Voltage Sensor Movement Research Articles

The Structural Basis of IKs Ion-Channel Activation: Mechanistic Insights from Molecular Simulations

Relating ion channel (iCh) structural dynamics to physiological function remains a challenge. Current experimental and computational techniques have limited ability to explore this relationship in atomistic detail over physiological timescales. A framework associating iCh structure to function is necessary for elucidating normal and disease mechanisms. We formulated a modeling schema that overcomes the limitations of current methods through applications of artificial intelligence machine learning. Using this approach, we studied molecular processes that underlie human IKs voltage-mediated gating. IKs malfunction underlies many debilitating and life-threatening diseases. Molecular components of IKs that underlie its electrophysiological function include KCNQ1 (a pore-forming tetramer) and KCNE1 (an auxiliary subunit). Simulations, using the IKs structure-function model, reproduced experimentally recorded saturation of gating-charge displacement at positive membrane voltages, two-step voltage sensor (VS) movement shown by fluorescence, iCh gating statistics, and current-voltage relationship. Mechanistic insights include the following: 1) pore energy profile determines iCh subconductance; 2) the entire protein structure, not limited to the pore, contributes to pore energy and channel subconductance; 3) interactions with KCNE1 result in two distinct VS movements, causing gating-charge saturation at positive membrane voltages and current activation delay; and 4) flexible coupling between VS and pore permits pore opening at lower VS positions, resulting in sequential gating. The new modeling approach is applicable to atomistic scale studies of other proteins on timescales of physiological function.

Gating interaction maps reveal a noncanonical electromechanical coupling mode in the Shaker K+ channel

Membrane potential regulates the activity of voltage-dependent ion channels via specialized voltage-sensing modules but the mechanisms involved in coupling voltage-sensor movement to pore opening remain unclear due to lack of resting state structures and robust methods to identify allosteric pathways. Here, using a newly developed interaction energy analysis, we probe the interfaces of the voltage-sensing and pore modules in the drosophila Shaker K+ channel. Our measurements reveal unexpectedly strong equilibrium gating interactions between contacts at the S4 and S5 helices in addition to those between S6 and S4–S5 linker. Network analysis of MD trajectories shows that the voltage-sensor and pore motions are linked by two distinct pathways- canonical one through the S4–S5 linker and a hitherto unknown pathway akin to rack and pinion coupling involving S4 and S5 helices. Our findings highlight the central role of the S5 helix in electromechanical transduction in the VGIC superfamily.

External Protons Accelerate Deactivation of hERG Channels by Destabilizing the Relaxed State of the Voltage-Sensor

External protons accelerate the deactivation rate of cardiac hERG channels by an unknown mechanism. Recently, we showed that the hERG voltage sensor displays pronounced mode-shift behaviour due to relaxation, which stabilizes the activated voltage sensor position and limits its return upon repolarization1. We hypothesized that protons accelerate deactivation by destabilizing the relaxed state of the voltage sensor. To test this, we used cut-open voltage clamp and voltage clamp fluorimetry in Xenopus oocytes to measure voltage sensor movement, in addition to measurements of pore gating using two-electrode voltage clamp. Using durations that capture the majority of charge movement, the voltage-dependence of off-gating charge movement at pH 7.4 (V1/2 = −75.5 ± 2.3 mV, n=4) was found to be −34 mV left-shifted from that of on-gating charge (V1/2 = −41.4 ± 3.5 mV, n=5) indicative of relaxation-induced stabilization of the activated voltage sensor. At pH 6.5 this mode-shift was reduced to −19 mV (p<0.002; off-gating charge V1/2 = −51.8 ± 3.3 mV, n=5; on-gating charge V1/2=-32.7 ± 4.2 mV, n=5). This finding was corroborated by fluorescence reports of voltage sensor movement, which showed that the mode-shift was reduced from −43 mV at pH 7.4 (n=5) to −23 mV at pH 6.5 (n=5). In both of these reports of voltage sensor mode-shift, acidic pH induced a large depolarizing shift in the voltage-dependence of voltage sensor return upon hyperpolarization. These data suggest that the relaxed state of the voltage sensor is destabilized by external protons and that this contributes to faster deactivation gating in hERG channels. 1Thouta et al., Biophys J.112:300-312.2017

