP binding is thought to sequester and immobilize a Kv4 N-terminal inactivation domain 14 , 22 , 23 , 29 , 46 , 49 , 51 , an involvement of N-type inactivation seemed rather unlikely beforehand. The virtual absence of an effect of a ten amino acid N-terminal truncation (Δ10) on Kv4.2 + DPP + 1b recovery kinetics confirmed this a priori assumption. High external K + caused an acceleration of Kv4.2 + DPP + 1b current decay kinetics, very similar to the results obtained by Kaulin and co-workers 30 , and an indication of the removal of the vestigial P/C-type inactivation to favor CSI in ternary Kv4.2 + DPP + 1b channels. Remarkably, however, high external K + also accelerated the slow component of the biphasic recovery process of wild-type and Δ10, and the monophasic recovery process of N408A ternary channels, reminiscent of the accelerated recovery kinetics observed previously under these conditions for the Shaker -related channels Kv1.3 32,33 and Kv1.4 34,35 . The striking similarity of these high K + effects on recovery kinetics supports the notion that the slow recovery component observed in Kv4 channels upon 1b co-expression is related to a Shaker -like P/C-type inactivation. Notably, our results suggest that 1b-independent vestigial P/C-type inactivation 30 and a more stable 1b-induced Shaker -like P/C-type inactivation may co-exist in Kv4 channels. This notion is also supported by our finding that TEA influenced current decay kinetics similar to high K + , but left Kv4.2 + DPP + 1b recovery kinetics largely unaffected (see Fig. S6). Apart from an immobilization of the Kv4 N-terminal inactivation domain caused by KChIP binding 14 , 22 , 23 , 29 , 46 , 49 , 51 , the structure-function relationships of Kv4 channel remodelling by KChIPs are largely unknown. In particular, The 11-amino-acid aromatic cluster, thought to be responsible for the special Kv4 remodelling by 1b 28 , is not included in the available Kv4/KChIP1 structures 22 , 23 , 46 , 49 – 51 (see also Fig. S2). Thus, a putative direct interaction between the aromatic cluster and the Kv4 α-subunit remains uncertain. One may speculate whether the clamping conformation adopted by the four KChIP1 molecules surrounding the gating-relevant 54 , 55 Kv4 T-domains 22 , 23 , 46 , 49 – 51 may be influenced by the aromatic cluster. It is intriguing in this context that a naturally occurring Kv1.5 Δ209 N-terminal truncation variant, which has lost its T-domain, exhibits, in addition to classical P/C-type inactivation, a form of CSI, resulting in biphasic kinetics of recovery from inactivation 56 . Recently high resolution cryo EM data have elucidated structural details of Kv1.2 P/C-type inactivation. The data suggest that P/C-type inactivation in these channels leads to a dilation rather than a constriction of the selectivity filter 57 , and that an isoleucine gate localized in S6, right below the selectivity filter plays a central role 58 . Cryo EM data have also elucidated structural details of Kv4.2 channel inactivation 14 , by capturing, in addition to an open state, two putative non-conducting states, referred to as "inactivated" and "intermediate". In both non-conducting states, an upper and a lower gate within the pore are expected to prevent the passage of K + ions. The lower gate is related to the dynamic coupling between voltage sensor and pore modules, allowing conformational rearrangements, that lead to a symmetry breakdown of S6-segments as the basis of CSI 13 , 14 . Intriguingly, the upper gate in Kv4.2 is homologous to the isoleucine gate, which mediates P/C-type inactivation in Kv1.2 58 . Based on these structural similarities, one may speculate that in the presence of the KChIP splice variant 1b, the upper (isoleucine) gate in Kv4.2 may evolve into a major inactivation gate, especially if S6-related CSI (lower gate) is unstable, as suggested by our experimental findings with the Kv4.2 S6 mutant N408A (see Fig. 5 ). Of note, a close inspection of the structures put forward by Kise and co-workers 22 and by Ma and co-workers 23 , containing Kv4.2 and Kv4.3, respectively, in different configurations with KChIP1 fragments lacking the 11-amino-acid aromatic cluster, suggest pore radii at the upper (isoleucine) gate between 4.7 and 7.7 Å, wide enough to let a hydrated K + ion pass. Thus, with the possible exceptions of the "intermediate" and "inactivated" structures put forward by Ye and co-workers, defining an upper (isoleucine) gate for Kv4.2 14 , putative P/C-type inactivated Kv4 channels have not been captured in 3D, yet. From a physiological point of view, the co-assembly of different Kv4.x channel subtypes with DPPs and a variety of KChIPs, including functionally distinct β-subunit splice variants like 1a and 1b, is expected to contribute to an immense diversity of I SA properties in different cell types, with a considerable impact on neuronal excitability and discharge behavior 59 . In the rat brain Kv4.2 is co-localized majorly with KChIP2 and KChIP4 in pyramidal neurons, whereas a high co-localization of Kv4.3 with KChIP1 is seen in large multipolar interneurons 60 . In fact, co-localization of Kv4.3 and KChIP1 is reliably found in a fraction of parvalbumin, calbindin, calretinin and somatostatin-positive hippocampal interneurons, such that Kv4.3/KChIP1 co-expression has been suggested to be used as a separate independent hippocampal interneuron marker 61 . Using siRNA knockdown of KChIP1 expression in a hippocampal preparation has been shown to specifically affect firing behavior of Kv4.3/KChIP1 co-expressing CA1 interneurons. KChIP1 knockdown in these interneurons caused an increase in firing frequency, reportedly due to a slowing of I SA recovery rather than a decrease in I SA amplitude 44 . Alternative splicing of KChIP1 with the effect of slowing I SA recovery may have a comparable effect. The distribution of KChIP1 transcripts, has been studied in human, rat and mouse tissues 25 – 27 . The findings suggest that the KChIP splice variants 1a and 1b are expressed at comparable amounts in the human brain. It should be noted, that the optional expression of the Kv4.3 splice variants S and L in combination with the KChIP splice variant 1a or 1b may allow for a fairly large spectrum of I SA properties in Kv4.3/KChIP1 co-expressing interneurons (see panels B and C in Figs. S3 - S5). Taken together, strong promotion of Shaker -like P/C-type inactivation features in Kv4 channels, especially by the KChIP splice variant 1b, may limit time-dependent I SA availability during repetitive firing, thereby increasing firing frequency, especially in large multipolar Kv4.3/KChIP1 co-expressing interneurons. Methods Plasmids and constructs In this study the human Kv4 channel clones Kv4.1, Kv4.2, and Kv4.3 62 were used. The long Kv4.3 splice variant (Kv4.3L) was a kind gift from Geoffrey Abbott (Department of Physiology and Biophysics, University of California, Irvine, USA). The human KChIP1a splice variant (referred to as 1a in the present paper) was kindly provided by Dirk Isbrandt (Center for Molecular Medicine, University of Cologne, Germany), and the human DPP6s splice variant (referred to as DPP in the present paper) by Nicole Schmitt (Department of Biomedical Sciences, Faculty of Health and Medical Sciences, Copenhagen, Denmark). In addition to Kv4.2 wild type, three previously studied Kv4.2 mutant constructs were used: In one construct the first ten amino acids had been deleted (Kv4.2 Δ10) 17 ; in the two other constructs individual residues in the distal S6 segment, leucine at position 400 or asparagine at position 408, had been replaced by alanine (Kv4.2 L400A and Kv4.2 N408A, respectively 13 ; see also Fig. S1 ). All cDNA clones were inserted into the multiple cloning site of pGEM-HE. In order to generate KChIP1b (referred to as 1b in the present paper), a 33 bp fragment was inserted into the 1a coding region by overlap PCR with appropriate primers (fwd: accagtatcagagagaTAAGATTGAAGATGAGCTGGAG; rev: aataccaccaggcgatgTCTTTCGAGGGTCGCCTT) in a back-to-back orientation, using the Q5 Site Directed Mutagenesis Kit (New England Biolabs). Successful mutagenesis was verified by Sanger sequencing of the complete coding region and flanking sequences. Transformed JM109 Eschericia coli cells (Promega) were grown in Luria Broth medium complemented with ampicillin, and plasmids were isolated using the QIAprep Spin Miniprep Kit (QIAGEN). Purified plasmids were linerarized using Not I (New England Biolabs), and the RiboMaxLargeScale RNA production system T7 (Promega) was utilized for the in vitro transcription of cRNA. Heterologous channel expression Kv4 channels and their auxiliary β-subunits were expressed in Xenopus laevis oocytes. Female frogs (Nasco) were anesthetized for 8–10 min in ethyl 3-aminobenzoate methanesulfonate (tricaine, Sigma; 1.2 g/l chlorine-free frog water containing 7.5 mM Tris-HCl). Part of the ovary lobes was surgically removed, and the wound was immediately sutured (monocryl 4 − 0, ethicon). For the final (sixth, intervals of ∼ one year) oocyte harvest, the frogs are euthanized by deep tricaine anesthesia (30 min) followed by decapitation. Animal care and experimental procedures related to the harvesting of Xenopus oocytes were conducted in accordance with the German Animal Welfare Act and were approved by the Authority for Justice and Consumer Protection of the City of Hamburg (approval # N 101/2023). All procedures comply with the ARRIVE guidelines. The obtained ovary tissue was mechanically dispersed using a pair of fine forceps and digested for 3–5 h under constant agitation in a calcium-free solution containing (in mM) 82.5 NaCl, 2 KCl, 1 MgCl 2 , 5 HEPES, and 1.3 mg/ml collagenase type II (Sigma); pH 7.5, NaOH. Defolliculated stage V–VI oocytes were selected one day after harvesting or later, and 25 or 50 nl cRNA solution were injected per oocyte using a Nanoliter 2000 microinjector (World Precision Instruments). Individual injections resulted in Kv4 cRNA amounts between 0.8 and 5 ng per oocyte, in the absence or presence of KChIP1 cRNA (between 2.5 and 20 ng per oocyte) and/or DPP cRNA (between 2.5 and 5 ng per oocyte). Injected oocytes were incubated at 16°C in a solution containing (in mM) 75 NaCl, 5 Na-pyruvate, 2 KCl, 2 CaCl 2 , 1 MgCl 2 , 5 HEPES, and 