Charged S4 Research Articles

Intracellular Ions Impede Voltage Sensor Return in Kv1.2 Channels

Voltage sensing in Kv channels originates from the coupling of movement between the charged S4 segment and the activation gate at the cytoplasmic region of the pore domain. The voltage sensor moves prior to the pore opening and the pore must shut before the voltage sensor returns to its resting state. However, gating current recordings from Kv channels indicate that frequently voltage sensors return more slowly after depolarisations that populate open states, indicating that the open pore exerts a resistance to S4 return. This process of pore closure therefore intrinsically regulates the deactivation kinetics of Kv channels. We observed slow voltage sensor return (IgOFF) in WT-Kv1.2 channels under non-permeant ionic conditions after depolarisations to voltages that caused channel openings. Using TEA+ and NMG+ internal solutions resulted in a slower IgOFF than internal Cs+, suggesting that the intracellular ionic composition was modulating IgOFF. A mutation in the pore lining S6 segment to enlarge the inner cavity (Kv1.2-I402C) removed the slowing of IgOFF in the presence of internal NMG+, suggesting that NMG+ interacted within the inner cavity of the WT channel to prevent pore closure through a ‘foot in the door’ mechanism. Gating currents of a non-conducting, P-type inactivated channel (Kv1.2-W366F, V381T), in the presence of intracellular K+ ions also displayed a slowing of IgOFF after depolarisations that would open the channel pore. These results suggest that internal K+ ions bound in the inner cavity can also slow activation gate closure. We propose that internal ions in the cavity of Kv1.2 allosterically regulate the voltage sensor deactivation kinetics by preventing pore closure and thus rate limiting the return of voltage sensors.

Phe233 in the Voltage-Sensor is Rate Limiting for Channel Closure but not for the Opening

During activation, the charged S4 segment in the voltage sensor domain (VSD) of voltage-gated ion channels is required to translate across a hydrophobic zone. This constitutes a thin aperture clearly separating the open/relaxed and closed/resting states. The nature of this barrier is critical for channel function, and has been the focus of much attention. Previously, we identified the single preserved residue F233 (F290 in Shaker) as a structural barrier for the gating charges, that uniquely determines intermediate state and substitutions modulate the deactivation barrier. A fundamental understanding of the S4 activation/deactivation barrier as well as kinetics is an important remaining challenge to decipher gating. Here, we study the free barrier and kinetics of the VSD through combining in-vitro and in-silico experiments. We used site-directed mutagenesis and measured the voltage-dependence in-vitro to study the effect of F290L compared to the wild-type (WT). In parallel, molecular dynamics simulations allowed us to identify residues interacting with the phenyl ring. Through in-silico mutations we show their impact on the barrier, as well as the structural and kinetics effects for the phenyl ring orientation during the first step of deactivation and the (reverse) last step of the activation. Strikingly, the channel closing transition shows a huge speedup from the F290L mutant (1ms, WT 10ms), in contrast to the opening that is completely unaffected. This indicates that F233 is only rate-limiting for the channel closure, but not opening and suggests different kinetics for the activation/deactivation barriers. Additionally the ring is clearly stabilized through vdW interactions with surrounding hydrophobic residues, and appears to always open by upward rotation both for activation/deactivation. This upward rotation suggests a model where the closing is possibly a mostly entropic process, while opening would be largely enthalpic.

Exploring the Energetics of Membrane Protein Stability using a Deformable Model of the Membrane

Experimental and computational studies have shown that cellular membranes deform to stabilize the inclusion of transmembrane (TM) proteins. Recent analysis suggests that membrane bending helps to expose charged and polar amino acids to the aqueous environment and polar phospholipid headgroups. Our previous study used elasticity theory to characterize the distortions of a membrane in the presence of a TM peptide. Here, we extend our method through the implementation of a search algorithm that identifies the membrane shape that minimizes the free energy of helix insertion. We show that our model robustly identifies the hydrophobic stretch of a TM protein and allows for large deformations to embed these stretches in the membrane. When applied to the MscL stretch activated channel, our model predicts local membrane thinning at the protein boundary that is corroborated by fluorescence and cysteine scanning experiments. We go on to show that the insertion energy for proteins containing multiple charged residues is non-additive, and the model provides a clear physical mechanism for this non-additivity, which is absent in other continuum membrane models. We predict that some highly charged S4 segments from voltage-gated K+ channels are stable in the bilayer in accordance with previous experiments and molecular dynamics simulations. Our method thus captures essential aspects of protein-membrane interactions at a small fraction of the cost of traditional molecular simulations.

