Immobilizing the moving parts of voltage-gated ion channels

Voltage-gated ion channels have at least two classes of moving parts, voltage sensors that respond to changes in the transmembrane potential and gates that create or deny permeant ions access to the conduction pathway. To explore the coupling between voltage sensors and gates, we have systematically immobilized each using a bifunctional photoactivatable cross-linker, benzophenone-4-carboxamidocysteine methanethiosulfonate, that can be tethered to cysteines introduced into the channel protein by mutagenesis. To validate the method, we first tested it on the inactivation gate of the sodium channel. The benzophenone-labeled inactivation gate of the sodium channel can be trapped selectively either in an open or closed state by ultraviolet irradiation at either a hyperpolarized or depolarized voltage, respectively. To verify that ultraviolet light can immobilize S4 segments, we examined its relative effects on ionic and gating currents in Shaker potassium channels, labeled at residue 359 at the extracellular end of the S4 segment. As predicted by the tetrameric stoichiometry of these potassium channels, ultraviolet irradiation reduces ionic current by approximately the fourth power of the gating current reduction, suggesting little cooperativity between the movements of individual S4 segments. Photocross-linking occurs preferably at hyperpolarized voltages after labeling residue 359, suggesting that depolarization moves the benzophenone adduct out of a restricted environment. Immobilization of the S4 segment of the second domain of sodium channels prevents channels from opening. By contrast, photocross-linking the S4 segment of the fourth domain of the sodium channel has effects on both activation and inactivation. Our results indicate that specific voltage sensors of the sodium channel play unique roles in gating, and suggest that movement of one voltage sensor, the S4 segment of domain 4, is at least a two-step process, each step coupled to a different gate.     

Reconstructing voltage sensor-pore interaction from a fluorescence scan of a voltage-gated K+ channel

X-ray crystallography has made considerable recent progress in providing static structures of ion channels. Here we describe a complementary method-systematic fluorescence scanning-that reveals the structural dynamics of a channel. Local protein motion was measured from changes in the fluorescent intensity of a fluorophore attached at one of 37 positions in the pore domain and in the S4 voltage sensor of the Shaker K+ channel. The local rearrangements that accompany activation and slow inactivation were mapped onto the homologous structure of the KcsA channel and onto models of S4. The results place clear constraints on S4 location, voltage-dependent movement, and the mechanism of coupling of S4 motion to the operation of the slow inactivation gate in the pore domain.     

Molecular coupling of S4 to a K(+) channel's slow inactivation gate

The mechanism by which physiological signals regulate the conformation of molecular gates that open and close ion channels is poorly understood. Voltage clamp fluorometry was used to ask how the voltage-sensing S4 transmembrane domain is coupled to the slow inactivation gate in the pore domain of the Shaker K(+) channel. Fluorophores attached at several sites in S4 indicate that the voltage-sensing rearrangements are followed by an additional inactivation motion. Fluorophores attached at the perimeter of the pore domain indicate that the inactivation rearrangement projects from the selectivity filter out to the interface with the voltage-sensing domain. Some of the pore domain sites also sense activation, and this appears to be due to a direct interaction with S4 based on the finding that S4 comes into close enough proximity to the pore domain for a pore mutation to alter the nanoenvironment of an S4-attached fluorophore. We propose that activation produces an S4-pore domain interaction that disrupts a bond between the S4 contact site on the pore domain and the outer end of S6. Our results indicate that this bond holds the slow inactivation gate open and, therefore, we propose that this S4-induced bond disruption triggers inactivation.     

S4 charges move close to residues in the pore domain during activation in a K channel.

