Molecular template for a voltage sensor in a novel K+ channel. I. Identification and functional characterization of KvLm, a voltage-gated K+ channel from Listeria monocytogenes

The fundamental principles underlying voltage sensing, a hallmark feature of electrically excitable cells, are still enigmatic and the subject of intense scrutiny and controversy. Here we show that a novel prokaryotic voltage-gated K(+) (Kv) channel from Listeria monocytogenes (KvLm) embodies a rudimentary, yet robust, sensor sufficient to endow it with voltage-dependent features comparable to those of eukaryotic Kv channels. The most conspicuous feature of the KvLm sequence is the nature of the sensor components: the motif is recognizable; it appears, however, to contain only three out of eight charged residues known to be conserved in eukaryotic Kv channels and accepted to be deterministic for folding and sensing. Despite the atypical sensor sequence, flux assays of KvLm reconstituted in liposomes disclosed a channel pore that is highly selective for K(+) and is blocked by conventional Kv channel blockers. Single-channel currents recorded in symmetric K(+) solutions from patches of enlarged Escherichia coli (spheroplasts) expressing KvLm showed that channel open probability sharply increases with depolarization, a hallmark feature of Kv channels. The identification of a voltage sensor module in KvLm with a voltage dependence comparable to that of other eukaryotic Kv channels yet encoded by a sequence that departs significantly from the consensus sequence of a eukaryotic voltage sensor establishes a molecular blueprint of a minimal sequence for a voltage sensor.     

Molecular template for a voltage sensor in a novel K+ channel. II. Conservation of a eukaryotic sensor fold in a prokaryotic K+ channel

KvLm, a novel bacterial depolarization-activated K(+) (Kv) channel isolated from the genome of Listeria monocytogenes, contains a voltage sensor module whose sequence deviates considerably from the consensus sequence of a Kv channel sensor in that only three out of eight conserved charged positions are present. Surprisingly, KvLm exhibits the steep dependence of the open channel probability on membrane potential that is characteristic of eukaryotic Kv channels whose sensor sequence approximates the consensus. Here we asked if the KvLm sensor shared a similar fold to that of Shaker, the archetypal eukaryotic Kv channel, by examining if interactions between conserved residues in Shaker known to mediate sensor biogenesis and function were conserved in KvLm. To this end, each of the five non-conserved residues in the KvLm sensor were mutated to their Shaker-like charged residues, and the impact of these mutations on the voltage dependence of activation was assayed by current recordings from excised membrane patches of Escherichia coli spheroplasts expressing the KvLm mutants. Conservation of pairwise interactions was investigated by comparison of the effect of single mutations to the impact of double mutations presumed to restore wild-type fold and voltage sensitivity. We observed significant functional coupling between sites known to interact in Shaker Kv channels, supporting the notion that the KvLm sensor largely retains the fold of its eukaryotic homologue.     

Hv1 Proton Channel Opening Is Preceded by a Voltage-independent Transition

The voltage sensing domain (VSD) of the voltage-gated proton channel Hv1 mediates a H⁺-selective conductance that is coordinately controlled by the membrane potential (V) and the transmembrane pH gradient (ΔpH). Allosteric control of Hv1 channel opening by ΔpH (V-ΔpH coupling) is manifested by a characteristic shift of approximately 40 mV per ΔpH unit in the activation. To further understand the mechanism for V-ΔpH coupling in Hv1, H⁺ current kinetics of activation and deactivation in excised membrane patches were analyzed as a function of the membrane potential and the pH in the intracellular side of the membrane (pH_I). In this study, it is shown for the first time to our knowledge that the opening of Hv1 is preceded by a voltage-independent transition. A similar process has been proposed to constitute the step involving coupling between the voltage-sensing and pore domains in tetrameric voltage-gated channels. However, for Hv1, the VSD functions as both the voltage sensor and the conduction pathway, suggesting that the voltage independent transition is intrinsic to the voltage-sensing domain. Therefore, this article proposes that the underlying mechanism for the activation of Hv1 involves a process similar to VSD relaxation, a process previously described for voltage-gated channels and voltage-controlled enzymes. Finally, deactivation seemingly occurs as a strictly voltage dependent process, implying that the kinetic event leading to opening of the proton conductance are different than those involved in the closing. Thus, from this work it is proposed that Hv1 activity displays hysteresis.     

