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 dipeptidyl-aminopeptidase-like protein 6 is an integral voltage sensor-interacting β-subunit of neuronal KV4.2 channels

Auxiliary β-subunits dictate the physiological properties of voltage-gated K⁺ (K_V) channels in excitable tissues. In many instances, however, the underlying mechanisms of action are poorly understood. The dipeptidyl-aminopeptidase-like protein 6 (DPP6) is a specific β-subunit of neuronal K_V4 channels, which may promote gating through interactions between the single transmembrane segment of DPP6 and the channel’s voltage sensing domain (VSD). A combination of gating current measurements and protein biochemistry (in-vitro translation and co-immunoprecipitations) revealed preferential physical interaction between the isolated K_V4.2-VSD and DPP6. Significantly weaker interactions were detected between DPP6 and K_V1.3 channels or the K_V4.2 pore domain. More efficient gating charge movement resulting from a direct interaction between DPP6 and the K_V4.2-VSD is unique among the known actions of K_V channel β-subunits. This study shows that the modular VSD of a K_V channel can be directly regulated by transmembrane protein-protein interactions involving an extrinsic β-subunit. Understanding these interactions may shed light on the pathophysiology of recently identified human disorders associated with mutations affecting the dpp6 gene.     

KCNQ1 Channels Do Not Undergo Concerted but Sequential Gating Transitions in Both the Absence and the Presence of KCNE1 Protein

The co-assembly of KCNQ1 with KCNE1 produces I_KS, a K⁺ current, crucial for the repolarization of the cardiac action potential. Mutations in these channel subunits lead to life-threatening cardiac arrhythmias. However, very little is known about the gating mechanisms underlying KCNQ1 channel activation. Shaker channels have provided a powerful tool to establish the basic gating mechanisms of voltage-dependent K⁺ channels, implying prior independent movement of all four voltage sensor domains (VSDs) followed by channel opening via a last concerted cooperative transition. To determine the nature of KCNQ1 channel gating, we performed a thermodynamic mutant cycle analysis by constructing a concatenated tetrameric KCNQ1 channel and by introducing separately a gain and a loss of function mutation, R231W and R243W, respectively, into the S4 helix of the VSD of one, two, three, and four subunits. The R231W mutation destabilizes channel closure and produces constitutively open channels, whereas the R243W mutation disrupts channel opening solely in the presence of KCNE1 by right-shifting the voltage dependence of activation. The linearity of the relationship between the shift in the voltage dependence of activation and the number of mutated subunits points to an independence of VSD movements, with each subunit incrementally contributing to channel gating. Contrary to Shaker channels, our work indicates that KCNQ1 channels do not experience a late cooperative concerted opening transition. Our data suggest that KCNQ1 channels in both the absence and the presence of KCNE1 undergo sequential gating transitions leading to channel opening even before all VSDs have moved.     

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.     

Modification of hERG1 channel gating by Cd2+

Each of the four subunits in a voltage-gated potassium channel has a voltage sensor domain (VSD) that is formed by four transmembrane helical segments (S1–S4). In response to changes in membrane potential, intramembrane displacement of basic residues in S4 produces a gating current. As S4 moves through the membrane, its basic residues also form sequential electrostatic interactions with acidic residues in immobile regions of the S2 and S3 segments. Transition metal cations interact with these same acidic residues and modify channel gating. In human ether-á-go-go–related gene type 1 (hERG1) channels, Cd²⁺ coordinated by D456 and D460 in S2 and D509 in S3 induces a positive shift in the voltage dependence of activation of ionic currents. Here, we characterize the effects of Cd²⁺ on hERG1 gating currents in Xenopus oocytes using the cut-open Vaseline gap technique. Cd²⁺ shifted the half-point (V_1/2) for the voltage dependence of the OFF gating charge–voltage (Q_OFF-V) relationship with an EC₅₀ of 171 µM; at 0.3 mM, V_1/2 was shifted by +50 mV. Cd²⁺ also induced an as of yet unrecognized small outward current (I_Cd-out) upon repolarization in a concentration- and voltage-dependent manner. We propose that Cd²⁺ and Arg residues in the S4 segment compete for interaction with acidic residues in S2 and S3 segments, and that the initial inward movement of S4 associated with membrane repolarization displaces Cd²⁺ in an outward direction to produce I_Cd-out. Co²⁺, Zn²⁺, and La³⁺ at concentrations that caused ∼+35-mV shifts in the Q_OFF-V relationship did not induce a current similar to I_Cd-out, suggesting that the binding site for these cations or their competition with basic residues in S4 differs from Cd²⁺. New Markov models of hERG1 channels were developed that describe gating currents as a noncooperative two-phase process of the VSD and can account for changes in these currents caused by extracellular Cd²⁺.     