The Isolated S4 Voltage Sensor Helix Translocates Spontaneously Across Membranes

The first complete structure of a voltage-gated potassium channel gave rise to a controversial model that, unexpectedly, placed the four cationic arginine residues in the S4 segment of the voltage sensor domain in direct contact with the membrane lipids. The S4 segment was hypothesized to move as much as 20 A across the membrane during channel gating, contradicting the expectation that highly charged peptides will be excluded from membranes. To assess whether the S4 sequence can insert into and move across a membrane without being chaperoned by any other part of the protein, we tested the ability of an isolated S4 peptide to spontaneously translocate across synthetic lipid vesicles. Translocation of S4 was compared to that of an arginine-containing peptide that we recently selected from a combinatorial peptide library specifically for its high translocation efficiency. Importantly, the arginines in the selected peptide are present in the same sequence motif as the arginines in S4, despite the unbiased nature of the library. While control peptides and polar dyes were excluded, the S4 sequence translocated into multi-bilayer vesicles with the same efficiency as the previously selected translocating peptide. The membrane hydrocarbon is not an effective barrier to the transbilayer movement of the highly charged S4 sequence. These results support the idea that the S4 segment in the sensor domain of voltage gated potassium channels interacts directly with the lipid bilayer and moves across the membrane during channel gating without being chaperoned by other parts of the voltage sensor domain.

Gating charge interactions with the S1 segment during activation of a Na + channel voltage sensor

Voltage-gated Na(+) channels initiate action potentials during electrical signaling in excitable cells. Opening and closing of the pore of voltage-gated ion channels are mechanically linked to voltage-driven outward movement of the positively charged S4 transmembrane segment in their voltage sensors. Disulfide locking of cysteine residues substituted for the outermost T0 and R1 gating-charge positions and a conserved negative charge (E43) at the extracellular end of the S1 segment of the bacterial Na(+) channel NaChBac detects molecular interactions that stabilize the resting state of the voltage sensor and define its conformation. Upon depolarization, the more inward gating charges R2 and R3 engage in these molecular interactions as the S4 segment moves outward to its intermediate and activated states. The R4 gating charge does not disulfide-lock with E43, suggesting an outer limit to its transmembrane movement. These molecular interactions reveal how the S4 gating charges are stabilized in the resting state and how their outward movement is catalyzed by interaction with negatively charged residues to effect pore opening and initiate electrical signaling.

Intermediate states of the Kv1.2 voltage sensor from atomistic molecular dynamics simulations

The response of a membrane-bound Kv1.2 ion channel to an applied transmembrane potential has been studied using molecular dynamics simulations. Channel deactivation is shown to involve three intermediate states of the voltage sensor domain (VSD), and concomitant movement of helix S4 charges 10-15 Å along the bilayer normal; the latter being enabled by zipper-like sequential pairing of S4 basic residues with neighboring VSD acidic residues and membrane-lipid head groups. During the observed sequential transitions S4 basic residues pass through the recently discovered charge transfer center with its conserved phenylalanine residue, F(233). Analysis indicates that the local electric field within the VSD is focused near the F(233) residue and that it remains essentially unaltered during the entire process. Overall, the present computations provide an atomistic description of VSD response to hyperpolarization, add support to the sliding helix model, and capture essential features inferred from a variety of recent experiments.