Analysis of Voltage Sensor Movement in KCNQ1-KCNE1 Channels by Voltage Clamp Fluorometry

Analysis of Voltage Sensor Movement in KCNQ1-KCNE1 Channels by Voltage Clamp Fluorometry

Calcium binding and voltage gating in Cx46 hemichannels

The opening of connexin (Cx) hemichannels in the membrane is tightly regulated by calcium (Ca2+) and membrane voltage. Electrophysiological and atomic force microscopy experiments indicate that Ca2+ stabilizes the hemichannel closed state. However, structural data show that Ca2+ binding induces an electrostatic seal preventing ion transport without significant structural rearrangements. In agreement with the closed-state stabilization hypothesis, we found that the apparent Ca2+ sensitivity is increased as the voltage is made more negative. Moreover, the voltage and Ca2+ dependence of the channel kinetics indicate that the voltage sensor movement and Ca2+ binding are allosterically coupled. An allosteric kinetic model in which the Ca2+ decreases the energy necessary to deactivate the voltage sensor reproduces the effects of Ca2+ and voltage in Cx46 hemichannels. In agreement with the model and suggesting a conformational change that narrows the pore, Ca2+ inhibits the water flux through Cx hemichannels. We conclude that Ca2+ and voltage act allosterically to stabilize the closed conformation of Cx46 hemichannels.

Threading the biophysics of mammalian Slo1 channels onto structures of an invertebrate Slo1 channel.

For those interested in the machinery of ion channel gating, the Ca2+ and voltage-activated BK K+ channel provides a compelling topic for investigation, by virtue of its dual allosteric regulation by both voltage and intracellular Ca2+ and because its large-single channel conductance facilitates detailed kinetic analysis. Over the years, biophysical analyses have illuminated details of the allosteric regulation of BK channels and revealed insights into the mechanism of BK gating, e.g., inner cavity size and accessibility and voltage sensor-pore coupling. Now the publication of two structures of an Aplysia californica BK channel-one liganded and one metal free-promises to reinvigorate functional studies and interpretation of biophysical results. The new structures confirm some of the previous functional inferences but also suggest new perspectives regarding cooperativity between Ca2+-binding sites and the relationship between voltage- and Ca2+-dependent gating. Here we consider the extent to which the two structures explain previous functional data on pore-domain properties, voltage-sensor motions, and divalent cation binding and activation of the channel.

Gating mechanisms underlying deactivation slowing by two KCNQ1 atrial fibrillation mutations

KCNQ1 is a voltage-gated potassium channel that is modulated by the beta-subunit KCNE1 to generate IKs, the slow delayed rectifier current, which plays a critical role in repolarizing the cardiac action potential. Two KCNQ1 gain-of-function mutations that cause a genetic form of atrial fibrillation, S140G and V141M, drastically slow IKs deactivation. However, the underlying gating alterations remain unknown. Voltage clamp fluorometry (VCF) allows simultaneous measurement of voltage sensor movement and current through the channel pore. Here, we use VCF and kinetic modeling to determine the effects of mutations on channel voltage-dependent gating. We show that in the absence of KCNE1, S140G, but not V141M, directly slows voltage sensor movement, which indirectly slows current deactivation. In the presence of KCNE1, both S140G and V141M slow pore closing and alter voltage sensor-pore coupling, thereby slowing current deactivation. Our results suggest that KCNE1 can mediate changes in pore movement and voltage sensor-pore coupling to slow IKs deactivation and provide a key step toward developing mechanism-based therapies.