50 mg/ml gentamicin (Sigma), pH 7.5, NaOH; and used for recordings 1–10 days (d1 - d10) after cRNA injection (see Tables S1 - S4 for cRNA amounts per oocyte and days of recording for individual experiments). Electrophysiology Currents were recorded at room temperature (20–22°C) under two electrode voltage-clamp, using a TurboTec-3 amplifier (npi electronics) controlled by PatchMaster software (HEKA). The ND96 bath solution contained (in mM) 96 NaCl, 2 KCl, 1 CaCl 2 , 1 MgCl 2 and 5 HEPES (pH 7.4, NaOH). In some experiments variations of this solution were used, in which either NaCl was replaced by KCl (high K + solution, 98 mM K + ) or NaCl and KCl were replaced by tetraethylammonium-Cl (TEA solution, 98 mM TEA). Glas microelectrodes were filled with 3 M KCl and had tip resistances of 0.2–0.5 MΩ in standard ND96 bath solution. The holding voltage was − 80 mV. For the study of macroscopic inactivation a 2.5 s test pulse to + 40 mV was applied following a 2 s conditioning pulse to -100 mV, in order to activate and immediately inactivate a large fraction of channels (see Fig. 1 A, B). Recovery from inactivation was measured at -80 mV using a double-pulse protocol with a 3 s control pulse and a brief test pulse to + 40 mV, separated by interpulse intervals (Δ t ), which lasted between 10 ms and ∼ 41 s (iteration factor 2, see Fig. 2 A, B). Voltage step protocols were applied to study the voltage dependence of gating. For the voltage dependence of activation, test pulses to voltages between − 90 and + 70 mV (10 mV increments, see Fig. 3 A, inset) were applied following a 2 s conditioning pulse to -100 mV. For the voltage dependence of steady-state inactivation a double-pulse protocol was used, in which after an initial 2 s conditioning pulse to -120 mV and a subsequent brief control pulse to + 40 mV, brief test pulses to + 40 mV were applied following an interpulse interval of 10 s with conditioning voltages between − 120 and 0 mV (10 mV increment, see Fig. 3 A, inset). Capacitive current transients were not compensated. Current measurements at -95 mV (approximate E rev for K + currents in standard ND96 bath solution), before each voltage pulse protocol, were used to calculate the leak current at any other voltage in order to correct peak current amplitudes. Alternatively, a prepulse-inactivation subtraction protocol was used 36 to leak-subtract entire current traces. Data analysis The data were analysed using FitMaster (HEKA) and Kaleidagraph (Synergy Software). Macroscopic inactivation (i.e., current decay) kinetics (at + 40 mV) were described by the sum of three (if possible) or two exponential functions. The kinetics of recovery from inactivation (I test / I control plotted against interpulse duration) were described by a single-exponential function or the sum of two exponential functions, as required. The relative amplitudes of the individual time constants, obtained with multi-exponential fitting, are given in %. The voltage dependences of activation (cord conductance calculation with E rev = -95 mV) and steady-state inactivation (I test / I control ) were analysed with appropriate Boltzmann-functions, as described previously 13 . Fit results (i.e., time constants, relative amplitudes, V 1/2 values and slope factors) are given as mean ± SD (Fig. 1 C, D; Fig. 2 E, F; Fig. 3 B, D; Fig. 4 A, B; Fig. 5 A, B; see also Tables S1 - S5). Normalized current amplitudes in pooled analysis plots containing the fitting curves are given as mean ± SEM (Fig. 2 C, D; Fig. 3 A, C; Fig. 4 B, D, F; Fig. 5 B, D, F). Statistical analyses for two groups were performed based on unpaired (comparison of 1a and 1b effects) or paired (effects of solution change while recording from individual oocyes) Student's t -tests. For multiple groups (1a and ab co-expression effects relative to Kv4.x alone or relative to Kv4.x + DPP) one-way analysis of variance (ANOVA) with Dunnett’s posthoc testing was used (see also Tables S1 – S5). Data availability The data are summarized in Tables S1 - S5; datasets generated and analyzed during the present study are available from the corresponding author on reasonable request. Declarations Acknowledgements We thank Annett Hasse for technical assistance and Christiane K. Bauer and Stefan Kindler for helpful discussion. Author contributions R.B. designed the experiments. W.C. and G.T. executed the experiments. W.C., G.T., and R.B. analyzed the data. W.C., G.T. and R.B. interpreted the data. R.B. wrote the manuscript. W.C., G.T., and R.B. critically reviewed and approved the manuscript. Funding This work was supported by the Deutsche Forschungsgemsinschaft (DFG; Ba2055/6), and by a grant from the China Scholarship Council (No. 202106090277) to Wuyou Cao. Additional information Supplementary information: The online version contains supplementary material (Figs. S1 - S6; Tables S1 - S5) available at xxxxx. Competing interests: The authors declare that they have no competing interests. References Hille, B. Ion Channels of Excitable Membranes . 3 edn, (Sinauer Associates, Inc., 2001). Pak, M. D. et al. mShal , a subfamily of A-type K + channel cloned from mammalian brain. Proc Natl Acad Sci U S A 88, 4386-4390 (1991). Wei, A. et al. K + current diversity is produced by an extended gene family conserved in Drosophila and mouse. Science 248, 599-603 (1990). Hoffman, D. A., Magee, J. 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05:06:09","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115803,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7356877/v1/1b245449057a38cbe2ff578c.png"},{"id":92300445,"identity":"73a69d62-60ac-4ae1-876b-0653c29295f7","added_by":"auto","created_at":"2025-09-27 05:06:09","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":167039,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7356877/v1/6b8b3d282eb8190dd3534a12.png"},{"id":92300843,"identity":"e03114b2-5646-4a27-b995-7e031d7dff9b","added_by":"auto","created_at":"2025-09-27 05:14:09","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":173209,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7356877/v1/d9b1fd5a38708dae03a7f449.png"},{"id":92300447,"identity":"481c1194-60a7-4372-abe5-e698d98035c8","added_by":"auto","created_at":"2025-09-27 05:06:09","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":148577,"visible":true,"origin":"","legend":"","description":"","filename":"d0464a18fb214a3282825160ca18813b1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7356877/v1/764a5b8a75bb7ae5267928d8.xml"},{"id":92300448,"identity":"ea14053e-2814-4609-a114-09fe3665015a","added_by":"auto","created_at":"2025-09-27 05:06:10","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":159188,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7356877/v1/636da23417056efa1069692b.html"},{"id":92300427,"identity":"e89df40c-fd9e-4a25-9d90-a0377e810d06","added_by":"auto","created_at":"2025-09-27 05:06:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":276591,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModulation of Kv4.2 channel macroscopic inactivation by the KChIP splice variants 1a and 1b.\u003c/strong\u003e \u0026nbsp;\u003cstrong\u003eA,\u003c/strong\u003e \u003cstrong\u003eB \u003c/strong\u003e\u0026nbsp;Representative current traces obtained for Kv4.2 alone (grey, black) or co-expressed with 1a (blue) or 1b (red) in \u003cem\u003eXenopus\u003c/em\u003e oocytes in the absence (A) or presence (B) of DPP. Currents were normalized to peak and superimposed to demonstrate the effects of KChIP1 splice variant co-expression on current decay kinetics; horizontal dotted lines represent zero current level. Note the typical crossover of normalized current traces in the absence and presence of KChIP1, respectively\u003csup\u003e18\u003c/sup\u003e. \u0026nbsp;\u003cstrong\u003eC,\u003c/strong\u003e \u003cstrong\u003eD\u003c/strong\u003e\u0026nbsp; Inactivation time constants and their relative amplitudes (%); different amounts of Kv4.2, KChIP1 and DPP cRNA (ng per oocyte) and number of oocytes (n) indicated; data pooled from different days after cRNA injction (see Table S2). Significant differences seen with 1a or 1b co-expression (compared to Kv4.2 alone or Kv4.2 + DPP) are indicated with * (p\u0026lt;0.05) or ** (p\u0026lt;0.0001). Significant differences seen with 1b co-expression (compared to 1a co-expression for comparable amounts of KChIP1 cRNA) are indicated with § (p\u0026lt;0.05) or §§ (p\u0026lt;0.0001). Statistical significance symbols represent the highest degree of significance found in ≥ 1 of 6 parameters analysed (see Table S2 for details).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7356877/v1/0530dc6995e905b40e6f366c.png"},{"id":92300428,"identity":"f77a9100-32df-4e77-90c8-0a6b69a5e5a8","added_by":"auto","created_at":"2025-09-27 05:06:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":404076,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModulation of Kv4.2 channel recovery kinetics by the KChIP splice variants 1a and 1b. \u0026nbsp;A,\u003c/strong\u003e \u003cstrong\u003eB\u003c/strong\u003e \u0026nbsp;Recordings obtained with a recovery protocol from oocytes expressing Kv4.2 alone (A, left; grey), Kv4.2 + 1b (A, right; red),\u003cstrong\u003e \u003c/strong\u003eKv4.2 + DPP (B, left; black) or Kv4.2 + DPP + 1b (B, right; red); control current amplitudes were normalized; horizontal dotted lines represent zero current level. \u0026nbsp;\u003cstrong\u003eC,\u003c/strong\u003e \u003cstrong\u003eD\u003c/strong\u003e Relative current amplitudes were plotted against interpulse duration, and the data were described by exponential functions (see also enveloping curves along the test current peaks in A and B). Dotted blue lines in C and D: single-exponential function applied to the data obtained with 1a co-expression; grey line without symbols in D: fitting curve obtained for Kv4.2 alone (C). \u0026nbsp;\u003cstrong\u003eE,\u003c/strong\u003e \u003cstrong\u003eF\u003c/strong\u003e Recovery time constants including their relative amplitudes (%); different amounts of cRNA (ng per oocyte) and number of oocytes (n) indicated; data pooled from different days after cRNA injction. Significant differences seen with 1a or 1b co-expression (compared to Kv4.2 alone or Kv4.2 + DPP): * (p\u0026lt;0.05) or ** (p\u0026lt;0.0001); significant differences seen with 1b co-expression (compared to 1a co-expression): § (p\u0026lt;0.05) or §§ (p\u0026lt;0.0001). Symbols represent the highest degree of significance found in ≥ 1 of 4 parameters analysed (see Table S2 for details).