Voltage-gated ion channels respond to changes in the transmembrane voltage by opening or closing their ion conducting pore. The positively charged fourth transmembrane segment (S4) has been identified as the main voltage sensor, but the mechanisms of coupling between the voltage sensor and the gates are still unknown. Obtaining information about the location and the exact motion of S4 is an important step toward an understanding of these coupling mechanisms. In previous studies we have shown that the extracellular end of S4 is located close to segment 5 (S5). The purpose of the present study is to estimate the location of S4 charges in both resting and activated states. We measured the modification rates by differently charged methanethiosulfonate regents of two residues in the extracellular end of S5 in the Shaker K channel (418C and 419C). When S4 moves to its activated state, the modification rate by the negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) increases significantly more than the modification rate by the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate, bromide (MTSET(+)). This indicates that the positive S4 charges are moving close to 418C and 419C in S5 during activation. Neutralization of the most external charge of S4 (R362), shows that R362 in its activated state electrostatically affects the environment at 418C by 19 mV. In contrast, R362 in its resting state has no effect on 418C. This suggests that, during activation of the channel, R362 moves from a position far away (>20 A) to a position close (8 A) to 418C. Despite its close approach to E418, a residue shown to be important in slow inactivation, R362 has no effect on slow inactivation or the recovery from slow inactivation. This refutes previous models for slow inactivation with an electrostatic S4-to-gate coupling. Instead, we propose a model with an allosteric mechanism for the S4-to-gate coupling.     

The cooperative voltage sensor motion that gates a potassium channel

The four arginine-rich S4 helices of a voltage-gated channel move outward through the membrane in response to depolarization, opening and closing gates to generate a transient ionic current. Coupling of voltage sensing to gating was originally thought to operate with the S4s moving independently from an inward/resting to an outward/activated conformation, so that when all four S4s are activated, the gates are driven to open or closed. However, S4 has also been found to influence the cooperative opening step (Smith-Maxwell et al., 1998a), suggesting a more complex mechanism of coupling. Using fluorescence to monitor structural rearrangements in a Shaker channel mutant, the ILT channel (Ledwell and Aldrich, 1999), that energetically isolates the steps of activation from the cooperative opening step, we find that opening is accompanied by a previously unknown and cooperative movement of S4. This gating motion of S4 appears to be coupled to the internal S6 gate and to two forms of slow inactivation. Our results suggest that S4 plays a direct role in gating. While large transmembrane rearrangements of S4 may be required to unlock the gating machinery, as proposed before, it appears to be the gating motion of S4 that drives the gates to open and close.     

Cooperative interactions between R531 and acidic residues in the voltage sensing module of hERG1 channels.

HERG1 K(+) channels are critical for modulating the duration of the cardiac action potential. The role of hERG1 channels in maintaining electrical stability in the heart derives from their unusual gating properties: slow activation and fast inactivation. HERG1 channel inactivation is intrinsically voltage sensitive and is not coupled to activation in the same way as in the Shaker family of K(+) channels. We recently proposed that the S4 transmembrane domain functions as the primary voltage sensor for hERG1 activation and inactivation and that distinct regions of S4 contribute to each gating process. In this study, we tested the hypothesis that S4 rearrangements underlying activation and inactivation gating may be associated with distinct cooperative interactions between a key residue in the S4 domain (R531) and acidic residues in neighboring regions (S1 - S3 domains) of the voltage sensing module. Using double-mutant cycle analysis, we found that R531 was energetically coupled to all acidic residues in S1-S3 during activation, but was coupled only to acidic residues near the extracellular portion of S2 and S3 (D456, D460 and D509) during inactivation. We propose that hERG1 activation involves a cooperative conformational change involving the entire voltage sensing module, while inactivation may involve a more limited interaction between R531 and D456, D460 and D509.     

A permanent ion binding site located between two gates of the Shaker K+ channel.

K+ channels can be occupied by multiple permeant ions that appear to bind at discrete locations in the conduction pathway. Neither the molecular nature of the binding sites nor their relation to the activation or inactivation gates that control ion flow are well understood. We used the permeant ion Ba2+ as a K+ analog to probe for K+ ion binding sites and their relationship to the activation and inactivation gates. Our data are consistent with the existence of three single-file permeant-ion binding sites: one deep site, which binds Ba2+ with high affinity, and two more external sites whose occupancy influences Ba2+ movement to and from the deep site. All three sites are accessible to the external solution in channels with a closed activation gate, and the deep site lies between the activation gate and the C-type inactivation gate. We identify mutations in the P-region that disrupt two of the binding sites, as well as an energy barrier between the sites that may be part of the selectivity filter.     

