S3-S4 Linker Length Modulates the Relaxed State of a Voltage-Gated Potassium Channel

Voltage-sensing domains (VSDs) are membrane protein modules found in ion channels and enzymes that are responsible for a large number of fundamental biological tasks, such as neuronal electrical activity. The VSDs switch from a resting to an active conformation upon membrane depolarization, altering the activity of the protein in response to voltage changes. Interestingly, numerous studies describe the existence of a third distinct state, called the relaxed state, also populated at positive potentials. Although some physiological roles for the relaxed state have been suggested, little is known about the molecular determinants responsible for the development and modulation of VSD relaxation. Several lines of evidence have suggested that the linker (S3-S4 linker) between the third (S3) and fourth (S4) transmembrane segments of the VSD alters the equilibrium between resting and active conformations. By measuring gating currents from the Shaker potassium channel, we demonstrate here that shortening the S3-S4 linker stabilizes the relaxed state, whereas lengthening the linker or splitting it and coinjecting two fragments of the channel have little effect. We propose that natural variations of the length of the S3-S4 linker in various VSD-containing proteins may produce differential VSD relaxation in vivo.     

Molecular dynamics investigation of the ω-current in the Kv1.2 voltage sensor domains

Voltage sensor domains (VSD) are transmembrane proteins that respond to changes in membrane voltage and modulate the activity of ion channels, enzymes, or in the case of proton channels allow permeation of protons across the cell membrane. VSDs consist of four transmembrane segments, S1-S4, forming an antiparallel helical bundle. The S4 segment contains several positively charged residues, mainly arginines, located at every third position along the helix. In the voltage-gated Shaker K(+) channel, the mutation of the first arginine of S4 to a smaller uncharged amino acid allows permeation of cations through the VSD. These currents, known as ω-currents, pass through the VSD and are distinct from K(+) currents passing through the main ion conduction pore. Here we report molecular dynamics simulations of the ω-current in the resting-state conformation for Kv1.2 and for four of its mutants. The four tested mutants exhibit various degrees of conductivity for K(+) and Cl(-) ions, with a slight selectivity for K(+) over Cl(-). Analysis of the ion permeation pathway, in the case of a highly conductive mutant, reveals a negatively charged constriction region near the center of the membrane that might act as a selectivity filter to prevent permeation of anions through the pore. The residues R1 in S4 and E1 in S2 are located at the narrowest region of the ω-pore for the resting state conformation of the VSD, in agreement with experiments showing that the largest increase in current is produced by the double mutation E1D and R1S.     

Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels

Alternative splicing of the skeletal muscle Ca_V1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant Ca_V1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3–S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (Ca_V1.1 null) myotubes reconstituted with Ca_V1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3–S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3–S4 linker is sufficient to confer Ca_V1.1a-like voltage dependence to the I VSD and that the IS3–S4 linker plus IS4 is sufficient to transfer Ca_V1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3–S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, Ca_V1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively.     

Solution Structure and Phospholipid Interactions of the Isolated Voltage-Sensor Domain from KvAP

Voltage-sensor domains (VSDs) are specialized transmembrane segments that confer voltage sensitivity to many proteins such as ion channels and enzymes. The activities of these domains are highly dependent on both the chemical properties and the physical properties of the surrounding membrane environment. To learn about VSD-lipid interactions, we used nuclear magnetic resonance spectroscopy to determine the structure and phospholipid interface of the VSD from the voltage-dependent K⁺ channel KvAP (prokaryotic Kv from Aeropyrum pernix). The solution structure of the KvAP VSD solubilized within phospholipid micelles is similar to a previously determined crystal structure solubilized by a nonionic detergent and complexed with an antibody fragment. The differences observed include a previously unidentified short amphipathic α-helix that precedes the first transmembrane helix and a subtle rigid-body repositioning of the S3-S4 voltage-sensor paddle. Using ¹⁵N relaxation experiments, we show that much of the VSD, including the pronounced kink in S3 and the S3-S4 paddle, is relatively rigid on the picosecond-to-nanosecond timescale. In contrast, the kink in S3 is mobile on the microsecond-to-millisecond timescale and may act as a hinge in the movement of the paddle during channel gating. We characterized the VSD-phospholipid micelle interactions using nuclear Overhauser effect spectroscopy and showed that the micelle uniformly coats the KvAP VSD and approximates the chemical environment of a phospholipid bilayer. Using paramagnetically labeled phospholipids, we show that bilayer-forming lipids interact with the S3 and S4 helices more strongly than with S1 and S2.     