Contribution of S4 Charges to Gating Mechanism in H v Channels

Voltage-gated proton (HV) channels have been shown to play an essential role in immune system function. They are homologous to the voltage-sensing domain (VSD) of voltage-gated potassium (Kv) channels. In contrast to the tetramer structure of Kv channels, we found that Hv channels are dimers with just two S4 segments. Recently, we showed that the total effective gating charges are 5.9±0.4e0 in the dimer and 2.7±0.1e0 in the monomer and that S4 movement (containing 3 arginines) precedes channel opening. However, which specific arginine from S4 segment contributes to the effective gating charge is still unknown. To answer this question, we replaced each arginine residue with asparagines separately in Ciona intestinalis Hv channels. Patch-clamp recordings in Xenopus oocytes showed a dramatic decrease of total gating charges when measured by the limiting slope method: 1.9, 2.9 and 2.2 e0 for R255, R258 and R261, respectively. In addition, our cysteine accessibility measurements are consistent with an outward movement of these three S4 charges during channel opening. According to our findings, we conclude that the S4 segment moves and functions as the voltage sensor and all S4 charges contribute to voltage gating in Hv channels.

Mode Shift of the Voltage Sensors in Shaker K + Channels is Caused by Energetic Coupling to the Pore Domain

The voltage sensors (S1-S4) of Kv channels are known to undergo a conformational change when triggered by membrane depolarisation that subsequently leads to opening of the central pore (S5-S6) and ion conduction. The electromechanical coupling between these domains is mediated by two well conserved regions: the C-terminus of the S6 segment and the S4-S5 linker. Upon change in the membrane potential, the structural rearrangements induced by the charged S4 segment can be measured as gating currents. During prolonged depolarization, voltage-gated ion channels present a behaviour called the “mode-shift”. This shift causes the QV to change towards negative potentials and essentially affects the energy required by the system to bring all sensors back to their resting state. To understand the structural cause of this process, we investigated the coupling between the pore and voltage sensor and its influence on the development of the mode shift. In our approach we used the cut-open voltage clamp fluorometry method to simultaneously measure gating or ionic currents and conformational changes of the S4 segment. We identified mutations that fully uncouple voltage sensors from the pore domain thus eliminating the mutual influence of these two domains on one another. The results show that, when eliminating the coupling between these domains, the voltage sensors require less energy to move. At the same time the mode shift occurring during prolonged depolarization is abolished. By instead preventing open state stabilization, voltage sensors are kept in the opposite mode. We also found that the mode shift is accompanied by a conformational change in the S4 segment. We propose that the pore influences the voltage sensor by the presence of a ‘’mechanical load’’ and allosterically induces a conformational change in the S4.

Membrane Insertion of a Voltage Sensor Helix

Most membrane proteins contain a transmembrane (TM) domain made up of a bundle of lipid-bilayer-spanning α-helices. TM α-helices are generally composed of a core of largely hydrophobic amino acids, with basic and aromatic amino acids at each end of the helix forming interactions with the lipid headgroups and water. In contrast, the S4 helix of ion channel voltage sensor (VS) domains contains four or five basic (largely arginine) side chains along its length and yet adopts a TM orientation as part of an independently stable VS domain. Multiscale molecular dynamics simulations are used to explore how a charged TM S4 α-helix may be stabilized in a lipid bilayer, which is of relevance in the context of mechanisms of translocon-mediated insertion of S4. Free-energy profiles for insertion of the S4 helix into a phospholipid bilayer suggest that it is thermodynamically favorable for S4 to insert from water to the center of the membrane, where the helix adopts a TM orientation. This is consistent with crystal structures of Kv channels, biophysical studies of isolated VS domains in lipid bilayers, and studies of translocon-mediated S4 helix insertion. Decomposition of the free-energy profiles reveals the underlying physical basis for TM stability, whereby the preference of the hydrophobic residues of S4 to enter the bilayer dominates over the free-energy penalty for inserting charged residues, accompanied by local distortion of the bilayer and penetration of waters. We show that the unique combination of charged and hydrophobic residues in S4 allows it to insert stably into the membrane.