Direct Interaction between the Voltage Sensors Produces Cooperative Sustained Deactivation in Voltage-gated H+ Channel Dimers

The voltage-gated H⁺ channel (Hv) is a voltage sensor domain-like protein consisting of four transmembrane segments (S1–S4). The native Hv structure is a homodimer, with the two channel subunits functioning cooperatively. Here we show that the two voltage sensor S4 helices within the dimer directly cooperate via a π-stacking interaction between Trp residues at the middle of each segment. Scanning mutagenesis showed that Trp situated around the original position provides the slow gating kinetics characteristic of the dimer''s cooperativity. Analyses of the Trp mutation on the dimeric and monomeric channel backgrounds and analyses with tandem channel constructs suggested that the two Trp residues within the dimer are functionally coupled during Hv deactivation but are less so during activation. Molecular dynamics simulation also showed direct π-stacking of the two Trp residues. These results provide new insight into the cooperative function of voltage-gated channels, where adjacent voltage sensor helices make direct physical contact and work as a single unit according to the gating process.     

Modal gating of human CaV2.1 (P/Q-type) calcium channels: I. The slow and the fast gating modes and their modulation by beta subunits

The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels and their modulation by the auxiliary beta1b, beta2e, beta3a, and beta4a subunits were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing human CaV2.1 channels. These calcium channels showed a complex modal gating, which is described in this and the following paper (Fellin, T., S. Luvisetto, M. Spagnolo, and D. Pietrobon. 2004. J. Gen. Physiol. 124:463-474). Here, we report the characterization of two modes of gating of human CaV2.1 channels, the slow mode and the fast mode. A channel in the two gating modes differs in mean closed times and latency to first opening (both longer in the slow mode), in voltage dependence of the open probability (larger depolarizations are necessary to open the channel in the slow mode), in kinetics of inactivation (slower in the slow mode), and voltage dependence of steady-state inactivation (occurring at less negative voltages in the slow mode). CaV2.1 channels containing any of the four beta subtypes can gate in either the slow or the fast mode, with only minor differences in the rate constants of the transitions between closed and open states within each mode. In both modes, CaV2.1 channels display different rates of inactivation and different steady-state inactivation depending on the beta subtype. The type of beta subunit also modulates the relative occurrence of the slow and the fast gating mode of CaV2.1 channels; beta3a promotes the fast mode, whereas beta4a promotes the slow mode. The prevailing mode of gating of CaV2.1 channels lacking a beta subunit is a gating mode in which the channel shows shorter mean open times, longer mean closed times, longer first latency, a much larger fraction of nulls, and activates at more positive voltages than in either the fast or slow mode.     

Cross talk between activation and slow inactivation gates of Shaker potassium channels

This study addresses the energetic coupling between the activation and slow inactivation gates of Shaker potassium channels. To track the status of the activation gate in inactivated channels that are nonconducting, we used two functional assays: the accessibility of a cysteine residue engineered into the protein lining the pore cavity (V474C) and the liberation by depolarization of a Cs(+) ion trapped behind the closed activation gate. We determined that the rate of activation gate movement depends on the state of the inactivation gate. A closed inactivation gate favors faster opening and slower closing of the activation gate. We also show that hyperpolarization closes the activation gate long before a channel recovers from inactivation. Because activation and slow inactivation are ubiquitous gating processes in potassium channels, the cross talk between them is likely to be a fundamental factor in controlling ion flux across membranes.     

A conserved glutamate is important for slow inactivation in K+ channels

Voltage-gated ion channels undergo slow inactivation during prolonged depolarizations. We investigated the role of a conserved glutamate at the extracellular end of segment 5 (S5) in slow inactivation by mutating it to a cysteine (E418C in Shaker). We could lock the channel in two different conformations by disulfide-linking 418C to two different cysteines, introduced in the Pore-S6 (P-S6) loop. Our results suggest that E418 is normally stabilizing the open conformation of the slow inactivation gate by forming hydrogen bonds with the P-S6 loop. Breaking these bonds allows the P-S6 loop to rotate, which closes the slow inactivation gate. Our results also suggest a mechanism of how the movement of the voltage sensor can induce slow inactivation by destabilizing these bonds.     

KCNK?: opening and closing the 2-P-domain potassium leak channel entails "C-type" gating of the outer pore.