S1 constrains S4 in the voltage sensor domain of Kv7.1 K+ channels

Voltage-gated K(+) channels comprise a central pore enclosed by four voltage-sensing domains (VSDs). While movement of the S4 helix is known to couple to channel gate opening and closing, the nature of S4 motion is unclear. Here, we substituted S4 residues of Kv7.1 channels by cysteine and recorded whole-cell mutant channel currents in Xenopus oocytes using the two-electrode voltage-clamp technique. In the closed state, disulfide and metal bridges constrain residue S225 (S4) nearby C136 (S1) within the same VSD. In the open state, two neighboring I227 (S4) are constrained at proximity while residue R228 (S4) is confined close to C136 (S1) of an adjacent VSD. Structural modeling predicts that in the closed to open transition, an axial rotation (approximately 190 degrees) and outward translation of S4 (approximately 12 A) is accompanied by VSD rocking. This large sensor motion changes the intra-VSD S1-S4 interaction to an inter-VSD S1-S4 interaction. These constraints provide a ground for cooperative subunit interactions and suggest a key role of the S1 segment in steering S4 motion during Kv7.1 gating.     

Relative transmembrane segment rearrangements during BK channel activation resolved by structurally assigned fluorophore-quencher pairing

Voltage-activated proteins can sense, and respond to, changes in the electric field pervading the cell membrane by virtue of a transmembrane helix bundle, the voltage-sensing domain (VSD). Canonical VSDs consist of four transmembrane helices (S1-S4) of which S4 is considered a principal component because it possesses charged residues immersed in the electric field. Membrane depolarization compels the charges, and by extension S4, to rearrange with respect to the field. The VSD of large-conductance voltage- and Ca-activated K(+) (BK) channels exhibits two salient inconsistencies from the canonical VSD model: (1) the BK channel VSD possesses an additional nonconserved transmembrane helix (S0); and (2) it exhibits a "decentralized" distribution of voltage-sensing charges, in helices S2 and S3, in addition to S4. Considering these unique features, the voltage-dependent rearrangements of the BK VSD could differ significantly from the standard model of VSD operation. To understand the mode of operation of this unique VSD, we have optically tracked the relative motions of the BK VSD transmembrane helices during activation, by manipulating the quenching environment of site-directed fluorescent labels with native and introduced Trp residues. Having previously reported that S0 and S4 diverge during activation, in this work we demonstrate that S4 also diverges from S1 and S2, whereas S2, compelled by its voltage-sensing charged residues, moves closer to S1. This information contributes spatial constraints for understanding the BK channel voltage-sensing process, revealing the structural rearrangements in a non-canonical VSD.     

Microscopic origin of gating current fluctuations in a potassium channel voltage sensor

Voltage-dependent ion channels open and close in response to changes in membrane electrical potential due to the motion of their voltage-sensing domains (VSDs). VSD charge displacements within the membrane electric field are observed in electrophysiology experiments as gating currents preceding ionic conduction. The elementary charge motions that give rise to the gating current cannot be observed directly, but appear as discrete current pulses that generate fluctuations in gating current measurements. Here we report direct observation of gating-charge displacements in an atomistic molecular dynamics simulation of the isolated VSD from the KvAP channel in a hydrated lipid bilayer on the timescale (10-μs) expected for elementary gating charge transitions. The results reveal that gating-charge displacements are associated with the water-catalyzed rearrangement of salt bridges between the S4 arginines and a set of conserved acidic side chains on the S1-S3 transmembrane segments in the hydrated interior of the VSD.     

The S4-S5 linker acts as a signal integrator for HERG K+ channel activation and deactivation gating

Human ether-à-go-go-related gene (hERG) K(+) channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4-S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4-S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4-S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4-S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4-S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4-S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel.     