Sodium Channel as a Gate for Molecular Arrhythmology: A Historical Overview

Ions carry transsarcolemmal currents that contribute importantly to electrical activity in cardiac myocytes, where the influx of Naþ ions plays an especially unique and critical role. The sodium current (INa), is not only responsible for initial cardiac action-potential depolarization, but also the influx of Naþ ions into the cytoplasm, which helps to generate the prolonged depolarization characteristics of the action-potential plateau. The magnitude and kinetics of INa are controlled in a complex manner by both membrane potential and intracellular metabolites. Such Naþ-preferring voltage-dependent channels are present in a wide variety of electrically excitable membranes of cardiac and skeletal muscles, nerves, and other tissues. The voltage-gated cardiac Naþ channel has been extensively studied by electrophysiological approaches, so that we have accumulated a large amount of information on the biophysical properties of this channel during the last thirty years. Mutations of the human voltage-gated cardiac Naþ-channel gene that cause several forms of inherited diseases have been identified. The underlying defects in the Naþ-channel-related disease state include aberrant regulation of channel function, resulting in lethal ventricular tachyarrhythmias and so on. This review article will cover certain aspects involved in the structure and regulation of the cardiac sarcolemmal voltage-gated Naþ channel in physiological and particularly in pathophysiological conditions. In 1984, in order to clarify the primary structure of the Naþ channel, Numa and colleagues used partial sequences from purified eel electroplax Naþ channels to clone the cDNA for the eel Naþ-channel -subunit. Using this probe, they cloned three Naþ-channel -subunits from the rat brain. The rat heart isoform (rHt) and its human counterpart (hHt) were cloned thereafter. The -subunit of the cardiac Naþ channel has been reported to be 240 kDa. A schematic diagram of the proposed membrane topology for the -subunit of the cardiac Naþ channel is shown in Figure 1. The -subunit consists of four large homologous membrane domains (I-IV). Each domain is comprised of six putative -helices (S1-S6). The channel’s pore is assumed to extend between segments S5 and S6. Studies with molecular-biological techniques, particularly site-directed mutagenesis of Naþ-channel proteins and functional expression mutant channels have pinpointed regions that are involved in different functions of the channel. Between each of the six transmembrane segments and particularly between each of the four larger domains are extensive regions that fold out from the membrane. Highly charged S4 segments of each domain transduce the transmembrane electrical voltage by moving across the transmembrane electrical field. This movement causes conformational changes that result in an opening or gating of the Naþ-conducting pathway. The opening of the channel, also called ‘‘activation’’, allows Naþ ions to passively enter the cell down the Naþconcentration gradient to depolarize the membrane. The Nand C-termini, as well as the linkers between segments alternate between intracellular and extracellular surfaces. The ball and chain mechanism was first proposed for the explanation of the sodium channel inactivation (Figure 1). The idea of the ball and chain inactivation in the Naþ channel is simple: when the channel is open, a peptide ball (composed of amino acid residues), tethered on the membrane by a chain, wanders randomly around the channel pore. Further studies have shown that the inactivation of sodium channels involves rather a hinged lid than a ball on chain, but this did not decrease the significance of the model.

State-dependent electrostatic interactions of S4 arginines with E1 in S2 during Kv7.1 activation

The voltage-sensing domain of voltage-gated channels is comprised of four transmembrane helices (S1–S4), with conserved positively charged residues in S4 moving across the membrane in response to changes in transmembrane voltage. Although it has been shown that positive charges in S4 interact with negative countercharges in S2 and S3 to facilitate protein maturation, how these electrostatic interactions participate in channel gating remains unclear. We studied a mutation in Kv7.1 (also known as KCNQ1 or KvLQT1) channels associated with long QT syndrome (E1K in S2) and found that reversal of the charge at E1 eliminates macroscopic current without inhibiting protein trafficking to the membrane. Pairing E1R with individual charge reversal mutations of arginines in S4 (R1–R4) can restore current, demonstrating that R1–R4 interact with E1. After mutating E1 to cysteine, we probed E1C with charged methanethiosulfonate (MTS) reagents. MTS reagents could not modify E1C in the absence of KCNE1. With KCNE1, (2-sulfonatoethyl) MTS (MTSES)− could modify E1C, but [2-(trimethylammonium)ethyl] MTS (MTSET)+ could not, confirming the presence of a positively charged environment around E1C that allows approach by MTSES− but repels MTSET+. We could change the local electrostatic environment of E1C by making charge reversal and/or neutralization mutations of R1 and R4, such that MTSET+ modified these constructs depending on activation states of the voltage sensor. Our results confirm the interaction between E1 and the fourth arginine in S4 (R4) predicted from open-state crystal structures of Kv channels and reveal an E1–R1 interaction in the resting state. Thus, E1 engages in electrostatic interactions with arginines in S4 sequentially during the gating movement of S4. These electrostatic interactions contribute energetically to voltage-dependent gating and are important in setting the limits for S4 movement.