Essential to nerve and muscle function, little is known about how potassium leak channels operate. KCNK? opens and closes in a kinase-dependent fashion. Here, the transition is shown to correspond to changes in the outer aspect of the ion conduction pore. Voltage-gated potassium (VGK) channels open and close via an internal gate; however, they also have an outer pore gate that produces "C-type" inactivation. While KCNK? does not inactivate, KCNK? and VGK channels respond in like manner to outer pore blockers, potassium, mutations, and chemical modifiers. Structural relatedness is confirmed: VGK residues that come close during C-type gating predict KCNK? sites that crosslink (after mutation to cysteine) to yield channels controlled by reduction and oxidization. We conclude that similar outer pore gates mediate KCNK? opening and closing and VGK channel C-type inactivation despite their divergent structures and physiological roles.     

Role of Arginine Residues on the S4 Segment of the <Emphasis Type="Italic">Bacillus halodurans</Emphasis> Na<Superscript>+</Superscript> Channel in Voltage-sensing

The one-domain voltage-gated sodium channel of Bacillus halodurans (NaChBac) is composed of six transmembrane segments (S1–S6) comprising a pore-forming region flanked by segments S5 and S6 and a voltage-sensing element composed of segment S4. To investigate the role of the S4 segment in NaChBac channel activation, we used the cysteine mutagenesis approach where the positive charges of single and multiple arginine (R) residues of the S4 segment were replaced by the neutrally charged amino acid cysteine (C). To determine whether it was the arginine residue itself or its positive charge that was involved in channel activation, arginine to lysine (R to K) mutations were constructed. Wild-type (WT) and mutant NaChBac channels were expressed in tsA201 cells and Na⁺ currents were recorded using the whole-cell configuration of the patch-clamp technique. The current/voltage (I-V) and conductance/voltage (G-V) relationships steady-state inactivation (h _∞) and recovery from inactivation were evaluated to determine the effects of the S4 mutations on the biophysical properties of the NaChBac channel. R to C on the S4 segment resulted in a slowing of both activation and inactivation kinetics. Charge neutralization of arginine residues mostly resulted in a shift toward more positive potentials of G-V and h _∞ curves. The G-V curve shifts were associated with a decrease in slope, which may reflect a decrease in the gating charge involved in channel activation. Single neutralization of R114, R117, or R120 by C resulted in a very slow recovery from inactivation. Double neutralization of R111 and R129 confirmed the role of R111 in activation and suggested that R129 is most probably not part of the voltage sensor. Most of the R to K mutants retained WT-like current kinetics but exhibited an intermediate G-V curve, a steady-state inactivation shifted to more hyperpolarized potentials, and intermediate time constants of recovery from inactivation. This indicates that R, at several positions, plays an important role in channel activation. The data are consistent with the notion that the S4 is most probably the voltage sensor of the NaChBac channel and that both positive charges and the nature of the arginine residues are essential for channel activation.This revised version was published online in June 2005 with a corrected cover date.     

Regional specificity of human ether-a'-go-go-related gene channel activation and inactivation gating

Slow activation and rapid C-type inactivation produce inward rectification of the current-voltage relationship for human ether-a'-go-go-related gene (hERG) channels. To characterize the voltage sensor movement associated with hERG activation and inactivation, we performed an Ala scan of the 32 amino acids (Gly(514)-Tyr(545)) that comprise the S4 domain and the flanking S3-S4 and S4-S5 linkers. Gating and ionic currents of wild-type and mutant channels were measured using cut-open oocyte Vaseline gap and two microelectrode voltage clamp techniques to determine the voltage dependence of charge movement, activation, and inactivation. Mapping the position of the charge-perturbing mutations (defined as |DeltaDeltaG| > 1.0 kcal/mol) on a three-dimensional S4 homology model revealed a spiral pattern. As expected, mutation of these residues also altered activation. However, mutation of residues in the S3-S4 and S4-S5 linkers and the C-terminal end of S4 perturbed activation (|DeltaDeltaG| > 1.0 kcal/mol) without altering charge movement, suggesting that the native residues in these regions couple S4 movement to the opening of the activation gate or stabilize the open or closed state of the channel. Finally, mutation of a distinct set of residues impacted inactivation and mapped to a single face of the S4 helix that was devoid of activation-perturbing residues. These results define regions on the S4 voltage sensor that contribute differentially to hERG activation and inactivation gating.     