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7356877/v1/0ffd6bb7a0ac6a58f030798b.png"},{"id":92300840,"identity":"187c7a1f-e660-41db-af4b-e6796b1bc6f8","added_by":"auto","created_at":"2025-09-27 05:14:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":368672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of KChIP splice variants 1a and 1b on the voltage dependence of Kv4.2 channel gating. \u003c/strong\u003eRaw data not shown, but see insets in the upper left for voltage protocols. \u0026nbsp;\u003cstrong\u003eA,\u003c/strong\u003e \u003cstrong\u003eC \u003c/strong\u003e\u0026nbsp;Normalized data from steady-state inactivation protocols and normalized conductance values (G\u003csub\u003enorm\u003c/sub\u003e) from activation protocols obtained for Kv4.2 alone (grey, black) or co-expressed with 1a (blue) or 1b (red) in the absence (A) or presence (C) of DPP. The data were plotted against conditioning or test pulse voltage, respectively, in the same graph and fitted with appropriate Boltzmann-functions\u003csup\u003e13\u003c/sup\u003e (see Methods). \u0026nbsp;\u003cstrong\u003eB,\u003c/strong\u003e \u003cstrong\u003eD\u003c/strong\u003e\u0026nbsp; Voltages of halfmaximal activation (act, circles) and inactivation (inact, squares; vertical dashed lines represent values obtained in the absence of KChIP1) and corresponding slope factors (V\u003csub\u003e1/2\u003c/sub\u003e and s, respectively). Different amounts of cRNA (ng per oocyte) and number of oocytes (n) indicated; data pooled from different days after cRNA injection. Significant differences seen with 1a or 1b co-expression (compared to Kv4.2 alone or Kv4.2 + DPP): * (p\u0026lt;0.05) or ** (p\u0026lt;0.0001). Significant differences seen with 1b co-expression (compared to 1a co-expression): § (p\u0026lt;0.05) or §§ (p\u0026lt;0.0001; see Table S2 for details).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7356877/v1/566535836caa359e8e6c5310.png"},{"id":92300433,"identity":"c2b8916a-aaa2-4dd9-867e-656e483bf58a","added_by":"auto","created_at":"2025-09-27 05:06:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":609260,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMacroscopic inactivation and recovery kinetics of ternary Kv4.2 + DPP + 1b channels after N-terminal truncation and in the presence of elevated external K\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e. \u0026nbsp;A, C, E \u003c/strong\u003e\u0026nbsp;The kinetics of macroscopic inactivation were analysed for wild-type (red) and N-terminally truncated (Δ10, orange) ternary Kv4.2 channels in standard ND96 (2 K\u003csup\u003e+\u003c/sup\u003e) and high external K\u003csup\u003e+\u003c/sup\u003e solution (98 K\u003csup\u003e+\u003c/sup\u003e, purple; see Methods). Currents were normalized to peak and superimposed; horizontal dotted lines represent zero current level. Inactivation time constants and their relative amplitudes (%) are shown below. Current decay kinetics were approximated by a double-exponential function, describing a fast (f) and a slow (s) phase, in the solution exchange experiments (C and E, data pairs indicated). \u003cstrong\u003eB, D, F\u003c/strong\u003e\u0026nbsp; Recovery plots including double-exponential fits for wild-type and N-terminally truncated channels in standard ND96 and high external K\u003csup\u003e+\u003c/sup\u003e solution. Congruent single-exponential fitting curves (black and grey) without symbols: Kv4.2 + DPP and Kv4.2 Δ10 + DPP. Recovery time constants and their relative amplitudes (%) are shown below (data pairs of solution exchange experiments in D and F indicated); number of oocytes (n) indicated. Significance of both mutant effects and effects of switching to elevated external K\u003csup\u003e+\u003c/sup\u003e are indicated as *(p\u0026lt;0.05) or **(p\u0026lt;0.0001; see also Table S5).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7356877/v1/3c090a1ce1d68702cb6b2193.png"},{"id":92300439,"identity":"b871717c-1166-433b-9d7a-83642b562958","added_by":"auto","created_at":"2025-09-27 05:06:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":601552,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eK\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e dependence of macroscopic inactivation and recovery kinetics in ternary Kv4.2 + DPP + 1b channels with mutations in S6. A \u0026nbsp;\u003c/strong\u003eKinetics of macroscopic inactivation were analysed for wild-type Kv4.2 channels (red) and Kv4.2 channels with amino acid exchanges in S6 (L400A or N408A; bronze and green, respectively). \u0026nbsp;\u003cstrong\u003eC,\u003c/strong\u003e \u003cstrong\u003eE\u003c/strong\u003e \u0026nbsp;Effects of high external K\u003csup\u003e+\u003c/sup\u003e solution (98 K\u003csup\u003e+\u003c/sup\u003e, purple; see Methods) on the macroscopic inactivation kinetics of L400A (C) and N408A (E) ternary channels. In A, C and E currents were normalized to peak and superimposed, and horizontal dotted lines represent zero current level. Inactivation time constants and their relative amplitudes (%) are shown below. Current decay kinetics were approximated by a double-exponential function, describing a fast (f) and a slow (s) phase, in the solution exchange experiments (data pairs indicated in C and E). \u0026nbsp;\u003cstrong\u003eB\u0026nbsp; \u003c/strong\u003eRecovery plot including exponential fits for wild-type Kv4.2 channels and Kv4.2 channels with amino acid exchanges in S6 (L400A or N408A). \u003cstrong\u003e\u0026nbsp;D, F\u003c/strong\u003e\u0026nbsp; Effects of high external K\u003csup\u003e+\u003c/sup\u003e solution (98 K\u003csup\u003e+\u003c/sup\u003e; see Methods) on the recovery kinetics of L400A (D) and N408A (F) ternary channels. Recovery time constants and their relative amplitudes (%) are shown below (data pairs indicated). Single-exponential fitting curves without symbols: Kv4.2 + DPP (black) and Kv4.2 N408A + DPP (grey) in standard ND96. Significance of both mutant effects and effects of switching to elevated external K\u003csup\u003e+\u003c/sup\u003e are indicated as *(p\u0026lt;0.05) or **(p\u0026lt;0.0001; see also Table S5). The numbers of oocytes (n) are indicated.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7356877/v1/c791ad0ae6c718984a073f32.png"},{"id":100616122,"identity":"5eaecceb-301e-449c-8215-7a0cd686376a","added_by":"auto","created_at":"2026-01-19 17:40:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3434061,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7356877/v1/4f2dbd21-065b-4447-a8a6-153e0d6ab009.pdf"},{"id":92300430,"identity":"cb9a0a47-1655-41ce-ac26-b151462636d9","added_by":"auto","created_at":"2025-09-27 05:06:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4300764,"visible":true,"origin":"","legend":"","description":"","filename":"CaoetalSupplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7356877/v1/d1cdef3bc83eff7ed554e96c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"KChIP1 splice variants modulate Kv4 channels by promoting P/C-type inactivation features","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVoltage-gated potassium (Kv) channels control action potential generation, shape and frequency\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The Kv4 channel subfamily underlies a somatodendritic A-type (i.e., rapidly inactivating) current (\u003cem\u003eI\u003c/em\u003e\u003csub\u003eSA\u003c/sub\u003e)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, involved in the control of low frequency neuronal firing, dendritic excitation and synaptic plasticity\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Like all Kv channels, Kv4 channels are formed by the assembly of four α-subunits, surrounding a central ion conduction pathway (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Each α-subunit consists of six transmembrane segments (S1 - S6) with intracellular N- and C-termini and an N-terminal tetramerization (T) domain, which controls subfamily-specific assembly\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The transmembrane portions of the α-subunit can be divided into a voltage sensing (S1-S4) and a pore module (S5-S6) with a selectivity filter sequence located between S5 and S6 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The distal S6 segments constitute the gate of the tetrameric channel by forming an aperture, able to either constrict or splay apart in a voltage-dependent manner\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Blocking of the open channel from the cytoplasmic side by an intrinsic N-terminal inactivation domain (N-type inactivation)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and conformational rearrangements of the external pore mouth near the selectivity filter (P/C-type inactivation)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, two well-investigated mechanisms of Kv channel autoinhibition, are vestigial in Kv4 channels, which undergo preferential closed-state inactivation (CSI) based on dynamic S6 rearrangements\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eKv4 channels form complexes with two types of auxiliary β-subunits, membrane-spanning dipeptidyl amino peptidase-related proteins (DPPs)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e and cytoplasmic Kv channel interacting proteins (KChIPs\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e; Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Association of either type of β-subunit enhances Kv4 channel surface expression and modulates Kv4 channel inactivation gating in a specific manner. Typically, macroscopic inactivation of Kv4 channels is accelerated by DPPs and slowed by KChIPs, whereas recovery from inactivation is accelerated by both\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19 CR20\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Structures of ternary Kv4\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;KChIP complexes\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, a likely native channel configuration\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, indicate