Stochastic diffusion model of multistep activation in a voltage-dependent K channel

The energy barrier to the activated state for the S4 voltage sensor of a K channel is dependent on the electrostatic force between positively charged S4 residues and negatively charged groups on neighboring segments, the potential difference across the membrane, and the dielectric boundary force on the charged residues near the interface between the solvent and the low dielectric region of the membrane gating pore. The variation of the potential function with transverse displacement and rotation of the S4 sensor across the membrane may be derived from a solution of Poisson's equation for the electrostatic potential. By approximating the energy of an S4 sensor along a path between stationary states by a piecewise linear function of the transverse displacement, the dynamics of slow activation, in the millisecond range, may be described by the lowest frequency component of an analytical solution of interacting diffusion equations of Fokker-Planck type for resting and barrier regions. The solution of the Smoluchowski equations for an S4 sensor in an energy landscape with several barriers is in accord with an empirical master equation for multistep activation in a voltage-dependent K channel.

Role of the S4 Charges on Activation Gating of the Sodium Channel

The conformational changes in the S4 voltage-sensors of the sodium channel on depolarization of the membrane result in gating of the voltage-gated sodium channel. The movement of the positively charged residues of the voltage-sensors in the membrane electric field generates a measurable transient current referred to as the gating current. The four S4 voltage-sensors in the sodium channel are homologous, but non-identical and prior work supports the hypothesis that each of the voltage-sensors may have a different role in the processes of activation and inactivation. In an attempt to characterize the role of each voltage-sensor in the process of activation, we generated a series of mutants in which the first three extracellular charges of each voltage-sensor were concurrently mutated to the neutral amino acid glutamine (Q3 mutants). These mutants show a tetrodotoxin (TTX) insensitive current component at hyperpolarized potentials presumably due to current flow through the voltage-sensors. Charge neutralization of DII resulted in a reduced Cole-Moore shift compared to the wild type channels. Gating current measurements at 15 degrees Celsius show that the rate of charge movement is the most rapid for the DII-Q3 mutant compared to the WT, and other Q3 mutants. These experiments may provide insights into the mechanisms underlying activation gating of the sodium channel.

S4 Arginines Make Unique Contributions to Voltage Dependent Gating Due to Electrostatic Interactions and the Membrane Potential

Conserved positively charged arginines in the fourth transmembrane segment (S4) of Kv channels are responsible for imparting voltage sensitivity to the channel. There are several forces that may influence these arginines including the membrane potential and electrostatic interactions with countercharges. In Shaker channels, the first four arginines are the primary gating charges that sense the membrane potential. Kv7.1 has fewer positively charged S4 residues than Shaker, notably with the third arginine in Shaker replaced by a glutamine (Q3). Further loss of charge induced by charge reversal at R1 (R1E) in Kv7.1 results in constitutively activated channels, perhaps due to insufficient charge in S4. Consistent with this idea, introduction of a positive charge at Q3 (Q3R) can restore voltage dependent activation to R1E, suggesting that Q3R may substitute for the loss of gating charge at R1E. In a related study, we have demonstrated in Kv7.1 channels that residues corresponding to the first four arginines in Shaker channels (R1-R4) interact sequentially with the first conserved glutamate in S2 (E1) during gating. Here we show via intragenic suppression that S4 arginines also interact electrostatically with the second conserved glutamate in S2 (E2), and these electrostatic interactions play an important role in voltage sensing of S4. Therefore, a network of electrostatic interactions and the membrane potential act on S4 arginines, and the balance of these forces stabilize the conformation of the voltage sensor at different states. The combination of these interactions acts uniquely on each arginine such that each arginine plays a different role in voltage dependent gating. In Kv7.1, the first two arginines (R1, R2) stabilize the resting state while the last three charged residues (R4, H5, R6) stabilize the activated state.