The voltage-sensing domain of a phosphatase gates the pore of a potassium channel

The modular architecture of voltage-gated K⁺ (Kv) channels suggests that they resulted from the fusion of a voltage-sensing domain (VSD) to a pore module. Here, we show that the VSD of Ciona intestinalis phosphatase (Ci-VSP) fused to the viral channel Kcv creates Kv_Synth1, a functional voltage-gated, outwardly rectifying K⁺ channel. Kv_Synth1 displays the summed features of its individual components: pore properties of Kcv (selectivity and filter gating) and voltage dependence of Ci-VSP (V_1/2 = +56 mV; z of ∼1), including the depolarization-induced mode shift. The degree of outward rectification of the channel is critically dependent on the length of the linker more than on its amino acid composition. This highlights a mechanistic role of the linker in transmitting the movement of the sensor to the pore and shows that electromechanical coupling can occur without coevolution of the two domains.     

Interaction between residues in the Mg2+-binding site regulates BK channel activation

As a unique member of the voltage-gated potassium channel family, a large conductance, voltage- and Ca²⁺-activated K⁺ (BK) channel has a large cytosolic domain that serves as the Ca²⁺ sensor, in addition to a membrane-spanning domain that contains the voltage-sensing (VSD) and pore-gate domains. The conformational changes of the cytosolic domain induced by Ca²⁺ binding and the conformational changes of the VSD induced by membrane voltage changes trigger the opening of the pore-gate domain. Although some structural information of these individual functional domains is available, how the interactions among these domains, especially the noncovalent interactions, control the dynamic gating process of BK channels is still not clear. Previous studies discovered that intracellular Mg²⁺ binds to an interdomain binding site consisting of D99 and N172 from the membrane-spanning domain and E374 and E399 from the cytosolic domain. The bound Mg²⁺ at this narrow interdomain interface activates the BK channel through an electrostatic interaction with a positively charged residue in the VSD. In this study, we investigated the potential interdomain interactions between the Mg²⁺-coordination residues and their effects on channel gating. By introducing different charges to these residues, we discovered a native interdomain interaction between D99 and E374 that can affect BK channel activation. To understand the underlying mechanism of the interdomain interactions between the Mg²⁺-coordination residues, we introduced artificial electrostatic interactions between residues 172 and 399 from two different domains. We found that the interdomain interactions between these two positions not only alter the local conformations near the Mg²⁺-binding site but also change distant conformations including the pore-gate domain, thereby affecting the voltage- and Ca²⁺-dependent activation of the BK channel. These results illustrate the importance of interdomain interactions to the allosteric gating mechanisms of BK channels.     

Role of putative voltage-sensor countercharge D4 in regulating gating properties of CaV1.2 and CaV1.3 calcium channels

Voltage-dependent calcium channels (Ca_V) activate over a wide range of membrane potentials, and the voltage-dependence of activation of specific channel isoforms is exquisitely tuned to their diverse functions in excitable cells. Alternative splicing further adds to the stunning diversity of gating properties. For example, developmentally regulated insertion of an alternatively spliced exon 29 in the fourth voltage-sensing domain (VSD IV) of Ca_V1.1 right-shifts voltage-dependence of activation by 30 mV and decreases the current amplitude several-fold. Previously we demonstrated that this regulation of gating properties depends on interactions between positive gating charges (R1, R2) and a negative countercharge (D4) in VSD IV of Ca_V1.1. Here we investigated whether this molecular mechanism plays a similar role in the VSD IV of Ca_V1.3 and in VSDs II and IV of Ca_V1.2 by introducing charge-neutralizing mutations (D4N or E4Q) in the corresponding positions of Ca_V1.3 and in two splice variants of Ca_V1.2. In both channels the D4N (VSD IV) mutation resulted in a ?5 mV right-shift of the voltage-dependence of activation and in a reduction of current density to about half of that in controls. However in Ca_V1.2 the effects were independent of alternative splicing, indicating that the two modulatory processes operate by distinct mechanisms. Together with our previous findings these results suggest that molecular interactions engaging D4 in VSD IV contribute to voltage-sensing in all examined Ca_V1 channels, however its striking role in regulating the gating properties by alternative splicing appears to be a unique property of the skeletal muscle Ca_V1.1 channel.     