that subunit assembly occurs in a 4:4:4 stoichiometry (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThere are four human KChIP subtypes (KChIP1\u0026ndash;4; Fig. S2), encoded by four different genes (\u003cem\u003eKCNIP1\u0026ndash;4\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Utilizing alternative transcription start sites and alternative splicing, \u003cem\u003eKCNIP1\u003c/em\u003e yields different KChIP1 isoforms. The KChIP1a and KChIP1b splice variants differ by an N-terminal 11-amino-acid stretch, rich in aromatic side chains, only found in KChIP1b\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e (Fig. S2). This N-terminal aromatic cluster has been reported to be responsible for the differential channel modulation by KChIP1b when co-expressed by transient cDNA transfection of a stable Kv4.2-expressing cell line. In particular, KChIP1b was found to induce an extremely slow component of recovery from inactivation\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, when other KChIPs, including KChIP1a, usually accelerate the recovery of Kv4 channels from inactivation\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHere, we asked whether the peculiar KChIP1b feature of slowing recovery from inactivation is also observed in a ternary configuration with DPP and for other Kv4.x channels. Intriguingly, our data reveal the induction of a slow recovery component for both KChIP1a and KChIP1b (referred to as 1a and 1b for simplicity below) co-expression. The effect is consistently observed for all Kv4.x channels, both in a binary configuration and in a ternary configuration with DPP. These results suggest a functional role for the KChIP1-mediated slow recovery from inactivation, which was therefore addressed mechanistically.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eModulation of Kv4 channel-mediated A-type currents by the KChIP splice variants 1a and 1b\u003c/h2\u003e\u003cp\u003eTo study different aspects of Kv4 channel modulation by the two KChIP splice variants 1a and 1b, we expressed Kv4.x channels (Kv4.1, Kv4.2, Kv4.3S or Kv4.3L) in the following configurations (see Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e): 1. Kv4.x α-subunit alone; 2. Kv4.x α-subunit together with either 1a or 1b; 3. Kv4.x α-subunit together with DPP; 4. Kv4.x α-subunit together with DPP and either 1a or 1b. The effects of 1a or 1b co-expression on the following parameters were examined: 1. Macroscopic inactivation kinetics; 2. Kinetics of recovery from inactivation; 3. Voltage dependences of activation and steady-state inactivation (see Methods). The present study was primarily focussed on the effects of KChIP1 splice variant co-expression on Kv4.2 (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e), in order to complement previously published data obtained with a Kv4.2 stable Human Embryonic Kidney (HEK) 293 cell line, transiently transfected with 1a or 1b cDNA in the absence of DPP\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In the Supplements our results for Kv4.1, Kv4.3S and Kv4.3L are illustrated (Figs. S3 - S5), and the data for all Kv4.x channels, including statistical analysis results, are summarized (Tables S1 - S4).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe initially analysed the kinetics of macroscopic inactivation during prolonged depolarizing voltage pulses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B; see Methods). We obtained time constants of \u0026sim; 16, 68, 809 ms (81, 15, 4%) for Kv4.2 alone and \u0026sim; 9, 50, 1136 ms (86, 11, 3%) for Kv4.2\u0026thinsp;+\u0026thinsp;DPP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D; Table S2). Both in a binary and in a ternary configuration (see Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), the co-expression of the KChIP splice variants 1a and 1b caused the typical crossover of normalized current traces when overlayed with control traces obtained in the absence of KChIP1\u003csup\u003e18\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). This was due to a slowing of the initial and intermediate decay components, while the final decay component was unaffected or slightly accelerated by KChIP1 co-expression. In the binary configuration we obtained time constants of \u0026sim; 47, 113, 697 ms (18, 80, 2%) for Kv4.2\u0026thinsp;+\u0026thinsp;1a, and \u0026sim; 34, 118, 615 ms (42, 48, 10%) for Kv4.2\u0026thinsp;+\u0026thinsp;1b (data for 10 ng KChIP1 cRNA per oocyte, but the differences between 1a and 1b co-expression in a binary configuration did not depend on the amount of KChIP1 cRNA; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In the ternary configuration the time constants were \u0026sim; 32, 65, 531 ms (41, 57, 2%) for Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1a, and \u0026sim; 33, 85, 483 ms (48, 48, 4%) for Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD; Table S2). The effects of auxiliary subunit co-expression on Kv4.1, Kv4.3S and Kv4.3L-mediated current decay kinetics showed a considerable variability. However, the typical acceleration of the final cumulative component of macroscopic inactivation was consistently observed for both 1a and 1b co-expression and in either channel configuration (Fig. S3; Tables S1, S3 and S4).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e- Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e -\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eUnusual modulation of Kv4 channel recovery kinetics by the KChIP splice variants 1a and 1b\u003c/h3\u003e\n\u003cp\u003eIt has been reported previously that, unlike 1a, 1b causes biphasic kinetics of recovery from inactivation, resulting in a slowing of the overall recovery process in Kv4.2 channels\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. We found that in the absence of KChIP1 the recovery of Kv4.2 channels from inactivation followed a single-exponential time course, and the recovery kinetics were accelerated by DPP co-expression\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA - D). The recovery time constants were 563 ms for Kv4.2 alone and 117 ms for Kv4.2\u0026thinsp;+\u0026thinsp;DPP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F; Table S2). Surprisingly, we observed biphasic recovery kinetics for both 1a and 1b co-expression in either channel configuration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D), albeit the obtained time constants and their relative amplitudes differed for the two KChIP1 splice variants, with a more pronounced slowing for 1b. In the binary configuration, we obtained recovery time constants of 126 ms and \u0026sim;1.5 s (87 and 13%) for Kv4.2\u0026thinsp;+\u0026thinsp;1a, and 218 ms and \u0026sim;5.4 s, (56 and 44%) for Kv4.2\u0026thinsp;+\u0026thinsp;1b (data for 10 ng KChIP1 cRNA per oocyte, but the distinct remodelling features seen for 1a and 1b co-expression in a binary configuration did not depend on the amount of KChIP1 cRNA; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE; Table S2). In a ternary configuration, the recovery time constants were \u0026sim;24 and 395 ms (79 and 21%) for Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1a, and \u0026sim; 41 ms and 1.3 s (57 and 43%) for Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF; Table S2). The modulation of recovery kinetics caused by 1a and 1b co-expression observed for Kv4.1, Kv4.3S and Kv4.3L in a binary and a ternary configuration was very similar to the results obtained with Kv4.2 (Fig. S4; Tables S1, S3 and S4).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e- Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e -\u003c/p\u003e\u003cp\u003e\u003cb\u003eVoltage dependence of Kv4 channel steady-state inactivation is differentially modulated by KChIP splice variants 1a and 1b\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFinally, we examined the effects of 1a and 1b co-expression on the voltage dependences of activation and steady-state inactivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e; see Methods). For all channel configurations tested, the Kv4.2 activation and inactivation curves showed little overlap, characteristic for preferential CSI\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, and inactivation curves were steeper than activation curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, C). For Kv4.2 alone, halfmaximal activation occurred at +\u0026thinsp;5.5 mV with a slope factor of 33.8 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB; Table S2). DPP co-expression resulted in a negative shift of the activation curve\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, with halfmaximal activation at -28.5 mV (slope factor 23.2 mV) for Kv4.2\u0026thinsp;+\u0026thinsp;DPP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD; Table S2). In a binary configuration, co-expression of KChIP1 splice variants caused a steepening and a negative shift of activation curves with halfmaximal activation at -9.6 mV (slope factor 25.6 mV) and \u0026minus;\u0026thinsp;5.7 mV (slope factor 30.8 mV) for Kv4.2\u0026thinsp;+\u0026thinsp;1a and Kv4.2\u0026thinsp;+\u0026thinsp;1b, respectively (data for 10 ng KChIP1 cRNA per oocyte, but the differences between 1a and 1b co-expression in a binary configuration were not dependent on the amount of KChIP1 cRNA; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B; Table S2). In a ternary configuration with DPP, KChIP1 co-expression caused a positive shift of activation curves, with halfmaximal activation at -19.2 mV (slope factor 26.1 mV) and \u0026minus;\u0026thinsp;12.1 mV (slope factor 33.7 mV) for Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1a and Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D; Table S2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e- Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e -\u003c/p\u003e\u003cp\u003eThe analysis of steady-state inactivation revealed that halfmaximal inactivation occurred at -68.3 mV (slope factor 6.4 mV) for Kv4.2 alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Similar to the voltage dependence of activation, the inactivation curve was shifted negative by DPP co-expression\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, with halfmaximal inactivation at -73.5 mV (slope factor 4.7 mV) for Kv4.2\u0026thinsp;+\u0026thinsp;DPP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD; Table S2). The modulation of the voltage dependence of steady-state inactivation differed between 1a and 1b co-expression. While 1a caused virtually no effect, 1b shifted inactivation curves in the negative direction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, C; Table S2). In a binary configuration, halfmaximal inactivation occurred at -66.3 mV (slope factor 5.2 mV) for Kv4.2\u0026thinsp;+\u0026thinsp;1a and at -76.0 mV (slope factor 6.1 mV) for Kv4.2\u0026thinsp;+\u0026thinsp;1b (data for 10 ng KChIP1 cRNA per oocyte, but the differences between 1a and 1b co-expression in a binary configuration were not dependent on the amount of KChIP1 cRNA; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB; Table S2). In a ternary configuration with DPP, halfmaximal inactivation occurred at -74.4 mV (slope factor 4.3 mV) for Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1a and at -81.3 mV (slope factor 7.4 mV) for Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD; Table S2). The modulation of the voltage dependences of Kv4.1, Kv4.3S and Kv4.3L gating caused by 1a and 1b co-expression in a binary and a ternary configuration was very similar to the results described for Kv4.2. Although the results showed some variability among the different Kv4.x channels, binary and ternary channels containing 1b had the most negative inactivation curves (Fig. S5; Tables S1, S3 and S4).