Hv1 Proton Channel Opening Is Preceded by a Voltage-independent Transition

The voltage sensing domain (VSD) of the voltage-gated proton channel Hv1 mediates a H⁺-selective conductance that is coordinately controlled by the membrane potential (V) and the transmembrane pH gradient (ΔpH). Allosteric control of Hv1 channel opening by ΔpH (V-ΔpH coupling) is manifested by a characteristic shift of approximately 40 mV per ΔpH unit in the activation. To further understand the mechanism for V-ΔpH coupling in Hv1, H⁺ current kinetics of activation and deactivation in excised membrane patches were analyzed as a function of the membrane potential and the pH in the intracellular side of the membrane (pH_I). In this study, it is shown for the first time to our knowledge that the opening of Hv1 is preceded by a voltage-independent transition. A similar process has been proposed to constitute the step involving coupling between the voltage-sensing and pore domains in tetrameric voltage-gated channels. However, for Hv1, the VSD functions as both the voltage sensor and the conduction pathway, suggesting that the voltage independent transition is intrinsic to the voltage-sensing domain. Therefore, this article proposes that the underlying mechanism for the activation of Hv1 involves a process similar to VSD relaxation, a process previously described for voltage-gated channels and voltage-controlled enzymes. Finally, deactivation seemingly occurs as a strictly voltage dependent process, implying that the kinetic event leading to opening of the proton conductance are different than those involved in the closing. Thus, from this work it is proposed that Hv1 activity displays hysteresis.     

Coupling between voltage sensors and activation gate in voltage-gated K+ channels

Current through voltage-gated K+ channels underlies the action potential encoding the electrical signal in excitable cells. The four subunits of a voltage-gated K+ channel each have six transmembrane segments (S1-S6), whereas some other K+ channels, such as eukaryotic inward rectifier K+ channels and the prokaryotic KcsA channel, have only two transmembrane segments (M1 and M2). A voltage-gated K+ channel is formed by an ion-pore module (S5-S6, equivalent to M1-M2) and the surrounding voltage-sensing modules. The S4 segments are the primary voltage sensors while the intracellular activation gate is located near the COOH-terminal end of S6, although the coupling mechanism between them remains unknown. In the present study, we found that two short, complementary sequences in voltage-gated K+ channels are essential for coupling the voltage sensors to the intracellular activation gate. One sequence is the so called S4-S5 linker distal to the voltage-sensing S4, while the other is around the COOH-terminal end of S6, a region containing the actual gate-forming residues.     

Role of the S3-S4 Linker in Shaker Potassium Channel Activation

Structural models of voltage-gated channels suggest that flexibility of the S3-S4 linker region may be important in allowing the S4 region to undergo large conformational changes in its putative voltage-sensing function. We report here the initial characterization of 18 mutations in the S3-S4 linker of the Shaker channel, including deletions, insertions, charge changes, substitution of prolines, and chimeras replacing the 25-residue Shaker linker with 7- or 9-residue sequences from Shab, Shaw, or Shal. As measured in Xenopus oocytes with a two-microelectrode voltage clamp, each mutant construct yielded robust currents. Changes in the voltage dependence of activation were small, with activation voltage shifts of 13 mV or less. Substitution of linkers from the slowly activating Shab and Shaw channels resulted in a three- to fourfold slowing of activation and deactivation. It is concluded that the S3-S4 linker is unlikely to participate in a large conformational change during channel activation. The linker, which in some channel subfamilies has highly conserved sequences, may however be a determinant of activation kinetics in potassium channels, as previously has been suggested in the case of calcium channels.     