Bimodal regulation of an Elk subfamily K+ channel by phosphatidylinositol 4,5-bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates Shaker K⁺ channels and voltage-gated Ca²⁺ channels in a bimodal fashion by inhibiting voltage activation while stabilizing open channels. Bimodal regulation is conserved in hyperpolarization-activated cyclic nucleotide–gated (HCN) channels, but voltage activation is enhanced while the open channel state is destabilized. The proposed sites of PIP₂ regulation in these channels include the voltage-sensor domain (VSD) and conserved regions of the proximal cytoplasmic C terminus. Relatively little is known about PIP₂ regulation of Ether-á-go-go (EAG) channels, a metazoan-specific family of K⁺ channels that includes three gene subfamilies, Eag (Kv10), Erg (Kv11), and Elk (Kv12). We examined PIP₂ regulation of the Elk subfamily potassium channel human Elk1 to determine whether bimodal regulation is conserved within the EAG K⁺ channel family. Open-state stabilization by PIP₂ has been observed in human Erg1, but the proposed site of regulation in the distal C terminus is not conserved among EAG family channels. We show that PIP₂ strongly inhibits voltage activation of Elk1 but also stabilizes the open state. This stabilization produces slow deactivation and a mode shift in voltage gating after activation. However, removal of PIP₂ has the net effect of enhancing Elk1 activation. R347 in the linker between the VSD and pore (S4–S5 linker) and R479 near the S6 activation gate are required for PIP₂ to inhibit voltage activation. The ability of PIP₂ to stabilize the open state also requires these residues, suggesting an overlap in sites central to the opposing effects of PIP₂ on channel gating. Open-state stabilization in Elk1 requires the N-terminal eag domain (PAS domain + Cap), and PIP₂-dependent stabilization is enhanced by a conserved basic residue (K5) in the Cap. Our data shows that PIP₂ can bimodally regulate voltage gating in EAG family channels, as has been proposed for Shaker and HCN channels. PIP₂ regulation appears fundamentally different for Elk and KCNQ channels, suggesting that, although both channel types can regulate action potential threshold in neurons, they are not functionally redundant.     

Elucidating Relayed Proton Transfer through a His–Trp–His Triad of a Transmembrane Proton Channel by Solid-State NMR

Proton transfer through membrane-bound ion channels is mediated by both water and polar residues of proteins, but the detailed molecular mechanism is challenging to determine. The tetrameric influenza A and B virus M2 proteins form canonical proton channels that use an HxxxW motif for proton selectivity and gating. The BM2 channel also contains a second histidine (His), H27, equidistant from the gating tryptophan, which leads to a symmetric H¹⁹xxxW²³xxxH²⁷ motif. The proton-dissociation constants (pK_a's) of H19 in BM2 were found to be much lower than the pK_a's of H37 in AM2. To determine if the lower pK_a's result from H27-facilitated proton dissociation of H19, we have now investigated a H27A mutant of BM2 using solid-state NMR. ¹⁵N NMR spectra indicate that removal of the second histidine converted the protonation and tautomeric equilibria of H19 to be similar to the H37 behavior in AM2, indicating that the peripheral H27 is indeed the origin of the low pK_a's of H19 in wild-type BM2. Measured interhelical distances between W23 sidechains indicate that the pore constriction at W23 increases with the H19 tetrad charge but is independent of the H27A mutation. These results indicate that H27 both accelerates proton dissociation from H19 to increase the inward proton conductance and causes the small reverse conductance of BM2. The proton relay between H19 and H27 is likely mediated by the intervening gating tryptophan through cation–π interactions. This relayed proton transfer may exist in other ion channels and has implications for the design of imidazole-based synthetic proton channels.     