\u003c/p\u003e\n\u003ch3\u003eHigh external K\u003csup\u003e+\u003c/sup\u003e identifies P/C-type inactivation features of the 1b-mediated slow recovery component\u003c/h3\u003e\n\u003cp\u003eOur kinetic analyses of recovery from inactivation have corroborated the view that the biphasic nature of the recovery process represents an intrinsic feature of KChIP1-containing Kv4 channels. Moreover, our data suggest that, unlike 1a, 1b co-expression induces only negligible acceleration of recovery, but more or less exclusively adds a slow recovery component (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Therefore, we chose Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b ternary channels (1.7\u0026thinsp;+\u0026thinsp;5\u0026thinsp;+\u0026thinsp;10 ng cRNA per oocyte) to study the kinetics of recovery from inactivation in more mechanistic detail. To this end, we intended to experimentally interfere with different mechanisms of inactivation, normally vestigial in Kv4 channels\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, but possibly promoted by 1b co-expression, such as N-type inactivation\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e or P/C-type inactivation\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eVestigial N-type inactivation features of Kv4 channels are thought to be largely suppressed by KChIP binding\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Nevertheless, in order to abolish putative residual or newly generated N-type inactivation features in Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b channels, we used an N-terminally truncated version of Kv4.2, which lacks the first 10 amino acids (Δ10; Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This deletion removes the Kv4.2 N-terminal inactivation domain, but leaves KChIP binding intact\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. We found that macroscopic inactivation kinetics of the truncated channels were almost identical to wild-type (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Also, the kinetics of recovery from inactivation were still biphasic and very similar to wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; Table S5). Based on these results, residual or newly generated N-type inactivation as a possible mechanism underlying the slow recovery kinetics of 1b containing Kv4.2 channel complexes was excluded.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e- Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e -\u003c/p\u003e\u003cp\u003eNext, we intended to interfere with putative residual or newly generated P/C-type inactivation in Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b channels by applying a high K\u003csup\u003e+\u003c/sup\u003e solution (see Methods). Elevated external K\u003csup\u003e+\u003c/sup\u003e has been shown previously to slow current decay and to accelerate recovery kinetics by interfering with the classical P/C-type inactivation of \u003cem\u003eShaker\u003c/em\u003e-related (Kv1) channels\u003csup\u003e\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Under these conditions, the macroscopic inactivation of Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b channels was accelerated, in accordance with previous reports\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC; see Discussion). Intriguingly, high K\u003csup\u003e+\u003c/sup\u003e solution also affected the kinetics of recovery from inactivation by specifically accelerating the slow component (2.4-fold; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD; Table S5). High K\u003csup\u003e+\u003c/sup\u003e solution also influenced Kv4.2 Δ10\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b channels, in the same manner as wild-type ternary channels, albeit with a somewhat weaker effect on the slow recovery component (2.1-fold acceleration; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F; Table S5). Notably, superfusion with TEA solution (see Methods) had no obvious effects, except for an acceleration of current decay kinetics, which was more pronounced for Kv4.2 Δ10 than for Kv4.2 wild-type ternary channels (Fig. S6; Table S5).\u003c/p\u003e\u003cp\u003eFrom our experimental results with high K\u003csup\u003e+\u003c/sup\u003e solution we concluded that Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b channels may not only undergo CSI related to dynamic S6 rearrangements\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, from which recovery is thought to be fast. Rather, promoted by 1b co-expression, a fraction of channels may also undergo strong \u003cem\u003eShaker\u003c/em\u003e-like P/C type inactivation with slow recovery kinetics. Therefore, we finally intended to study Kv4.2 channel constructs in which S6-related CSI was specifically modified. For this purpose, we chose two previously characterized S6 mutants (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), in which recovery from inactivation had been found to be either drastically slowed (Kv4.2 L400A) or drastically accelerated (Kv4.2 N408A)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. We expected the putative newly generated P/C-type inactivation with slow recovery kinetics in the presence of 1b to be further augmented in Kv4.2 N408A, where the affinity towards S6-related CSI states is lowered\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e (see Discussion), but not in Kv4.2 L400A. The S6 mutations influenced both macroscopic inactivation and recovery from inactivation in Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b channels in a characteristic manner. While L400A caused an overall slowing, N408A caused a strong acceleration of current decay kinetics (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; Table S5). Kv4.2 L400A\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b recovery kinetics were very similar to wild-type ternary, whereas Kv4.2 N408A\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b recovery kinetics differed substantially: They followed a single exponential time course, apparently corresponding to the slow recovery components of wild-type and L400A ternary channels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; Table S5). Finally, we tested the two mutants in high K\u003csup\u003e+\u003c/sup\u003e solution. For both mutants, macroscopic inactivation was accelerated under these conditions, similar to wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, E; see also Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Also, the slow recovery component was accelerated in L400A ternary channels, as seen for wild-type, albeit only 1.4-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD; Table S5). Remarkably, in N408A ternary channels the recovery kinetics remained single-exponential, and were accelerated 2.2-fold in high K\u003csup\u003e+\u003c/sup\u003e solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF; Table S5), suggesting that Kv4.2 N408A\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b recovery kinetics fully reflect the recovery from a putative \u003cem\u003eShaker\u003c/em\u003e-like P/C type inactivation. Taken together, our findings support the notion that otherwise vestigial P/C-type inactivation features of Kv4.2 channels are strongly promoted by 1b and may co-exist with S6-related CSI.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e- Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e -\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present paper we set out to critically revise a previous report on the distinctive features of the KChIP splice variants 1a and 1b. The previously reported induction of a novel extremely slow component of recovery from inactivation by 1b is confirmed by our results, but they reveal that 1a co-expression can also cause biphasic recovery kinetics. KChIP1 co-expression effects in heterologous systems and the putative mechanism, including possible structure-function relationships, underlying the slow recovery component, will be discussed in a physiological context.