The Calcium Channel A2D Subunit Increases the Gating Charges of Cav1.2 Channels

Optically tracking the movement of individual voltage sensors (VSD) of human CaV1.2 revealed that association of α2δ-1 with α1C resulted in a substantial change in the intrinsic voltage-sensing properties of VSDs I-III (Savalli et al 2016, JGP 142, 147-159). Co-expression of α2δ-1 increased the sensitivity of activation for several VSDs indicating that this mostly extracellular subunit increased the electrical distances traversed by voltage-sensing charges. In this study we measured the slope of the voltage-dependence of the channel's probability of being open (Po) at extremely low probability using marcropatches of Xenopus oocytes expressing CaV1.2/CaVβ3 by themselves or in combination with α2δ-1. Patches containing from 50 to several 100 channels were recorded in 75 mM Ba2+ and in the presence of 1 µM (-) Bay K 8644 and held at several voltages ranging from −70 to −20 mV for 10 to 90 s. Po times the number of channels (NPo) were obtained by finding the most likely combination of normal distributions shifted by the single channel currents amplitude that described the data. The relative weight of the different distributions was then contrasted with what should be expected from a Poisson distribution of opening levels. The number of channels (N) was estimated from the noise analysis of hundreds of tail current traces at −40 mV following a depolarizing pulse to +80 mV. In the presence of α2δ-1, the limiting slope was about 4 elementary charges, close to the sum of the voltage-dependencies of VSDII and VSDIII activation and clearly less than the sum of all VSDs and thus consistent with the idea that only a subset of VSDs contribute to the effective charges for channel opening. When the α2δ-1 subunit was absent the limiting slope was reduced by about one elementary charge. This modest change can be accounted by a reduction in the electrical distance that gating charges need to cross for channel opening.Funding: FONDECYT 3140590 (GC) and 1120864 (AN), R01GM110276 (RO) and 16POST27250284 (NS).

Open and Closed States of the NaVAb Activation Gate

The bacterial voltage-gated sodium channel NavAb is a structural model for eukaryotic voltage-gated sodium channels, and it has been crystallized in both the pre-open and inactivated states (Payanadeh et al 2011, 2012). These structures revealed the general architecture of bacterial voltage-gated sodium channels and provided insight into mechanisms of voltage-sensing, electromechanical coupling, and the selective conductance of sodium ions. Comparison with structures of other bacterial sodium channels (Zhang et al 2012, Bagneris et al 2013) has also informed hypotheses of activation gating, an opening transition now thought to involve twisting movements of the S6 helix that ultimately result in an iris-like dilation of the intracellular ends of the four S6 segments. We sought to capture closed and open states in the same channel protein through x-ray crystallography and electrophysiological studies of mutations in the S6 of NavAb. Our studies show that paired mutations of hydrophobic residues in the S6 helix can uncouple voltage-sensor movement from pore opening and thereby lock the channel in a permanently closed state in which the intracellular ends of the four S6 helices interact tightly with each other. On the other hand, truncation of the intracellular extensions of the S6 segments results in an open conformation of the activation gate. These analyses allow for comparison of the closed, open, and inactivated states of NavAB's activation gate, a first for a voltage-gated sodium channel. Our results help confirm and sharpen the previously hypotheses for the pore-opening transition of sodium channels and reveal key conformational changes that connect the closed, open, and inactivated states of NavAb.

Independent movement of the voltage sensors in KV2.1/KV6.4 heterotetramers

Heterotetramer voltage-gated K+ (KV) channels KV2.1/KV6.4 display a gating charge-voltage (QV) distribution composed by two separate components. We use state dependent chemical accessibility to cysteines substituted in either KV2.1 or KV6.4 to assess the voltage sensor movements of each subunit. By comparing the voltage dependences of chemical modification and gating charge displacement, here we show that each gating charge component corresponds to a specific subunit forming the heterotetramer. The voltage sensors from KV6.4 subunits move at more negative potentials than the voltage sensors belonging to KV2.1 subunits. These results indicate that the voltage sensors from the tetrameric channels move independently. In addition, our data shows that 75% of the total charge is attributed to KV2.1, while 25% to KV6.4. Thus, the most parsimonious model for KV2.1/KV6.4 channels’ stoichiometry is 3:1.