The pore of the voltage-gated proton channel

In classical tetrameric voltage-gated ion channels four voltage-sensing domains (VSDs), one from each subunit, control one ion permeation pathway formed by four pore domains. The human Hv1 proton channel has a different architecture, containing?a VSD, but lacking a pore domain. Since its location is not known, we searched for the Hv permeation pathway. We find that mutation of the S4 segment's third arginine R211 (R3) compromises proton selectivity, enabling conduction of a metal cation and even of the large organic cation guanidinium, reminiscent of Shaker's omega pore. In the open state, R3 appears to interact with an aspartate (D112) that is situated in the middle of S1 and is unique to Hv channels. The double mutation of both residues further compromises cation selectivity. We propose that membrane depolarization reversibly positions R3 next to D112 in?the transmembrane VSD to form the ion selectivity filter in the channel's open conformation.     

Down-State Model of the Voltage-Sensing Domain of a Potassium Channel

Voltage-sensing domains (VSDs) of voltage-gated potassium (Kv) channels undergo a series of conformational changes upon membrane depolarization, from a down state when the channel is at rest to an up state, all of which lead to the opening of the channel pore. The crystal structures reported to date reveal the pore in an open state and the VSDs in an up state. To gain insights into the structure of the down state, we used a set of experiment-based restraints to generate a model of the down state of the KvAP VSD using molecular-dynamics simulations of the VSD in a lipid bilayer in excess water. The equilibrated VSD configuration is consistent with the biotin-avidin accessibility and internal salt-bridge data used to generate it, and with additional biotin-avidin accessibility data. In the model, both the S3b and S4 segments are displaced ∼10 Å toward the intracellular side with respect to the up-state configuration, but they do not move as a rigid body. Arginine side chains that carry the majority of the gating charge also make large excursions between the up and down states. In both states, arginines interact with water and participate in salt bridges with acidic residues and lipid phosphate groups. An important feature that emerges from the down-state model is that the N-terminal half of the S4 segment adopts a 3₁₀-helical conformation, which appears to be necessary to satisfy a complex salt-bridge network.     

The Molecular Basis of Polyunsaturated Fatty Acid Interactions with the Shaker Voltage-Gated Potassium Channel

Voltage-gated potassium (K_V) channels are membrane proteins that respond to changes in membrane potential by enabling K⁺ ion flux across the membrane. Polyunsaturated fatty acids (PUFAs) induce channel opening by modulating the voltage-sensitivity, which can provide effective treatment against refractory epilepsy by means of a ketogenic diet. While PUFAs have been reported to influence the gating mechanism by electrostatic interactions to the voltage-sensor domain (VSD), the exact PUFA-protein interactions are still elusive. In this study, we report on the interactions between the Shaker K_V channel in open and closed states and a PUFA-enriched lipid bilayer using microsecond molecular dynamics simulations. We determined a putative PUFA binding site in the open state of the channel located at the protein-lipid interface in the vicinity of the extracellular halves of the S3 and S4 helices of the VSD. In particular, the lipophilic PUFA tail covered a wide range of non-specific hydrophobic interactions in the hydrophobic central core of the protein-lipid interface, while the carboxylic head group displayed more specific interactions to polar/charged residues at the extracellular regions of the S3 and S4 helices, encompassing the S3-S4 linker. Moreover, by studying the interactions between saturated fatty acids (SFA) and the Shaker K_V channel, our study confirmed an increased conformational flexibility in the polyunsaturated carbon tails compared to saturated carbon chains, which may explain the specificity of PUFA action on channel proteins.     

The orientation and molecular movement of a k(+) channel voltage-sensing domain

Voltage-gated channels operate through the action of a voltage-sensing domain (membrane segments S1-S4) that controls the conformation of gates located in the pore domain (membrane segments S5-S6). Recent structural studies on the bacterial K(v)AP potassium channel have led to a new model of voltage sensing in which S4 lies in the lipid at the channel periphery and moves through the membrane as a unit with a portion of S3. Here we describe accessibility probing and disulfide scanning experiments aimed at determining how well the K(v)AP model describes the Drosophila Shaker potassium channel. We find that the S1-S3 helices have one end that is externally exposed, S3 does not undergo a transmembrane motion, and S4 lies in close apposition to the pore domain in the resting and activated state.     