Stochastic 16-state model of voltage gating of gap-junction channels enclosing fast and slow gates

Gap-junction (GJ) channels formed of connexin (Cx) proteins provide a direct pathway for electrical and metabolic cell-cell interaction. Each hemichannel in the GJ channel contains fast and slow gates that are sensitive to transjunctional voltage (Vj). We developed a stochastic 16-state model (S16SM) that details the operation of two fast and two slow gates in series to describe the gating properties of homotypic and heterotypic GJ channels. The operation of each gate depends on the fraction of Vj that falls across the gate (VG), which varies depending on the states of three other gates in series, as well as on parameters of the fast and slow gates characterizing 1), the steepness of each gate's open probability on VG; 2), the voltage at which the open probability of each gate equals 0.5; 3), the gating polarity; and 4), the unitary conductances of the gates and their rectification depending on VG. S16SM allows for the simulation of junctional current dynamics and the dependence of steady-state junctional conductance (gj,ss) on Vj. We combined global coordinate optimization algorithms with S16SM to evaluate the gating parameters of fast and slow gates from experimentally measured gj,ss-Vj dependencies in cells expressing different Cx isoforms and forming homotypic and/or heterotypic GJ channels.     

Calcium Channels: Unanswered Questions

Despite decades of intensive research, many questions remain unresolved regarding the structure and function of voltage-dependent calcium channels. This review considers some of those questions: Where is the activation gate? Where are the inactivation gates? How are the voltage sensors coupled to the gates? Are bacterial K⁺ channels good models for the Ca²⁺ channel pore? Are protein–protein interactions fundamental to Ca²⁺ channel function? Do voltage-dependent Ca²⁺ channels regulate basal intracellular Ca²⁺? Are N and P/Q channels specialized for fast neurotransmitter release?     

Conformational Changes and Slow Dynamics through Microsecond Polarized Atomistic Molecular Simulation of an Integral Kv1.2 Ion Channel

Structure and dynamics of voltage-gated ion channels, in particular the motion of the S4 helix, is a highly interesting and hotly debated topic in current membrane protein research. It has critical implications for insertion and stabilization of membrane proteins as well as for finding how transitions occur in membrane proteins—not to mention numerous applications in drug design. Here, we present a full 1 µs atomic-detail molecular dynamics simulation of an integral Kv1.2 ion channel, comprising 120,000 atoms. By applying 0.052 V/nm of hyperpolarization, we observe structural rearrangements, including up to 120° rotation of the S4 segment, changes in hydrogen-bonding patterns, but only low amounts of translation. A smaller rotation (∼35°) of the extracellular end of all S4 segments is present also in a reference 0.5 µs simulation without applied field, which indicates that the crystal structure might be slightly different from the natural state of the voltage sensor. The conformation change upon hyperpolarization is closely coupled to an increase in 3₁₀ helix contents in S4, starting from the intracellular side. This could support a model for transition from the crystal structure where the hyperpolarization destabilizes S4–lipid hydrogen bonds, which leads to the helix rotating to keep the arginine side chains away from the hydrophobic phase, and the driving force for final relaxation by downward translation is partly entropic, which would explain the slow process. The coordinates of the transmembrane part of the simulated channel actually stay closer to the recently determined higher-resolution Kv1.2 chimera channel than the starting structure for the entire second half of the simulation (0.5–1 µs). Together with lipids binding in matching positions and significant thinning of the membrane also observed in experiments, this provides additional support for the predictive power of microsecond-scale membrane protein simulations.     