Estimating Conformational Changes of KCNQ1 Channels During Gating Using Molecular Dynamics Simulations

Although the structure of an ion-channel protein may be known through crystallography or homology modeling, measuring its conformational changes during gating is extremely difficult if not impossible. Alternatively, molecular simulations is a powerful tool for estimating conformational changes. Movement of the positively charged S4 segment is a known conformational change of voltage-gated ion-channels, observed by measuring the accessibility of different amino-acid residues.A previously determined structure of KCNQ1 was used. Movement of the S4-S3 complex with 3 translational and 2 rotational degrees of freedom was assumed to be the major conformational change during gating. More than 1 million conformations were considered. This expanded configuration space (compared to 1 translational and 1 rotational degree of freedom and about 2000 conformations used previously) enabled more accurate analysis of stable conformations. Conformations with steric overlap were eliminated. The electrostatic energies of the remaining conformations for various membrane potentials were computed and used to determine the probability of the ion-channel residing in each conformation (state residency). Conformations with small state residencies were eliminated.Two regions in the configuration space with high state residency were identified: one associated with open conformations (outward position of S4-S3) and the other with closed conformations (inward position of S4-S3). These regions were separated by a narrow region with low state residency (an energy barrier). The open state consists of a large number of conformations while the energy barrier and its adjacent closed state (intermediate closed) consist of only few conformations. Channel conformations may branch from the intermediate closed conformation into two trajectories toward different subsets of closed conformations (deep closed). The simulated steady state open probabilities at various membrane potentials were consistent with experimental channel activation curves.

Molecular Requirements for Recognition of Brain Voltage-gated Sodium Channels by Scorpion α-Toxins

The scorpion alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus) is active at various mammalian voltage-gated sodium channels (Na(v)s) and is inactive at insect Na(v)s. To resolve the molecular basis of this preference we used the following strategy: 1) Lqh2 was expressed in recombinant form and key residues important for activity at the rat brain channel rNa(v)1.2a were identified by mutagenesis. These residues form a bipartite functional surface made of a conserved "core domain" (residues of the loops connecting the secondary structure elements of the molecule core), and a variable "NC domain" (five-residue turn and the C-tail) as was reported for other scorpion alpha-toxins. 2) The functional role of the two domains was validated by their stepwise construction on the similar scaffold of the anti-insect toxin LqhalphaIT. Analysis of the activity of the intermediate constructs highlighted the critical role of Phe(15) of the core domain in toxin potency at rNa(v)1.2a, and has suggested that the shape of the NC-domain is important for toxin efficacy. 3) Based on these findings and by comparison with other scorpion alpha-toxins we were able to eliminate the activity of Lqh2 at rNa(v)1.4 (skeletal muscle), hNa(v)1.5 (cardiac), and rNa(v)1.6 channels, with no hindrance of its activity at Na(v)1.1-1.3. These results suggest that by employing a similar approach the design of further target-selective sodium channel modifiers is imminent.

Sensitivity of HCN channel deactivation to cAMP is amplified by an S4 mutation combined with activation mode shift

Hyperpolarisation-activation of HCN ion channels relies on the movement of a charged S4 transmembrane helix, preferentially stabilising the open conformation of the ion pore gate. The open state is additionally stabilised, (a) when cyclic AMP (cAMP) is bound to a cytoplasmic C-terminal domain or (b) when the "mode I" open state formed initially by gate opening undergoes a "mode shift" into a "mode II" open state with a new S4 conformation. We isolated a mutation (lysine 381 to glutamate) in S4 of mouse HCN4; patch-clamp of homomeric channels in excised inside-out membranes revealed a conditional phenotype. When cAMP-liganded K381E channels are previously activated by hyperpolarisation, tens of seconds are required for complete deactivation at a weakly depolarised potential; this "ultra-sustained activation" is not observed without cAMP. Whilst cAMP slows deactivation of wild-type channels, the K381E mutation amplifies this effect to enable extraordinary kinetic stabilisation of the open state. K381E channels retain S4-gate coupling, with strong voltage dependence of the rate-limiting step for deactivation of mode II channels near -40 mV. At these voltages, the mode I deactivation pathway shows a different rate-limiting step, lacking strong voltage or cAMP dependence. Ultra-sustained activation thus reflects stabilisation of the mode II open state by the K381E mutation in synergistic combination with cAMP binding. Thus, the voltage-sensing domain is subject to strong functional coupling not only to the pore domain but also to the cytoplasmic cAMP-sensing domain in a manner specific to the voltage sensor conformation.