The Sensorless Pore Module of Voltage-gated K+ Channel Family 7 Embodies the Target Site for the Anticonvulsant Retigabine

KCNQ (voltage-gated K⁺ channel family 7 (Kv7)) channels control cellular excitability and underlie the K⁺ current sensitive to muscarinic receptor signaling (the M current) in sympathetic neurons. Here we show that the novel anti-epileptic drug retigabine (RTG) modulates channel function of pore-only modules (PMs) of the human Kv7.2 and Kv7.3 homomeric channels and of Kv7.2/3 heteromeric channels by prolonging the residence time in the open state. In addition, the Kv7 channel PMs are shown to recapitulate the single-channel permeation and pharmacological specificity characteristics of the corresponding full-length proteins in their native cellular context. A mutation (W265L) in the reconstituted Kv7.3 PM renders the channel insensitive to RTG and favors the conductive conformation of the PM, in agreement to what is observed when the Kv7.3 mutant is heterologously expressed. On the basis of the new findings and homology models of the closed and open conformations of the Kv7.3 PM, we propose a structural mechanism for the gating of the Kv7.3 PM and for the site of action of RTG as a Kv7.2/Kv7.3 K⁺ current activator. The results validate the modular design of human Kv channels and highlight the PM as a high-fidelity target for drug screening of Kv channels.     

Transfer of Kv3.1 Voltage Sensor Features to the Isolated Ci-VSP Voltage-Sensing Domain

Membrane proteins that respond to changes in transmembrane voltage are critical in regulating the function of living cells. The voltage-sensing domains (VSDs) of voltage-gated ion channels are extensively studied to elucidate voltage-sensing mechanisms, and yet many aspects of their structure-function relationship remain elusive. Here, we transplanted homologous amino acid motifs from the tetrameric voltage-activated potassium channel Kv3.1 to the monomeric VSD of Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) to explore which portions of Kv3.1 subunits depend on the tetrameric structure of Kv channels and which properties of Kv3.1 can be transferred to the monomeric Ci-VSP scaffold. By attaching fluorescent proteins to these chimeric VSDs, we obtained an optical readout to establish membrane trafficking and kinetics of voltage-dependent structural rearrangements. We found that motifs extending from 10 to roughly 100 amino acids can be readily transplanted from Kv3.1 into Ci-VSP to form engineered VSDs that efficiently incorporate into the plasma membrane and sense voltage. Some of the functional features of these engineered VSDs are reminiscent of Kv3.1 channels, indicating that these properties do not require interactions between Kv subunits or between the voltage sensing and the pore domains of Kv channels.     

Proline Scan of the hERG Channel S6 Helix Reveals the Location of the Intracellular Pore Gate

In Shaker-like channels, the activation gate is formed at the bundle crossing by the convergence of the inner S6 helices near a conserved proline-valine-proline motif, which introduces a kink that allows for electromechanical coupling with voltage sensor motions via the S4-S5 linker. Human ether-a-go-go-related gene (hERG) channels lack the proline-valine-proline motif and the location of the intracellular pore gate and how it is coupled to S4 movement is less clear. Here, we show that proline substitutions within the S6 of hERG perturbed pore gate closure, trapping channels in the open state. Performing a proline scan of the inner S6 helix, from Ile⁶⁵⁵ to Tyr⁶⁶⁷ revealed that gate perturbation occurred with proximal (I655P-Q664P), but not distal (R665P-Y667P) substitutions, suggesting that Gln⁶⁶⁴ marks the position of the intracellular gate in hERG channels. Using voltage-clamp fluorimetry and gating current analysis, we demonstrate that proline substitutions trap the activation gate open by disrupting the coupling between the voltage-sensing unit and the pore of the channel. We characterize voltage sensor movement in one such trapped-open mutant channel and demonstrate the kinetics of what we interpret to be intrinsic hERG voltage sensor movement.     

Functional diversity of potassium channel voltage-sensing domains

Voltage-gated potassium channels or Kv's are membrane proteins with fundamental physiological roles. They are composed of 2 main functional protein domains, the pore domain, which regulates ion permeation, and the voltage-sensing domain, which is in charge of sensing voltage and undergoing a conformational change that is later transduced into pore opening. The voltage-sensing domain or VSD is a highly conserved structural motif found in all voltage-gated ion channels and can also exist as an independent feature, giving rise to voltage sensitive enzymes and also sustaining proton fluxes in proton-permeable channels. In spite of the structural conservation of VSDs in potassium channels, there are several differences in the details of VSD function found across variants of Kvs. These differences are mainly reflected in variations in the electrostatic energy needed to open different potassium channels. In turn, the differences in detailed VSD functioning among voltage-gated potassium channels might have physiological consequences that have not been explored and which might reflect evolutionary adaptations to the different roles played by Kv channels in cell physiology.     