Impact of geometry changes in the channel pore by the gating movements on the channel’s conductance

Kv 1.2 are voltage-dependent potassium channels of great biological importance. Despite the existence of many reports considering structure – function relations of the Kv 1.2 channel's quantitative domains, some details of the voltage gating remain ambiguous, or even unknown. One of the examples is the range of the S4–S6 domains motions involved in channel activation and gating. Another important question is to what extent the channel geometry influences the observable channel conductance at different voltages, and what mechanism stands behind. Does the narrowing of the pore reduce the conductance by ohmic resistance growth? The answer is surprisingly negative. But it can be explained in an alternative way by considering the fluctuations.To address these problems, we formulate geometric models that mimic the generic features of voltage sensor movement and trigger the movement of the other domains involved in gating. We carry out a complete simulation of S4–S6 domains translations and tilts. The obtained pore profiles allow to estimate the (ohmic) conductance dependency on the voltage. From a family of analysed models, we choose the one most concurring with the experimental data. The results allow to suggest the most probable scenario of S4–S6 domains movement during channel activation by membrane depolarization.

Probing the S4-S5 Linker Movement During Activation in KV Channels

Numerous electrophysiological and fluorescence studies have been performed on the voltage sensor (VS) movement in Kv channels, while less have focused on the S4-S5 linker, mainly due to limitations in fluorophore labeling. Electromechanical coupling between the VS movement and pore opening in Kv channels is, in part, controlled by the S4-S5 linker and it is therefore crucial to understand the mechanism underlying S4-S5 linker movement. The question remains which part of the S4-S5 linker moves during independent voltage sensor activation and which part during cooperative pore opening. We also wanted to address with which of the major charge movements S4-S5 linker movements is associated. In order to address the movement of the S4-S5 linker, we have incorporated a fluorescent unnatural amino acid (Anap) at four positions in the linker of a Shaker Kv channel (L382, R387, K390 and A391) to probe conformational changes. Using voltage clamp fluorometry, we found that the entire linker moves during voltage sensor activation. Only the C-terminal part of the linker also moves during pore opening and is thus associated with pore opening. To separate the 1st and 2nd major gating charge movements, we inserted the F290A mutation. We found that the entire linker moves during both major charge movements. Based on these results, we suggest a model where the linker undergoes distinct conformational changes during each S4 movement throughout voltage sensor activation. The energy generated during voltage sensor activation is stored in the C-terminal S4-S5 linker that finally relaxes during pore opening.

Mechanisms of KCNE Beta Subunit Modulation of Voltage Sensing and Gating in KCNQ1 Channels

KCNQ1 (Kv7.1) channels are expressed in different tissues and exhibit different gating behaviors in these different tissues. In the heart, KCNQ1 channels are coexpressed with KCNE1 beta subunits and form slowly activating, voltage-gated potassium channels, whereas in epithelial cells KCNQ1 are coexpressed with KCNE2 or KCNE3 beta subunits and form potassium channels that appear voltage-independent. We have used voltage clamp fluorometry (VCF) to elucidate the gating mechanism in KCNQ1 channels and the effects of KCNE beta subunits on KCNQ1 gating. Voltage clamp fluorometry allows us to follow both voltage sensor movement and gate opening simultaneously, thereby better understand the relationship of voltage sensor movement and channel opening in these channels. We show that the voltage sensor S4 moves and the gate closes in KCNQ1/KCNE3 channels, but only at very negative voltages. By decoupling the voltage sensor and the gate, we show that KCNE3 mainly affects the voltage sensor and only indirectly affects the pore through the voltage-sensor-to-gate coupling. The KCNE3 seems to affect the voltage sensor S4 via an electrostatic interactions between negatively charged KCNE3 residues and positively charged S4 residues. Our data suggests that KCNE1 and KCNE3 affect different domains of KCNQ1, thereby resulting in very different modulations of KCNQ1 voltage gating. KCNE1 affects both the voltage-sensing domain and the pore-gate domain, whereas KCNE3 affects mainly the voltage-sensing domain. That different KCNE beta subunits affect different domains of KCNQ1 explains how two highly similar KCNE beta subunits have so different effects on KCNQ1 gating.