Cooperativity Between Voltage-sensing Domains in the Human BK Channel Revealed by Voltage-clamp Fluorometry.

Like other members of the voltage-gated K+ channel superfamily, BK channels are thought to derive voltage sensitivity from charge-possessing transmembrane segments S2-S4. Particularly in BK, S2 is thought to have a direct role in voltage sensing (Ma et al., JGP2006). We combined cut-open oocyte voltage-clamp with fluorometry, after labeling unique cysteines introduced in cysteine-less BK channels (hSlo) with TMRM (Savalli et al., PNAS2006 and JGP2007), to resolve voltage-dependent conformational rearrangements near the extracellular side of S2. The intensity of fluorescence emission (ΔF) was strongly voltage-dependent (FVhalf=-92±2.7mV, Fz=0.95±0.07, n=8), reporting protein rearrangements. To investigate voltage sensor function, we targeted two putative voltage-sensing residues: D153 (S2) and R213 (the single voltage-sensing residue in S4 -Ma et al., JGP2006). Neutralizing D153 in S2-labeled channels abolished voltage-evoked fluorescence deflections, strongly supporting the role of D153 in voltage activation of S2. Neutralizing R213 in the S4-labeled channel gave rise to a detectable but weakly voltage-dependent ΔF (Fz<0.2, n=2), perhaps arising from the S3 charge (D186, Ma et al., 2006). Cooperativity amongst voltage sensing transmembrane segments was evaluated by investigating ionic currents and ΔF from an S4 charge mutant labeled in the S2 and vice versa. In both cases, protein rearrangements were detected, albeit less voltage-dependent (Fz=0.21-0.23, n=5-6 respectively). These findings revealed that strong S2-S4 cooperativity underlies voltage sensing in the intact channel. In contrast to fluorescence experiments, the change in voltage sensitivity of ionic currents was smaller (Gz=0.70-0.86), supporting the view that the pore has intrinsic voltage dependence. An allosteric model of gating composed of two types of voltage sensing tetramers surrounding a single pore was used to provide a global fit of the experimental results.

S4-based voltage sensors have three major conformations

Voltage sensors containing the charged S4 membrane segment display a gating charge vs. voltage ( Q–V ) curve that depends on the initial voltage. The voltage-dependent phosphatase (Ci-VSP), which does not have a conducting pore, shows the same phenomenon and the Q–V recorded with a depolarized initial voltage is more stable by at least 3 RT . The leftward shift of the Q–V curve under prolonged depolarization was studied in the Ci-VSP by using electrophysiological and site-directed fluorescence measurements. The fluorescence shows two components: one that traces the time course of the charge movement between the resting and active states and a slower component that traces the transition between the active state and a more stable state we call the relaxed state. Temperature dependence shows a large negative enthalpic change when going from the active to the relaxed state that is almost compensated by a large negative entropic change. The Q–V curve midpoint measured for pulses that move the sensor between the resting and active states, but not long enough to evolve into the relaxed states, show a periodicity of 120°, indicating a 3 10 secondary structure of the S4 segment when determined under histidine scanning. We hypothesize that the S4 segment moves as a 3 10 helix between the resting and active states and that it converts to an α-helix when evolving into the relaxed state, which is most likely to be the state captured in the crystal structures.

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 (IKs) channels with an S6-domain mutation, F340W in KCNQ1, exhibit constitutive activation but voltage-dependent C-type inactivation. F340W-IKs inactivation was sensitive to extracellular cation concentration and species, and it altered ion selectivity, suggestive of pore constriction. The rate and extent of F340W-IKs 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, zi, of ∼1. Finally, double-mutant channels with a single S4 charge neutralization (R231A,F340W-IKs) exhibited constitutive C-type inactivation. The results suggest that F340W-IKs 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.