\u003c/p\u003e\u003cp\u003eCo-expression of an initially identified KChIP1, which is identical to the 1a splice variant used herein, with Kv4.2 in tissue culture cells and \u003cem\u003eXenopus\u003c/em\u003e oocytes defined the hallmarks of Kv4 channel modulation by KChIPs. These included an increase in current density, a slowing of macroscopic inactivation and an acceleration of recovery from inactivation\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The initially identified KChIP1 (=\u0026thinsp;1a) was used for a detailed analysis of Kv4 channel assembly and trafficking, to demonstrate the stabilizing effect of KChIP1 on Kv4.3 tetramers, as well as the KChIP1-mediated release of Kv4.2 ER retention, and the role of KChIP1-specific N-terminal myristoylation in subcellular targeting of the Kv4.2\u0026thinsp;+\u0026thinsp;KChIP1 complex to post-ER transport vesicles\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. These results provided an explanation for the observed increase in current density upon KChIP1 co-expression. The initially identified KChIP1 (=\u0026thinsp;1a) was also used for a detailed biophysical analysis of Kv4 channel gating modulation, showing similar remodelling of Kv4.1 and Kv4.3 channel inactivation\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The remodelling included the typical streamlining effect on macroscopic currents, with a crossover of normalized current traces obtained in the absence and presence of KChIP1, respectively (see also Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig. S3), as well as a shift of inactivation curves to more positive voltages, most likely reflecting the accelerated recovery from inactivation, upon KChIP1 co-expression\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Our experimental results obtained with Kv4.x\u0026thinsp;+\u0026thinsp;1a co-expression largely confirm these initial observations.\u003c/p\u003e\u003cp\u003eThe alternatively spliced KChIP variant 1b differs from 1a by an 11-amino-acid N-terminal insertion (residues 22\u0026ndash;33 in 1b; Fig. S2), rich in aromatic side chains\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The 1b splice variant has been reported previously to differ from 1a quite substantially, by inducing biphasic kinetics of recovery from inactivation with a newly generated extremely slow component\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Our critical revision of this previous report on the distinctive features of 1b was motivated by the fact that the authors had used transient transfection of a stable Kv4.2 cell-line with KChIP1 cDNA. We suspected that in this system, cell-to-cell variations in cDNA uptake and differences in the temporal expression profiles of α- (stable) and β-subunits (transient), may have caused two different Kv4.2 channel populations present at roughly equal amounts in the plasma membrane; i.e., Kv4.2\u0026thinsp;+\u0026thinsp;1b and Kv4.2 alone (stable), with fast and slow recovery kinetics, respectively. Therefore, we subjected the previous findings to a rigorous test by studying KChIP1 co-expression effects on Kv4.2 channel gating in a more quantitative manner in cRNA-injected \u003cem\u003eXenopus\u003c/em\u003e oocytes. With this approach the previously reported biphasic recovery kinetics were confirmed, irrespective of the amount of 1b cRNA injected (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Thus, our initial concerns regarding the previously used expression system and transfection procedure were clearly unfounded. We further asked whether the 1b effect on recovery kinetics is also observed with other Kv4.x subtypes, and whether it is still visible in a ternary configuration with DPP, which itself strongly accelerates the recovery of Kv4 channels from inactivation\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e and may therefore be able to mask the 1b effect. The results of our experiments performed with all Kv4.x channel subtypes co-expressed with excess amounts of 1b, both in a binary configuration and in a ternary configuration with DPP, support the notion that the induction of a slow recovery component represents an intrinsic feature of the KChIP splice variant 1b with general applicability.\u003c/p\u003e\u003cp\u003eUnexpectedly, we found that, similar to 1b, 1a is also capable of inducing a slow recovery component. It is remarkable that, for the most part, previous reports of KChIP1 (=\u0026thinsp;1a) co-expression effects on Kv4 channels, in both cDNA-transfected tissue culture cells\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan additionalcitationids=\"CR44 CR45\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e and cRNA-injected \u003cem\u003eXenopus\u003c/em\u003e oocytes\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan additionalcitationids=\"CR48 CR49 CR50 CR51\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, used a single-exponential function to describe the kinetics of recovery from inactivation. Also, previous reports on the recovery kinetics of ternary Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1a channels in different expression systems\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e used a single-exponential function for their analyses. Multi-exponential fitting of the 1a-induced recovery kinetics was not considered in these previous studies, despite the sometimes obvious inadequacy of the single-exponential fit. In some of the previously used experimental protocols the chosen interpulse durations may have been not long enough to reliably capture such a slow recovery component. Notably, in one study, performed in \u003cem\u003eXenopus\u003c/em\u003e oocytes with 2 mM standard external K\u003csup\u003e+\u003c/sup\u003e, like in the present study, the initially identified KChIP1 (=\u0026thinsp;1a) was reported to cause biphasic recovery kinetics for Kv4.1 and Kv4.2\u003csup\u003e53\u003c/sup\u003e. Double-exponential fitting in that study resulted in a numerical ratio of fast and slow recovery time constants and in fractional amplitudes very similar to our results. Van Hoorick and co-workers were able to convert biphasic into virtually monophasic recovery kinetics by mutating three of the five aromatic amino acid residues in 1b to alanine\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. However, our kinetic analyses suggest that additional structural determinants in KChIP1 may contribute to the special remodelling features.\u003c/p\u003e\u003cp\u003eWe have chosen Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b ternary channels to study the newly generated slow recovery component mechanistically. In addition to CSI, which is most prominent in Kv4 channels\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, Kv channels may undergo N-type inactivation and/or P/C-type inactivation, which are the major inactivation mechanisms of \u003cem\u003eShaker\u003c/em\u003e-related (Kv1) channels\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. These classical \u003cem\u003eShaker\u003c/em\u003e inactivation mechanisms are also present in Kv4 channels, but in vestigial forms\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Commonly used approaches; i.e., N-terminal truncation and application of high external K\u003csup\u003e+\u003c/sup\u003e, respectively, were applied in the present study to test for a possible enhancement of the classical \u003cem\u003eShaker\u003c/em\u003e inactivation mechanisms in Kv4 channels, caused by the KChIP splice variant 1b. Since KChIP binding is thought to sequester and immobilize a Kv4 N-terminal inactivation domain\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, an involvement of N-type inactivation seemed rather unlikely beforehand. The virtual absence of an effect of a ten amino acid N-terminal truncation (Δ10) on Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b recovery kinetics confirmed this \u003cem\u003ea priori\u003c/em\u003e assumption. High external K\u003csup\u003e+\u003c/sup\u003e caused an acceleration of Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b current decay kinetics, very similar to the results obtained by Kaulin and co-workers\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, and an indication of the removal of the vestigial P/C-type inactivation to favor CSI in ternary Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b channels. Remarkably, however, high external K\u003csup\u003e+\u003c/sup\u003e also accelerated the slow component of the biphasic recovery process of wild-type and Δ10, and the monophasic recovery process of N408A ternary channels, reminiscent of the accelerated recovery kinetics observed previously under these conditions for the \u003cem\u003eShaker\u003c/em\u003e-related channels Kv1.3\u003csup\u003e32,33\u003c/sup\u003e and Kv1.4\u003csup\u003e34,35\u003c/sup\u003e. The striking similarity of these high K\u003csup\u003e+\u003c/sup\u003e effects on recovery kinetics supports the notion that the slow recovery component observed in Kv4 channels upon 1b co-expression is related to a \u003cem\u003eShaker\u003c/em\u003e-like P/C-type inactivation. Notably, our results suggest that 1b-independent vestigial P/C-type inactivation\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and a more stable 1b-induced \u003cem\u003eShaker\u003c/em\u003e-like P/C-type inactivation may co-exist in Kv4 channels. This notion is also supported by our finding that TEA influenced current decay kinetics similar to high K\u003csup\u003e+\u003c/sup\u003e, but left Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;1b recovery kinetics largely unaffected (see Fig. S6).