Inter-Subunit Interactions across the Upper Voltage Sensing-Pore Domain Interface Contribute to the Concerted Pore Opening Transition of Kv Channels

The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.     

A Gating Model for the Archeal Voltage-Dependent K Channel KvAP in DPhPC and POPE:POPG Decane Lipid Bilayers

Voltage-dependent K⁺ (Kv) channels form the basis of the excitability of nerves and muscles. KvAP is a well-characterized archeal Kv channel that has been widely used to investigate many aspects of Kv channel biochemistry, biophysics, and structure. In this study, a minimal kinetic gating model for KvAP function in two different phospholipid decane bilayers is developed. In most aspects, KvAP gating is similar to the well-studied eukaryotic Shaker Kv channel: conformational changes occur within four voltage sensors, followed by pore opening. Unlike the Shaker Kv channel, KvAP possesses an inactivated state that is accessible from the pre-open state of the channel. Changing the lipid composition of the membrane influences multiple gating transitions in the model, but, most dramatically, the rate of recovery from inactivation. Inhibition by the voltage sensor toxin VSTx1 is most easily explained if VSTx1 binds only to the depolarized conformation of the voltage sensor. By delaying the voltage sensor's return to the hyperpolarized conformation, VSTx1 favors the inactivated state of KvAP.     

Voltage-dependent gating rearrangements in the intracellular T1-T1 interface of a K+ channel

The intracellular tetramerization domain (T1) of most eukaryotic voltage-gated potassium channels (Kv channels) exists as a "hanging gondola" below the transmembrane regions that directly control activation gating via the electromechanical coupling between the S4 voltage sensor and the main S6 gate. However, much less is known about the putative contribution of the T1 domain to Kv channel gating. This possibility is mechanistically intriguing because the T1-S1 linker connects the T1 domain to the voltage-sensing domain. Previously, we demonstrated that thiol-specific reagents inhibit Kv4.1 channels by reacting in a state-dependent manner with native Zn(2+) site thiolate groups in the T1-T1 interface; therefore, we concluded that the T1-T1 interface is functionally active and not protected by Zn(2+) (Wang, G., M. Shahidullah, C.A. Rocha, C. Strang, P.J. Pfaffinger, and M. Covarrubias. 2005. J. Gen. Physiol. 126:55-69). Here, we co-expressed Kv4.1 channels and auxiliary subunits (KChIP-1 and DPPX-S) to investigate the state and voltage dependence of the accessibility of MTSET to the three interfacial cysteines in the T1 domain. The results showed that the average MTSET modification rate constant (k(MTSET)) is dramatically enhanced in the activated state relative to the resting and inactivated states (approximately 260- and approximately 47-fold, respectively). Crucially, under three separate conditions that produce distinct activation profiles, k(MTSET) is steeply voltage dependent in a manner that is precisely correlated with the peak conductance-voltage relations. These observations strongly suggest that Kv4 channel gating is tightly coupled to voltage-dependent accessibility changes of native T1 cysteines in the intersubunit Zn(2+) site. Furthermore, cross-linking of cysteine pairs across the T1-T1 interface induced substantial inhibition of the channel, which supports the functionally dynamic role of T1 in channel gating. Therefore, we conclude that the complex voltage-dependent gating rearrangements of eukaryotic Kv channels are not limited to the membrane-spanning core but must include the intracellular T1-T1 interface. Oxidative stress in excitable tissues may perturb this interface to modulate Kv4 channel function.     

Coupling between Residues on S4 and S1 Defines the Voltage-Sensor Resting Conformation in NaChBac

The voltage sensor is a four-transmembrane helix bundle (S1-S4) that couples changes in membrane potential to conformational alterations in voltage-gated ion channels leading to pore opening and ion conductance. Although the structure of the voltage sensor in activated potassium channels is available, the conformation of the voltage sensor at rest is still obscure, limiting our understanding of the voltage-sensing mechanism. By employing a heterologously expressed Bacillus halodurans sodium channel (NaChBac), we defined constraints that affect the positioning and depolarization-induced outward motion of the S4 segment. We compared macroscopic currents mediated by NaChBac and mutants in which E43 on the S1 segment and the two outermost arginines (R1 and R2) on S4 were substituted. Neutralization of the negatively charged E43 (E43C) had a significant effect on channel gating. A double-mutant cycle analysis of E43 and R1 or R2 suggested changes in pairing during channel activation, implying that the interaction of E43 with R1 stabilizes the voltage sensor in its closed/available state, whereas interaction of E43 with R2 stabilizes the channel open/unavailable state. These constraints on S4 dynamics that define its stepwise movement upon channel activation and positioning at rest are novel, to the best of our knowledge, and compatible with the helical-screw and electrostatic models of S4 motion.     