Molecular mechanism for depolarization-induced modulation of Kv channel closure

Voltage-dependent potassium (Kv) channels provide the repolarizing power that shapes the action potential duration and helps control the firing frequency of neurons. The K⁺ permeation through the channel pore is controlled by an intracellularly located bundle-crossing (BC) gate that communicates with the voltage-sensing domains (VSDs). During prolonged membrane depolarizations, most Kv channels display C-type inactivation that halts K⁺ conduction through constriction of the K⁺ selectivity filter. Besides triggering C-type inactivation, we show that in Shaker and Kv1.2 channels (expressed in Xenopus laevis oocytes), prolonged membrane depolarizations also slow down the kinetics of VSD deactivation and BC gate closure during the subsequent membrane repolarization. Measurements of deactivating gating currents (reporting VSD movement) and ionic currents (BC gate status) showed that the kinetics of both slowed down in two distinct phases with increasing duration of the depolarizing prepulse. The biphasic slowing in VSD deactivation and BC gate closure was strongly correlated in time and magnitude. Simultaneous recordings of ionic currents and fluorescence from a probe tracking VSD movement in Shaker directly demonstrated that both processes were synchronized. Whereas the first slowing originates from a stabilization imposed by BC gate opening, the subsequent slowing reflects the rearrangement of the VSD toward its relaxed state (relaxation). The VSD relaxation was observed in the Ciona intestinalis voltage-sensitive phosphatase and in its isolated VSD. Collectively, our results show that the VSD relaxation is not kinetically related to C-type inactivation and is an intrinsic property of the VSD. We propose VSD relaxation as a general mechanism for depolarization-induced slowing of BC gate closure that may enable Kv1.2 channels to modulate the firing frequency of neurons based on the depolarization history.     

Capturing distinct KCNQ2 channel resting states by metal ion bridges in the voltage-sensor domain

Although crystal structures of various voltage-gated K⁺ (Kv) and Na⁺ channels have provided substantial information on the activated conformation of the voltage-sensing domain (VSD), the topology of the VSD in its resting conformation remains highly debated. Numerous studies have investigated the VSD resting state in the Kv Shaker channel; however, few studies have explored this issue in other Kv channels. Here, we investigated the VSD resting state of KCNQ2, a K⁺ channel subunit belonging to the KCNQ (Kv7) subfamily of Kv channels. KCNQ2 can coassemble with the KCNQ3 subunit to mediate the I_M current that regulates neuronal excitability. In humans, mutations in KCNQ2 are associated with benign neonatal forms of epilepsy or with severe epileptic encephalopathy. We introduced cysteine mutations into the S4 transmembrane segment of the KCNQ2 VSD and determined that external application of Cd²⁺ profoundly reduced the current amplitude of S4 cysteine mutants S195C, R198C, and R201C. Based on reactivity with the externally accessible endogenous cysteine C106 in S1, we infer that each of the above S4 cysteine mutants forms Cd²⁺ bridges to stabilize a channel closed state. Disulfide bonds and metal bridges constrain the S4 residues S195, R198, and R201 near C106 in S1 in the resting state, and experiments using concatenated tetrameric constructs indicate that this occurs within the same VSD. KCNQ2 structural models suggest that three distinct resting channel states have been captured by the formation of different S4–S1 Cd²⁺ bridges. Collectively, this work reveals that residue C106 in S1 can be very close to several N-terminal S4 residues for stabilizing different KCNQ2 resting conformations.     

Calculation of the Gating Charge for the Kv1.2 Voltage-Activated Potassium Channel

The atomic models of the Kv1.2 potassium channel in the active and resting state, originally presented elsewhere, are here refined using molecular dynamics simulations in an explicit membrane-solvent environment. With a minor adjustment of the orientation of the first arginine along the S4 segment, the total gating charge of the channel determined from >0.5 μs of molecular dynamics simulation is ∼12-12.7 e, in good accord with experimental estimates for the Shaker potassium channel, indicating that the final models offer a realistic depiction of voltage-gating. In the resting state of Kv1.2, the S4 segment in the voltage-sensing domain (VSD) spontaneously converts into a 3₁₀ helix over a stretch of 10 residues. The 3₁₀ helical conformation orients the gating arginines on S4 toward a water-filled crevice within the VSD and allows salt-bridge interactions with negatively charged residues along S2 and S3. Free energy calculations of the fractional transmembrane potential, acting upon key charged residues of the VSD, reveals that the applied field varies rapidly over a narrow region of 10-15 Å corresponding to the outer leaflet of the bilayer. The focused field allows the transfer of a large gating charge without translocation of S4 across the membrane.     