\u003c/p\u003e\u003cp\u003eApart from an immobilization of the Kv4 N-terminal inactivation domain caused by KChIP binding\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, the structure-function relationships of Kv4 channel remodelling by KChIPs are largely unknown. In particular, The 11-amino-acid aromatic cluster, thought to be responsible for the special Kv4 remodelling by 1b\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, is not included in the available Kv4/KChIP1 structures\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e (see also Fig. S2). Thus, a putative direct interaction between the aromatic cluster and the Kv4 α-subunit remains uncertain. One may speculate whether the clamping conformation adopted by the four KChIP1 molecules surrounding the gating-relevant\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e Kv4 T-domains\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e may be influenced by the aromatic cluster. It is intriguing in this context that a naturally occurring Kv1.5 Δ209 N-terminal truncation variant, which has lost its T-domain, exhibits, in addition to classical P/C-type inactivation, a form of CSI, resulting in biphasic kinetics of recovery from inactivation\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRecently high resolution cryo EM data have elucidated structural details of Kv1.2 P/C-type inactivation. The data suggest that P/C-type inactivation in these channels leads to a dilation rather than a constriction of the selectivity filter\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, and that an isoleucine gate localized in S6, right below the selectivity filter plays a central role\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Cryo EM data have also elucidated structural details of Kv4.2 channel inactivation\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, by capturing, in addition to an open state, two putative non-conducting states, referred to as \"inactivated\" and \"intermediate\". In both non-conducting states, an upper and a lower gate within the pore are expected to prevent the passage of K\u003csup\u003e+\u003c/sup\u003e ions. The lower gate is related to the dynamic coupling between voltage sensor and pore modules, allowing conformational rearrangements, that lead to a symmetry breakdown of S6-segments as the basis of CSI\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Intriguingly, the upper gate in Kv4.2 is homologous to the isoleucine gate, which mediates P/C-type inactivation in Kv1.2\u003csup\u003e58\u003c/sup\u003e. Based on these structural similarities, one may speculate that in the presence of the KChIP splice variant 1b, the upper (isoleucine) gate in Kv4.2 may evolve into a major inactivation gate, especially if S6-related CSI (lower gate) is unstable, as suggested by our experimental findings with the Kv4.2 S6 mutant N408A (see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Of note, a close inspection of the structures put forward by Kise and co-workers\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and by Ma and co-workers\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, containing Kv4.2 and Kv4.3, respectively, in different configurations with KChIP1 fragments lacking the 11-amino-acid aromatic cluster, suggest pore radii at the upper (isoleucine) gate between 4.7 and 7.7 \u0026Aring;, wide enough to let a hydrated K\u003csup\u003e+\u003c/sup\u003e ion pass. Thus, with the possible exceptions of the \"intermediate\" and \"inactivated\" structures put forward by Ye and co-workers, defining an upper (isoleucine) gate for Kv4.2\u003csup\u003e14\u003c/sup\u003e, putative P/C-type inactivated Kv4 channels have not been captured in 3D, yet.\u003c/p\u003e\u003cp\u003eFrom a physiological point of view, the co-assembly of different Kv4.x channel subtypes with DPPs and a variety of KChIPs, including functionally distinct β-subunit splice variants like 1a and 1b, is expected to contribute to an immense diversity of \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSA\u003c/sub\u003e properties in different cell types, with a considerable impact on neuronal excitability and discharge behavior\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. In the rat brain Kv4.2 is co-localized majorly with KChIP2 and KChIP4 in pyramidal neurons, whereas a high co-localization of Kv4.3 with KChIP1 is seen in large multipolar interneurons\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. In fact, co-localization of Kv4.3 and KChIP1 is reliably found in a fraction of parvalbumin, calbindin, calretinin and somatostatin-positive hippocampal interneurons, such that Kv4.3/KChIP1 co-expression has been suggested to be used as a separate independent hippocampal interneuron marker\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Using siRNA knockdown of KChIP1 expression in a hippocampal preparation has been shown to specifically affect firing behavior of Kv4.3/KChIP1 co-expressing CA1 interneurons. KChIP1 knockdown in these interneurons caused an increase in firing frequency, reportedly due to a slowing of \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSA\u003c/sub\u003e recovery rather than a decrease in \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSA\u003c/sub\u003e amplitude\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Alternative splicing of KChIP1 with the effect of slowing \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSA\u003c/sub\u003e recovery may have a comparable effect. The distribution of KChIP1 transcripts, has been studied in human, rat and mouse tissues\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The findings suggest that the KChIP splice variants 1a and 1b are expressed at comparable amounts in the human brain. It should be noted, that the optional expression of the Kv4.3 splice variants S and L in combination with the KChIP splice variant 1a or 1b may allow for a fairly large spectrum of \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSA\u003c/sub\u003e properties in Kv4.3/KChIP1 co-expressing interneurons (see panels B and C in Figs. S3 - S5).\u003c/p\u003e\u003cp\u003eTaken together, strong promotion of \u003cem\u003eShaker\u003c/em\u003e-like P/C-type inactivation features in Kv4 channels, especially by the KChIP splice variant 1b, may limit time-dependent \u003cem\u003eI\u003c/em\u003e\u003csub\u003eSA\u003c/sub\u003e availability during repetitive firing, thereby increasing firing frequency, especially in large multipolar Kv4.3/KChIP1 co-expressing interneurons.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePlasmids and constructs\u003c/h2\u003e\u003cp\u003eIn this study the human Kv4 channel clones Kv4.1, Kv4.2, and Kv4.3\u003csup\u003e62\u003c/sup\u003e were used. The long Kv4.3 splice variant (Kv4.3L) was a kind gift from Geoffrey Abbott (Department of Physiology and Biophysics, University of California, Irvine, USA). The human KChIP1a splice variant (referred to as 1a in the present paper) was kindly provided by Dirk Isbrandt (Center for Molecular Medicine, University of Cologne, Germany), and the human DPP6s splice variant (referred to as DPP in the present paper) by Nicole Schmitt (Department of Biomedical Sciences, Faculty of Health and Medical Sciences, Copenhagen, Denmark). In addition to Kv4.2 wild type, three previously studied Kv4.2 mutant constructs were used: In one construct the first ten amino acids had been deleted (Kv4.2 Δ10)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e; in the two other constructs individual residues in the distal S6 segment, leucine at position 400 or asparagine at position 408, had been replaced by alanine (Kv4.2 L400A and Kv4.2 N408A, respectively\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e; see also Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). All cDNA clones were inserted into the multiple cloning site of pGEM-HE. In order to generate KChIP1b (referred to as 1b in the present paper), a 33 bp fragment was inserted into the 1a coding region by overlap PCR with appropriate primers (fwd: accagtatcagagagaTAAGATTGAAGATGAGCTGGAG; rev: aataccaccaggcgatgTCTTTCGAGGGTCGCCTT) in a back-to-back orientation, using the Q5 Site Directed Mutagenesis Kit (New England Biolabs). Successful mutagenesis was verified by Sanger sequencing of the complete coding region and flanking sequences. Transformed JM109 \u003cem\u003eEschericia coli\u003c/em\u003e cells (Promega) were grown in Luria Broth medium complemented with ampicillin, and plasmids were isolated using the QIAprep Spin Miniprep Kit (QIAGEN). Purified plasmids were linerarized using \u003cem\u003eNot\u003c/em\u003eI (New England Biolabs), and the RiboMaxLargeScale RNA production system T7 (Promega) was utilized for the \u003cem\u003ein vitro\u003c/em\u003e transcription of cRNA.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eHeterologous channel expression\u003c/h3\u003e\n\u003cp\u003eKv4 channels and their auxiliary β-subunits were expressed in \u003cem\u003eXenopus laevis\u003c/em\u003e oocytes. Female frogs (Nasco) were anesthetized for 8\u0026ndash;10 min in ethyl 3-aminobenzoate methanesulfonate (tricaine, Sigma; 1.2 g/l chlorine-free frog water containing 7.5 mM Tris-HCl). Part of the ovary lobes was surgically removed, and the wound was immediately sutured (monocryl 4\u0026thinsp;\u0026minus;\u0026thinsp;0, ethicon). For the final (sixth, intervals of \u0026sim; one year) oocyte harvest, the frogs are euthanized by deep tricaine anesthesia (30 min) followed by decapitation. Animal care and experimental procedures related to the harvesting of \u003cem\u003eXenopus\u003c/em\u003e oocytes were conducted in accordance with the German Animal Welfare Act and were approved by the Authority for Justice and Consumer Protection of the City of Hamburg (approval # N 101/2023). All procedures comply with the ARRIVE guidelines. The obtained ovary tissue was mechanically dispersed using a pair of fine forceps and digested for 3\u0026ndash;5 h under constant agitation in a calcium-free solution containing (in mM) 82.5 NaCl, 2 KCl, 1 MgCl\u003csub\u003e2\u003c/sub\u003e, 5 HEPES, and 1.3 mg/ml collagenase type II (Sigma); pH 7.5, NaOH. Defolliculated stage V\u0026ndash;VI oocytes were selected one day after harvesting or later, and 25 or 50 nl cRNA solution were injected per oocyte using a Nanoliter 2000 microinjector (World Precision Instruments). Individual injections resulted in Kv4 cRNA amounts between 0.8 and 5 ng per oocyte, in the absence or presence of KChIP1 cRNA (between 2.5 and 20 ng per oocyte) and/or DPP cRNA (between 2.5 and 5 ng per oocyte). Injected oocytes were incubated at 16\u0026deg;C in a solution containing (in mM) 75 NaCl, 5 Na-pyruvate, 2 KCl, 2 CaCl\u003csub\u003e2\u003c/sub\u003e, 1 MgCl\u003csub\u003e2\u003c/sub\u003e, 5 HEPES, and 50 mg/ml gentamicin (Sigma), pH 7.5, NaOH; and used for recordings 1\u0026ndash;10 days (d1 - d10) after cRNA injection (see Tables S1 - S4 for cRNA amounts per oocyte and days of recording for individual experiments).