Channel gating pore: a new therapeutic target

Each subunit of voltage-gated cation channels comprises a voltage-sensing domain and a pore region. In a paper recently published in Cell Research, Li et al. showed that the gating charge pathway of the voltage sensor of the KCNQ2 K⁺ channel can accommodate small opener molecules and offer a new target to treat hyperexcitability disorders.Voltage-gated cation channels (VGCCs) are key players of many vital functions and their genetic defects in humans can lead to severe diseases, called “channelopathies”¹. Each channel α subunit possesses two main transmembrane modules, a voltage-sensing domain (VSD) and a pore region. VSDs are membrane protein modules comprising four membrane-spanning segments (S1-S4) endowed with charged amino acids, also called gating charges². Although the precise nature and extent of the conformational rearrangement of the VSD is still debated, it is commonly recognized that four highly conserved arginine residues along S4 (R1, R2, R3, and R4) mainly contribute to the voltage-driven gating charge transfer during channel activation³. The gating charges reside in aqueous crevices, and they translocate across a focused electric field spanned by a short distance where hydrophobic residues form a hydrophobic plug occluding a “gating pore”. Along this narrow hydrophobic region, the positive charges in S4 are stabilized by electrostatic interactions with negative countercharges in segments S2 and S3, water in the crevices and negatively charged phospholipids³. In voltage-gated K⁺ channels, a highly conserved phenylalanine residue located at the bottom of the S2 segment faces the intracellular side of the hydrophobic plug⁴. This aromatic residue forms the extracellular lid of an occluded site that separates the extracellular and intracellular water-filled crevices of the VSD and forms the charge-transfer center that catalyzes movement of the gating charges⁵.Molecules that target ion channel proteins have been very instrumental in adding drugs to the medicinal therapeutic arsenal as well as in providing tools to dissect the mechanisms of ion channel gating. However, so far, the pharmacological toolbox has focused only on the pore and gate regions of ion channels, both from a fundamental biophysical perspective and from a therapeutic outlook. In contrast, the VSD was virtually not targeted with small ligand molecules neither for therapeutic purposes nor for deciphering ion channel gating, though it is the target of various toxins. A recent study in Cell Research by Li et al.⁶ showed that the gating charge pathway or “gating pore” of the therapeutically relevant voltage-dependent K⁺ channel KCNQ2 could accommodate small opener molecules, thereby offering a new target to treat hyperexcitability disorders.Using a comprehensive approach employing homology modeling, molecular docking, molecular dynamics (MD) simulation, mutagenesis and electrophysiology, Li et al.⁶ identified an activator-binding pocket in the occluded gating pore of KCNQ2. First, a small opener molecule ztz240 recently discovered by the same group was used as a probe to determine by scanning mutagenesis the binding model of ligands in the KCNQ2 gating charge pathway (Figure 1). Among the mutational hits, several VSD mutants in S2 and S4 dramatically decreased the opener activity of ztz240, including the mutant of the conserved phenylalanine (F137A) in S2 forming the hydrophobic plug of the KCNQ2 gating pore. Exploiting the mutational constraints and using a flexible docking program, Li and co-workers built a docking model for the opener ztz240 onto a structural homology model of KCNQ2 that was based on the open state structure of Kv1.2 channel. They could precisely determine the orientation of the ligand into the binding pocket by wisely synthesizing two chemical derivatives of ztz240 and testing them on KCNQ2 channel activity. Next, they further optimized the docking model by MD simulation of the ligand-channel complex embedded into phospholipids. The docking model defined a broad pocket, spreading from the extracellular entrance of the VSD groove to the bottom of the gating pore with the ligand engaged in a wide array of hydrophobic, H-bonding and electrostatic interactions. Adopting a very elegant strategy, Li et al. set out to screen a structure-based virtual library of about 200 000 chemicals that were selected to fit the newly identified ligand-binding pocket by a docking approach. The purpose was to discover new KCNQ2 channel openers and eventually provide lead optimization (Figure 1). Out of 25 hits selected by bioassays, nine compounds showed significant KCNQ2 opener activity with EC₅₀ in the micromolar range. Remarkably, as an ultimate validation, these newly discovered KCNQ2 channel openers demonstrated an excellent anti-epileptic activity in two different murine models of epilepsy.Open in a separate windowFigure 1Cartoon summarizing the strategy used to discover new channel opener molecules. Following synthesis of an initial lead compound, a scanning channel mutagenesis and subsequent electrophysiological testing of the lead are performed on the mutants. This step allows identification of crucial residues for lead activity. Next, flexible docking and MD simulations are carried out to define the ligand-binding pocket. Then, a screen of a structure-based virtual library is performed where chemicals are selected to fit the newly identified ligand-binding pocket by a docking approach. Following this stage, the hits are validated in vitro by electrophysiology, which allows discovery of new compounds and lead optimization. The novel active compounds are tested for validation in vivo using animal models. This strategy could be applied to the discovery of any modulator in any kind of ion channel.The study of Li et al. identifies a new therapeutic target, a ligand-binding site in the gating pore of KCNQ2 channels at the heart of the gating machinery where the electric field is highly focused. The opener-binding pocket with a volume of about 170 ų extends deeply inside the VSD and is different from the site of another compound NH29, previously reported to locate in a more superficial region of the VSD⁷. The clever approach of Li and co-workers provides a 36% hit rate of virtual screening, which is much higher than hit rates of cell-based high throughput screening for discovering channel activators. By targeting the gating pore as a novel channel site for new opener molecules, this work provides a tool to dissect the basic biophysical mechanisms underlying gating of VGCCs. From a translational viewpoint, it offers novel therapeutic strategies for the treatment of hyperexcitability disorders, such as epilepsy or neuropathic pain.A number of exciting issues will certainly stimulate future investigations. Knowing the adaptability and modular nature of the VSD, could the gating pore of other voltage-gated Na⁺, Ca²⁺ and K⁺ channels accommodate small ligands and be the target of novel molecules? If so, would it be possible to trap the VSD in the resting or activated conformation and thereby design new inhibitors or openers? To what extent the gating pore shares common attributes among different VGCCs and how the selectivity of the compounds could be preserved? From a fundamental perspective, it will be important to examine the impact of these new molecules on gating currents and the effects of the surrounding lipid on their pharmacological sensitivities.     