Long α helices projecting from the membrane as the dimer interface in the voltage-gated H+ channel

The voltage-gated H⁺ channel (Hv) is a H⁺-permeable voltage-sensor domain (VSD) protein that consists of four transmembrane segments (S1–S4). Hv assembles as a dimeric channel and two transmembrane channel domains function cooperatively, which is mediated by the coiled-coil assembly domain in the cytoplasmic C terminus. However, the structural basis of the interdomain interactions remains unknown. Here, we provide a picture of the dimer configuration based on the analyses of interactions among two VSDs and a coiled-coil domain. Systematic mutations of the linker region between S4 of VSD and the coiled-coil showed that the channel gating was altered in the helical periodicity with the linker length, suggesting that two domains are linked by helices. Cross-linking analyses revealed that the two S4 helices were situated closely in the dimeric channel. The interaction interface between the two S4 and the assembly interface of the coiled-coil domain were aligned in the same direction based on the phase angle calculation along α helices. Collectively, we propose that continuous helices stretching from the transmembrane to the cytoplasmic region in the dimeric interface regulate the channel activation in the Hv dimer.     

In search of a consensus model of the resting state of a voltage-sensing domain

Voltage-sensing domains (VSDs) undergo conformational changes in response to the membrane potential and are the critical structural modules responsible for the activation of voltage-gated channels. Structural information about the key conformational states underlying voltage activation is currently incomplete. Through the use of experimentally determined residue-residue interactions as structural constraints, we determine and refine a model of the Kv channel VSD in the resting conformation. The resulting structural model is in broad agreement with results that originate from various labs using different techniques, indicating the emergence of a consensus for the structural basis of voltage sensing.     

Coupling between the voltage-sensing and pore domains in a voltage-gated potassium channel

Voltage-dependent potassium (Kv), sodium (Nav), and calcium channels open and close in response to changes in transmembrane (TM) potential, thus regulating cell excitability by controlling ion flow across the membrane. An outstanding question concerning voltage gating is how voltage-induced conformational changes of the channel voltage-sensing domains (VSDs) are coupled through the S4-S5 interfacial linking helices to the opening and closing of the pore domain (PD). To investigate the coupling between the VSDs and the PD, we generated a closed Kv channel configuration from Aeropyrum pernix (KvAP) using atomistic simulations with experiment-based restraints on the VSDs. Full closure of the channel required, in addition to the experimentally determined TM displacement, that the VSDs be displaced both inwardly and laterally around the PD. This twisting motion generates a tight hydrophobic interface between the S4-S5 linkers and the C-terminal ends of the pore domain S6 helices in agreement with available experimental evidence.     

Intermediate state trapping of a voltage sensor

Voltage sensor domains (VSDs) regulate ion channels and enzymes by undergoing conformational changes depending on membrane electrical signals. The molecular mechanisms underlying the VSD transitions are not fully understood. Here, we show that some mutations of I241 in the S1 segment of the Shaker Kv channel positively shift the voltage dependence of the VSD movement and alter the functional coupling between VSD and pore domains. Among the I241 mutants, I241W immobilized the VSD movement during activation and deactivation, approximately halfway between the resting and active states, and drastically shifted the voltage activation of the ionic conductance. This phenotype, which is consistent with a stabilization of an intermediate VSD conformation by the I241W mutation, was diminished by the charge-conserving R2K mutation but not by the charge-neutralizing R2Q mutation. Interestingly, most of these effects were reproduced by the F244W mutation located one helical turn above I241. Electrophysiology recordings using nonnatural indole derivatives ruled out the involvement of cation-Π interactions for the effects of the Trp inserted at positions I241 and F244 on the channel’s conductance, but showed that the indole nitrogen was important for the I241W phenotype. Insight into the molecular mechanisms responsible for the stabilization of the intermediate state were investigated by creating in silico the mutations I241W, I241W/R2K, and F244W in intermediate conformations obtained from a computational VSD transition pathway determined using the string method. The experimental results and computational analysis suggest that the phenotype of I241W may originate in the formation of a hydrogen bond between the indole nitrogen atom and the backbone carbonyl of R2. This work provides new information on intermediate states in voltage-gated ion channels with an approach that produces minimum chemical perturbation.     

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.