\u003c/p\u003e\n\u003ch3\u003eElectrophysiology\u003c/h3\u003e\n\u003cp\u003eCurrents were recorded at room temperature (20\u0026ndash;22\u0026deg;C) under two electrode voltage-clamp, using a TurboTec-3 amplifier (npi electronics) controlled by PatchMaster software (HEKA). The ND96 bath solution contained (in mM) 96 NaCl, 2 KCl, 1 CaCl\u003csub\u003e2\u003c/sub\u003e, 1 MgCl\u003csub\u003e2\u003c/sub\u003e and 5 HEPES (pH 7.4, NaOH). In some experiments variations of this solution were used, in which either NaCl was replaced by KCl (high K\u003csup\u003e+\u003c/sup\u003e solution, 98 mM K\u003csup\u003e+\u003c/sup\u003e) or NaCl and KCl were replaced by tetraethylammonium-Cl (TEA solution, 98 mM TEA). Glas microelectrodes were filled with 3 M KCl and had tip resistances of 0.2\u0026ndash;0.5 MΩ in standard ND96 bath solution. The holding voltage was \u0026minus;\u0026thinsp;80 mV. For the study of macroscopic inactivation a 2.5 s test pulse to +\u0026thinsp;40 mV was applied following a 2 s conditioning pulse to -100 mV, in order to activate and immediately inactivate a large fraction of channels (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Recovery from inactivation was measured at -80 mV using a double-pulse protocol with a 3 s control pulse and a brief test pulse to +\u0026thinsp;40 mV, separated by interpulse intervals (Δ\u003cem\u003et\u003c/em\u003e), which lasted between 10 ms and \u0026sim; 41 s (iteration factor 2, see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Voltage step protocols were applied to study the voltage dependence of gating. For the voltage dependence of activation, test pulses to voltages between \u0026minus;\u0026thinsp;90 and +\u0026thinsp;70 mV (10 mV increments, see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, inset) were applied following a 2 s conditioning pulse to -100 mV. For the voltage dependence of steady-state inactivation a double-pulse protocol was used, in which after an initial 2 s conditioning pulse to -120 mV and a subsequent brief control pulse to +\u0026thinsp;40 mV, brief test pulses to +\u0026thinsp;40 mV were applied following an interpulse interval of 10 s with conditioning voltages between \u0026minus;\u0026thinsp;120 and 0 mV (10 mV increment, see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, inset). Capacitive current transients were not compensated. Current measurements at -95 mV (approximate E\u003csub\u003erev\u003c/sub\u003e for K\u003csup\u003e+\u003c/sup\u003e currents in standard ND96 bath solution), before each voltage pulse protocol, were used to calculate the leak current at any other voltage in order to correct peak current amplitudes. Alternatively, a prepulse-inactivation subtraction protocol was used\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e to leak-subtract entire current traces.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eThe data were analysed using FitMaster (HEKA) and Kaleidagraph (Synergy Software). Macroscopic inactivation (i.e., current decay) kinetics (at +\u0026thinsp;40 mV) were described by the sum of three (if possible) or two exponential functions. The kinetics of recovery from inactivation (I\u003csub\u003etest\u003c/sub\u003e / I\u003csub\u003econtrol\u003c/sub\u003e plotted against interpulse duration) were described by a single-exponential function or the sum of two exponential functions, as required. The relative amplitudes of the individual time constants, obtained with multi-exponential fitting, are given in %. The voltage dependences of activation (cord conductance calculation with E\u003csub\u003erev\u003c/sub\u003e = -95 mV) and steady-state inactivation (I\u003csub\u003etest\u003c/sub\u003e / I\u003csub\u003econtrol\u003c/sub\u003e) were analysed with appropriate Boltzmann-functions, as described previously\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Fit results (i.e., time constants, relative amplitudes, V\u003csub\u003e1/2\u003c/sub\u003e values and slope factors) are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, D; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B; see also Tables S1 - S5). Normalized current amplitudes in pooled analysis plots containing the fitting curves are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, C; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, D, F; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, D, F). Statistical analyses for two groups were performed based on unpaired (comparison of 1a and 1b effects) or paired (effects of solution change while recording from individual oocyes) Student's \u003cem\u003et\u003c/em\u003e-tests. For multiple groups (1a and ab co-expression effects relative to Kv4.x alone or relative to Kv4.x\u0026thinsp;+\u0026thinsp;DPP) one-way analysis of variance (ANOVA) with Dunnett\u0026rsquo;s posthoc testing was used (see also Tables S1 \u0026ndash; S5).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe data are summarized in Tables S1 - S5; datasets generated and analyzed during the present study are available from the corresponding author on reasonable request.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Annett Hasse for technical assistance and Christiane K. Bauer and Stefan Kindler for helpful discussion.\u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.B. designed the experiments. W.C. and G.T. executed the experiments. W.C., G.T., and R.B. analyzed the data. W.C., G.T. and R.B. interpreted the data. R.B. wrote the manuscript. W.C., G.T., and R.B. critically reviewed and approved the manuscript.\u0026nbsp;\u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Deutsche Forschungsgemsinschaft (DFG; Ba2055/6), and by a grant from the China Scholarship Council (No. 202106090277) to Wuyou Cao.\u0026nbsp;\u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information:\u0026nbsp;\u003c/strong\u003eThe online version contains supplementary material (Figs. S1 - S6; Tables S1 - S5) available at xxxxx.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHille, B. \u003cem\u003eIon Channels of Excitable Membranes\u003c/em\u003e. 3 edn, (Sinauer Associates, Inc., 2001).\u003c/li\u003e\n\u003cli\u003ePak, M. 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Dendritic A-type potassium channel subunit expression in CA1 hippocampal interneurons. \u003cem\u003eNeuroscience\u003c/em\u003e \u003cstrong\u003e154,\u003c/strong\u003e 953-964 (2008). \u003c/li\u003e\n\u003cli\u003eIsbrandt, D.\u003cem\u003e et al.\u003c/em\u003e Gene structures and expression profiles of three human KCND (Kv4) potassium channels mediating A-type currents I\u003csub\u003eTO\u003c/sub\u003e and I\u003csub\u003eSA\u003c/sub\u003e. \u003cem\u003eGenomics\u003c/em\u003e \u003cstrong\u003e64,\u003c/strong\u003e 144-154 (2000).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"A-type current, repetitive firing, recovery from inactivation, exponential fitting, Xenopus oocytes, two-electrode voltage-clamp","lastPublishedDoi":"10.21203/rs.3.rs-7356877/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7356877/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eKv4 channels mediate a somatodendritic A-type (i.e., rapidly inactivating) potassium current, which controls neuronal excitability and firing frequency. Kv4 channels form complexes with auxiliary DPPs and KChIPs, which modify channel gating, including an acceleration of recovery from inactivation. Although ternary Kv4\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;KChIP complexes represent a likely native channel configuration, little is known about the concerted Kv4 channel modulation by DPPs and KChIPs. Here, we studied the modulatory effects of two functionally distinct KChIP1 splice variants (1a and 1b), utilizing two-electrode voltage-clamp in \u003cem\u003eXenopus\u003c/em\u003e oocytes. We tested Kv4.1, Kv4.2, Kv4.3S, and Kv4.3L, co-expressed with either KChIP1 splice variant in binary Kv4\u0026thinsp;+\u0026thinsp;KChIP1 and ternary Kv4\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;KChIP1 channel configurations. For all Kv4.x channels, we observed an extremely slow component of recovery from inactivation upon co-expression of either KChIP1 splice variant, which persisted in a ternary configuration with DPP. Our results suggest a special functional role of KChIP1b, limiting the time-dependent availability of the somatodendritic A-type current. Our mechanistic investigations of ternary Kv4.2\u0026thinsp;+\u0026thinsp;DPP\u0026thinsp;+\u0026thinsp;KChIP1b channels revealed a strong enhancement of P/C-type inactivation features, which are normally vestigial in Kv4 channels, but may co-exist with preferential closed-state inactivation in the presence of KChIP1b.\u003c/p\u003e","manuscriptTitle":"KChIP1 splice variants modulate Kv4 channels by promoting P/C-type inactivation features","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-27 05:06:04","doi":"10.21203/rs.3.rs-7356877/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-03T02:50:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-31T20:07:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-30T21:17:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-30T14:54:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169075543890826438362698025419743617369","date":"2025-10-16T20:06:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152938309789285129062995688927374756480","date":"2025-10-16T01:06:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"142997502035455532376010318680854703051","date":"2025-10-15T21:06:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-03T02:13:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52296106618709384762915079240824375611","date":"2025-09-17T21:52:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-17T19:43:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-22T12:47:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-19T05:45:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-14T11:12:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-14T11:08:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5ac0975a-0972-43dc-a21c-8290198a6dcf","owner":[],"postedDate":"September 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55162050,"name":"Biological sciences/Biophysics"},{"id":55162051,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-01-19T17:04:40+00:00","versionOfRecord":{"articleIdentity":"rs-7356877","link":"https://doi.org/10.1038/s41598-026-35770-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-16 16:29:00","publishedOnDateReadable":"January 16th, 2026"},"versionCreatedAt":"2025-09-27 05:06:04","video":"","vorDoi":"10.1038/s41598-026-35770-5","vorDoiUrl":"https://doi.org/10.1038/s41598-026-35770-5","workflowStages":[]},"version":"v1","identity":"rs-7356877","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7356877","identity":"rs-7356877","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}