Gating charge immobilization in Kv4.2 channels: the basis of closed-state inactivation

Kv4 channels mediate the somatodendritic A-type K+ current (I(SA)) in neurons. The availability of functional Kv4 channels is dynamically regulated by the membrane potential such that subthreshold depolarizations render Kv4 channels unavailable. The underlying process involves inactivation from closed states along the main activation pathway. Although classical inactivation mechanisms such as N- and P/C-type inactivation have been excluded, a clear understanding of closed-state inactivation in Kv4 channels has remained elusive. This is in part due to the lack of crucial information about the interactions between gating charge (Q) movement, activation, and inactivation. To overcome this limitation, we engineered a charybdotoxin (CTX)-sensitive Kv4.2 channel, which enabled us to obtain the first measurements of Kv4.2 gating currents after blocking K+ conduction with CTX (Dougherty and Covarrubias. 2006J. Gen. Physiol. 128:745-753). Here, we exploited this approach further to investigate the mechanism that links closed-state inactivation to slow Q-immobilization in Kv4 channels. The main observations revealed profound Q-immobilization at steady-state over a range of hyperpolarized voltages (-110 to -75 mV). Depolarization in this range moves <5% of the observable Q associated with activation and is insufficient to open the channels significantly. The kinetics and voltage dependence of Q-immobilization and ionic current inactivation between -153 and -47 mV are similar and independent of the channel's proximal N-terminal region (residues 2-40). A coupled state diagram of closed-state inactivation with a quasi-absorbing inactivated state explained the results from ionic and gating current experiments globally. We conclude that Q-immobilization and closed-state inactivation at hyperpolarized voltages are two manifestations of the same process in Kv4.2 channels, and propose that inactivation in the absence of N- and P/C-type mechanisms involves desensitization to voltage resulting from a slow conformational change of the voltage sensors, which renders the channel's main activation gate reluctant to open.     

Voltage-dependent C-type inactivation in a constitutively open K+ channel

Most voltage-gated potassium (Kv) channels undergo C-type inactivation during sustained depolarization. The voltage dependence and other mechanistic aspects of this process are debated, and difficult to elucidate because of concomitant voltage-dependent activation. Here, we demonstrate that MinK-KCNQ1 (I_Ks) channels with an S6-domain mutation, F340W in KCNQ1, exhibit constitutive activation but voltage-dependent C-type inactivation. F340W-I_Ks inactivation was sensitive to extracellular cation concentration and species, and it altered ion selectivity, suggestive of pore constriction. The rate and extent of F340W-I_Ks inactivation and recovery from inactivation were voltage-dependent with physiologic intracellular ion concentrations, and in the absence or presence of external K⁺, with an estimated gating charge, z_i, of ∼1. Finally, double-mutant channels with a single S4 charge neutralization (R231A,F340W-I_Ks) exhibited constitutive C-type inactivation. The results suggest that F340W-I_Ks channels exhibit voltage-dependent C-type inactivation involving S4, without the necessity for voltage-dependent opening, allosteric coupling to voltage-dependent S6 transitions occurring during channel opening, or voltage-dependent changes in ion occupancy. The data also identify F340 as a critical hub for KCNQ1 gating processes and their modulation by MinK, and present a unique system for further mechanistic studies of the role of coupling of C-type inactivation to S4 movement, without contamination from voltage-dependent activation.     

The S4-S5 linker directly couples voltage sensor movement to the activation gate in the human ether-a'-go-go-related gene (hERG) K+ channel

A key unresolved question regarding the basic function of voltage-gated ion channels is how movement of the voltage sensor is coupled to channel opening. We previously proposed that the S4-S5 linker couples voltage sensor movement to the S6 domain in the human ether-a'-go-go-related gene (hERG) K+ channel. The recently solved crystal structure of the voltage-gated Kv1.2 channel reveals that the S4-S5 linker is the structural link between the voltage sensing and pore domains. In this study, we used chimeras constructed from hERG and ether-a'-go-go (EAG) channels to identify interactions between residues in the S4-S5 linker and S6 domain that were critical for stabilizing the channel in a closed state. To verify the spatial proximity of these regions, we introduced cysteines in the S4-S5 linker and at the C-terminal end of the S6 domain and then probed for the effect of oxidation. The D540C-L666C channel current decreased in an oxidizing environment in a state-dependent manner consistent with formation of a disulfide bond that locked the channel in a closed state. Disulfide bond formation also restricted movement of the voltage sensor, as measured by gating currents. Taken together, these data confirm that the S4-S5 linker directly couples voltage sensor movement to the activation gate. Moreover, rather than functioning simply as a mechanical lever, these findings imply that specific interactions between the S4-S5 linker and the activation gate stabilize the closed channel conformation.