Using Voltage Clamp Fluorometry to Understand KCNQ Channel Pharmacology

With the emergence of retigabine, the first-in-class potassium channel activator used for treatment of epilepsy, KCNQ channels have entered the spotlight for the development of targeted therapeutics to treat excitability disorders. The detailed mechanisms of novel KCNQ activators are largely unexplored, and many fundamental questions regarding their effects on channel function remain unanswered. For instance - how/why do some drugs specifically modulate channel open probability, while others impact voltage sensitivity, or both? To investigate these questions we have employed voltage clamp fluorometry to measure retigabine effects on voltage sensor movements in KCNQ3 channels. Fluorescence reports from channels labelled at position Q218C (in the S3-S4 linker) indicate tight coupling between the voltage sensor domain (VSD) and pore domains (PD) in KCNQ3 channels, with both processes displaying similar voltage dependence and kinetics. In response to the prototype KCNQ activator retigabine, VSD conformational changes and pore gating are equally shifted to hyperpolarized potentials, reflecting the ability of retigabine to stabilize the channel active state. To separate possible effects of retigabine on the VSD versus PD, we have co-expressed voltage sensitive phosphatases (CiVSP and DrVSP) to deplete membrane PIP2. VSP-mediated PIP2 hydrolysis causes pore closure and rundown of KCNQ3 current, but does not alter the voltage-sensor fluorescence report, suggesting that pore gating has been dissociated from VSD motions. This experimental system will allow investigation of whether retigabine effects require intact coupling between the VSD and PD. Our findings reveal unique aspects of VSD-PD coupling in KCNQ3 channels, and highlight the role of PIP2 as an important co-factor for channel activity and drug sensitivity of KCNQ channels.

Regulation of Voltage Sensing Structures of CaV1.2 Calcium Channels by the Auxiliary β-Subunit (β3)

High Voltage-Activated channels (HVA) are composed of four non-identical repeats with distinct voltage-sensing domain (VSD). The cardiac HVA CaV1.2 is modulated by at least two auxiliary subunits, α2δ and β. The β-subunit is known to facilitate channel opening without altering gating currents or single channel conductance. By tracking the movement of individual voltage sensors in human CaV1.2 channels using voltage clamp fluorometry we learned that individual voltage-sensors contribute to channel opening and gating currents to a different degree (Pantazis et al. 2014, PNAS). Taking advantage that rabbit CaV1.2 can be expressed without auxiliary subunits, we explored the impact that β subunits exert on voltage-sensing or coupling and found that β3 regulates Voltage-sensor I (VSD I) activity. Voltage-sensors of all members of the voltage-gated ion channel family have three main conformations: resting, active and relaxed. VSD transition from the resting to the active conformation is thought to be coupled to channel opening, while the relaxed configuration is populated during prolonged depolarizations. Once in the relaxed state, strong hyperpolarizations are necessary to return to the resting state. When rabbit CaV1.2 channels are co-expressed without β subunit the voltage-dependency of VSD I indicates that near 50% of VSD I are in the relaxed state even at −90mV holding potential. In contrast, with β3 present, VSD I dwells between resting and active state. The augmented preference of VSD I for the relaxed state in the absence of β3 may explain why CaV1.2 channels lacking the β-subunit are less willing to open and together with our results with human CaV2.1 suggest that VSD I participates in inactivation rather than activation. We hypothesize that when in the relaxed state, VSD I keeps channels from opening or in an “unwilling” state.

Effect of Small-Molecules on S4 Movement and Gating of Normal and Mutated IKM Channels