The role of S4 charges in voltage-dependent and voltage-independent KCNQ1 potassium channel complexes

Voltage-gated potassium (Kv) channels extend their functional repertoire by coassembling with MinK-related peptides (MiRPs). MinK slows the activation of channels formed with KCNQ1 alpha subunits to generate the voltage-dependent I(Ks) channel in human heart; MiRP1 and MiRP2 remove the voltage dependence of KCNQ1 to generate potassium "leak" currents in gastrointestinal epithelia. Other Kv alpha subunits interact with MiRP1 and MiRP2 but without loss of voltage dependence; the mechanism for this disparity is unknown. Here, sequence alignments revealed that the voltage-sensing S4 domain of KCNQ1 bears lower net charge (+3) than that of any other eukaryotic voltage-gated ion channel. We therefore examined the role of KCNQ1 S4 charges in channel activation using alanine-scanning mutagenesis and two-electrode voltage clamp. Alanine replacement of R231, at the N-terminal side of S4, produced constitutive activation in homomeric KCNQ1 channels, a phenomenon not observed with previous single amino acid substitutions in S4 of other channels. Homomeric KCNQ4 channels were also made constitutively active by mutagenesis to mimic the S4 charge balance of R231A-KCNQ1. Loss of single S4 charges at positions R231 or R237 produced constitutively active MinK-KCNQ1 channels and increased the constitutively active component of MiRP2-KCNQ1 currents. Charge addition to the CO2H-terminal half of S4 eliminated constitutive activation in MiRP2-KCNQ1 channels, whereas removal of homologous charges from KCNQ4 S4 produced constitutively active MiRP2-KCNQ4 channels. The results demonstrate that the unique S4 charge paucity of KCNQ1 facilitates its unique conversion to a leak channel by ancillary subunits such as MiRP2.     

Voltage sensitivity and gating charge in Shaker and Shab family potassium channels.

The members of the voltage-dependent potassium channel family subserve a variety of functions and are expected to have voltage sensors with different sensitivities. The Shaker channel of Drosophila, which underlies a transient potassium current, has a high voltage sensitivity that is conferred by a large gating charge movement, approximately 13 elementary charges. A Shaker subunit's primary voltage-sensing (S4) region has seven positively charged residues. The Shab channel and its homologue Kv2.1 both carry a delayed-rectifier current, and their subunits have only five positively charged residues in S4; they would be expected to have smaller gating-charge movements and voltage sensitivities. We have characterized the gating currents and single-channel behavior of Shab channels and have estimated the charge movement in Shaker, Shab, and their rat homologues Kv1.1 and Kv2.1 by measuring the voltage dependence of open probability at very negative voltages and comparing this with the charge-voltage relationships. We find that Shab has a relatively small gating charge, approximately 7.5 e(o). Surprisingly, the corresponding mammalian delayed rectifier Kv2.1, which has the same complement of charged residues in the S2, S3, and S4 segments, has a gating charge of 12.5 e(o), essentially equal to that of Shaker and Kv1.1. Evidence for very strong coupling between charge movement and channel opening is seen in two channel types, with the probability of voltage-independent channel openings measured to be below 10(-9) in Shaker and below 4 x 10(-8) in Kv2.1.     