Abnormal or synchronous neuronal activity in the brain leads to epileptic seizures that, when repeated or prolonged, can cause neuronal damage resulting in delayed psychomotor development, intellectual disability and other neurological disorders. One of the major potassium current in neurons is the muscarine-regulated M-current. The M-current, formed by the heteromerization of KCNQ2 and KCNQ3 channels (IKM), activates in the time frame of action potential initiation, thereby its crucial role in controlling neuronal excitability. The slow kinetics of activation and deactivation of the IKM (KCNQ2/KCNQ3) channel regulates the membrane potential and impede repetitive neuronal firing. Inherited mutations in the IKM channel cause a wide spectrum of early-onset epileptic disorders ranging from benign familial neonatal seizures to severe epileptic encephalopathies. I here study the molecular mechanisms by which a set of epileptic-inducing mutations in KCNQ2 and KCNQ3 cause malfunction of the IKM channel. I use a fluorescence assay, voltage clamp fluorometry (VCF), to measure simultaneously voltage sensor movement and gate opening during IKM channel activation in these mutations. The IKM channel is an attractive pharmacological target to treat epilepsy, because increasing the M-current stabilizes the resting and subthreshold membrane potential, thereby reducing membrane excitability. Lipophilic compounds, such as polyunsaturated fatty acids (PUFAs), have been shown to favorably affect neuronal function. In particular, PUFAs have been shown to improve the outcome of epilepsy, therefore constituting very promising anti-epileptic agents. However, the molecular mechanism of action of PUFAs is unknown. Based on the molecular mechanism for each epileptic-inducing IKM channel mutation, I show that different PUFAs shift the voltage dependence of the IKM channel mutations, suggesting that PUFAs should, at least partly, reverse the effects of some epilepsy-causing mutations and restore wild type-like IKM currents in these mutations.

N-Arachidonoyl Taurine Rescues Diverse Long QT Syndrome-Associated Mutations in the Cardiac I←Ks Channel

The cardiac I←Ks channel is important for cardiomyocyte repolarization. More than 300 loss-of-function mutations in the genes encoding I←Ks have been identified in patients with Long QT syndrome. These mutations cause cardiac arrhythmias, such as torsades de pointes and ventricular fibrillation. How specific mutations cause arrhythmia is, however, not known in most cases and there is no approved I←Ks channel activator for treatment of arrhythmia. In this work, we study the biophysical properties and mechanism of loss of function of Long QT syndrome-associated I←Ks channel mutations expressed in Xenopus laevis oocytes. We next test the ability of the fatty acid analogue N-arachidonoyl taurine to restore the function of these mutants. Using two-electrode voltage clamp, we find that a number of Long QT syndrome-associated, loss-of-function I←Ks channel mutations shift the voltage dependence of opening towards positive voltages and speed up channel closing. Using voltage clamp fluorometry, we show that these alterations are caused by either disrupted voltage sensor movement or gate opening. We also find that the fatty acid analogue N-arachidonoyl taurine activates all tested Long QT syndrome-associated I←Ks channel mutations by shifting their voltage dependence of activation towards more negative voltages and altering the kinetics of channel opening and closing. Thus, N-arachidonoyl taurine restores the function of these mutants and compensates for the loss of function induced by these mutations, whether the mutations disrupted voltage sensor movement or gate opening. N-AT also shortens a drug-induced prolonged QT interval in isolated perfused guinea pig hearts in Langendorff preparation back to a normal range. N-AT is therefore a promising future I←Ks channel activator that may restore a physiological QT interval by enhancing the function of diverse Long QT syndrome I←Ks mutants, independent of the mutational defect.

NS1643 Interacts around L529 of hERG to Alter Voltage Sensor Movement on the Path to Activation

Activators of hERG1 such as NS1643 are being developed for congenital/acquired long QT syndrome. Previous studies identify the neighborhood of L529 around the voltage-sensor as a putative interacting site for NS1643. With NS1643, the V1/2 of activation of L529I (−34 ± 4 mV) is similar to wild-type (WT) (−37 ± 3 mV; P > 0.05). WT and L529I showed no difference in the slope factor in the absence of NS1643 (8 ± 0 vs. 9 ± 0) but showed a difference in the presence of NS1643 (9 ± 0.3 vs. 22 ± 1; P < 0.01). Voltage-clamp-fluorimetry studies also indicated that in L529I, NS1643 reduces the voltage-sensitivity of S4 movement. To further assess mechanism of NS1643 action, mutations were made in this neighborhood. NS1643 shifts the V1/2 of activation of both K525C and K525C/L529I to hyperpolarized potentials (−131 ± 4 mV for K525C and −120 ± 21 mV for K525C/L529I). Both K525C and K525C/K529I had similar slope factors in the absence of NS1643 (18 ± 2 vs. 34 ± 5, respectively) but with NS1643, the slope factor of K525C/L529I increased from 34 ± 5 to 71 ± 10 (P < 0.01) whereas for K525C the slope factor did not change (18 ± 2 at baseline and 16 ± 2 for NS1643). At baseline, K525R had a slope factor similar to WT (9 vs. 8) but in the presence of NS1643, the slope factor of K525R was increased to 24 ± 4 vs. 9 ± 0 mV for WT (P < 0.01). Molecular modeling indicates that L529I induces a kink in the S4 voltage-sensor helix, altering a salt-bridge involving K525. Moreover, docking studies indicate that NS1643 binds to the kinked structure induced by the mutation with a higher affinity. Combining biophysical, computational, and electrophysiological evidence, a mechanistic principle governing the action of some activators of hERG1 channels is proposed.