Functional Rescue of Kv4.3 Channel Tetramerization Mutants by KChIP4a

KChIP4a shows a high homology with other members of the family of Kv channel-interacting proteins (KChIPs) in the conserved C-terminal core region, but exhibits a unique modulation of Kv4 channel gating and surface expression. Unlike KChIP1, the KChIP4 splice variant KChIP4a has been shown to inhibit surface expression and function as a suppressor of channel inactivation of Kv4. In this study, we sought to determine whether the multitasking KChIP4a modulates Kv4 function in a clamping fashion similar to that shown by KChIP1. Injection of Kv4.3 T1 zinc mutants into Xenopus oocytes resulted in the nonfunctional expression of Kv4.3 channels. Coexpression of Kv4.3 zinc mutants with WT KChIP4a gave rise to the functional expression of Kv4.3 current. Oocyte surface labeling results confirm the correlation between functional rescue and enhanced surface expression of zinc mutant proteins. Chimeric mutations that replace the Kv4.3 N-terminus with N-terminal KChIP4a or N-terminal deletion of KChIP4a further demonstrate that the functional rescue of Kv4.3 channel tetramerization mutants depends on the KChIP4a core region, but not its N-terminus. Structure-guided mutation of two critical residues of core KChIP4a attenuated functional rescue and tetrameric assembly. Moreover, size exclusion chromatography combined with fast protein liquid chromatography showed that KChIP4a can drive zinc mutant monomers to assemble as tetramers. Taken together, our results show that KChIP4a can rescue the function of tetramerization-defective Kv4 monomers. Therefore, we propose that core KChIP4a functions to promote tetrameric assembly and enhance surface expression of Kv4 channels by a clamping action, whereas its N-terminus inhibits surface expression of Kv4 by a mechanism that remains elusive.     

Ethanol inhibits Kv7.2/7.3 channel open probability by reducing the PI(4,5)P2 sensitivity of Kv7.2 subunit

Ethanol often causes critical health problems by altering the neuro-nal activities of the central and peripheral nerve systems. One of the cellular targets of ethanol is the plasma membrane proteins including ion channels and receptors. Recently, we reported that ethanol elevates membrane excitability in sympathetic neurons by inhibiting Kv7.2/7.3 channels in a cell type-specific manner. Even though our studies revealed that the inhibitory effects of ethanol on the Kv7.2/7.3 channel was diminished by the increase of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI (4,5)P₂), the molecular mechanism of ethanol on Kv7.2/7.3 channel inhibition remains unclear. By investigating the kinetics of Kv7.2/7.3 current in high K⁺ solution, we found that ethanol inhibited Kv7.2/7.3 channels through a mechanism distinct from that of tetraethylammonium (TEA) which enters into the pore and blocks the gate of the channels. Using a non-stationary noise analysis (NSNA), we demonstrated that the inhibitory effect of ethanol is the result of reduction of open probability (P_O) of the Kv7.2/7.3 channel, but not of a single channel current (i) or channel number (N). Finally, ethanol selectively facilitated the kinetics of Kv7.2 current suppression by voltage-sensing phosphatase (VSP)-induced PI(4,5)P₂ depletion, while it slowed down Kv7.2 current recovery from the VSP-induced inhibition. Together our results suggest that ethanol regulates neuronal activity through the reduction of open probability and PI(4,5)P₂ sensitivity of Kv7.2/7.3 channels.     

Proline Scan of the hERG Channel S6 Helix Reveals the Location of the Intracellular Pore Gate

In Shaker-like channels, the activation gate is formed at the bundle crossing by the convergence of the inner S6 helices near a conserved proline-valine-proline motif, which introduces a kink that allows for electromechanical coupling with voltage sensor motions via the S4-S5 linker. Human ether-a-go-go-related gene (hERG) channels lack the proline-valine-proline motif and the location of the intracellular pore gate and how it is coupled to S4 movement is less clear. Here, we show that proline substitutions within the S6 of hERG perturbed pore gate closure, trapping channels in the open state. Performing a proline scan of the inner S6 helix, from Ile⁶⁵⁵ to Tyr⁶⁶⁷ revealed that gate perturbation occurred with proximal (I655P-Q664P), but not distal (R665P-Y667P) substitutions, suggesting that Gln⁶⁶⁴ marks the position of the intracellular gate in hERG channels. Using voltage-clamp fluorimetry and gating current analysis, we demonstrate that proline substitutions trap the activation gate open by disrupting the coupling between the voltage-sensing unit and the pore of the channel. We characterize voltage sensor movement in one such trapped-open mutant channel and demonstrate the kinetics of what we interpret to be intrinsic hERG voltage sensor movement.