Sequence of Gating Charge Movement and Pore Gating in hERG Activation and Deactivation Pathways

KV11.1 voltage-gated K+ channels are noted for unusually slow activation, fast inactivation, and slow deactivation kinetics, which tune channel activity to provide vital repolarizing current during later stages of the cardiac action potential. The bulk of charge movement in human ether-a-go-go-related gene (hERG) is slow, as is return of charge upon repolarization, suggesting that the rates of hERG channel opening and, critically, that of deactivation might be determined by slow voltage sensor movement, and also by a mode-shift after activation. To test these ideas, we compared the kinetics and voltage dependence of ionic activation and deactivation with gating charge movement. At 0 mV, gating charge moved ∼threefold faster than ionic current, which suggests the presence of additional slow transitions downstream of charge movement in the physiological activation pathway. A significant voltage sensor mode-shift was apparent by 24 ms at +60 mV in gating currents, and return of charge closely tracked pore closure after pulses of 100 and 300 ms duration. A deletion of the N-terminus PAS domain, mutation R4AR5A or the LQT2-causing mutation R56Q gave faster-deactivating channels that displayed an attenuated mode-shift of charge. This indicates that charge movement is perturbed by N- and C-terminus interactions, and that these domain interactions stabilize the open state and limit the rate of charge return. We conclude that slow on-gating charge movement can only partly account for slow hERG ionic activation, and that the rate of pore closure has a limiting role in the slow return of gating charges.

Two KCNQ1 Mutations Associated with Familial Atrial Fibrillation, S140G and V141M, Demonstrate Distinct Voltage Sensor Phenotypes

KCNQ1 is a voltage-dependent potassium channel that is expressed in the heart with the β-subunit KCNE1 to generate the slowly activating IKs current that plays a critical role in cardiac impulse conduction by allowing cardiac repolarization. Mutations in KCNQ1 leading to slowing of channel deactivation have been linked to arrhythmias including Short QT syndrome and familial atrial fibrillation. Two adjacent disease-linked mutations located in the voltage sensing domain (S1-S4) of KCNQ1, S140G and V141M, have been shown to drastically slow current deactivation. While their effects on IKs current deactivation kinetics are similar, their mechanisms may differ. For example, in the absence of KCNE1, S140G but not V141M slows current deactivation. Moreover, crosslinking studies suggest that while V141M can directly interact with KCNE1, S140G cannot. We explore the hypothesis that S140G and V141M, while exhibiting similar effects in IKs current gating, demonstrate distinct phenotypes on voltage sensor movement. Using voltage clamp fluorometry, we studied voltage sensor movement simultaneously with channel current. We found that in the absence of KCNE1, S140G but not V141M slows voltage sensor deactivation, consistent with their effects on current. Furthermore, in the presence of KCNE1, S140G slows voltage sensor movement, but V141M does not slow voltage sensor deactivation. This work shows that while S140G slows both the current deactivation and voltage sensor movement in the presence and absence of KCNE1, in contrast, V141M slows current deactivation only in the presence of KCNE1, without significantly slowing the kinetics of voltage sensor movement. This suggests that these two mutations slow the deactivation of KCNQ1/KCNE1 channels by different mechanisms, an observation made possible by the simultaneous measurement of both voltage sensor movement and channel current.