Arachidonic acid effect on the allosteric gating mechanism of BK (Slo1) channels associated with the β1 subunit

Arachidonic acid (AA) is a fatty acid involved in the modulation of several ion channels. Previously, we reported that AA activates the high conductance Ca²⁺- and voltage-dependent K⁺ channel (BK) in vascular smooth muscle depending on the expression of the auxiliary β1 subunit. Here, using the patch-clamp technique on BK channel co-expressed with β1 subunit in a heterologous cell expression system, we analyzed whether AA modifies the three functional modules involved in the channel gating: the voltage sensor domain (VSD), the pore domain (PD), and the intracellular calcium sensor domain (CSD). We present evidence that AA activates BK channel in a direct way, inducing VSD stabilization on its active configuration observed as a significant left shift in the Q-V curve obtained from gating currents recordings. Moreover, AA facilitates the channel opening transitions when VSD are at rest, and the CSD are unoccupied. Furthermore, the activation was independent of the intracellular Ca²⁺ concentration and reduced when the BK channel was co-expressed with the Y74A mutant of the β1 subunit. These results allow us to present new insigths in the mechanism by which AA modulates BK channels co-expressed with its auxiliary β1 subunit.     

Ion-pair interactions between voltage-sensing domain IV and pore domain I regulate CaV1.1 gating

The voltage-gated calcium channel Ca_V1.1 belongs to the family of pseudo-heterotetrameric cation channels, which are built of four structurally and functionally distinct voltage-sensing domains (VSDs) arranged around a common channel pore. Upon depolarization, positive gating charges in the S4 helices of each VSD are moved across the membrane electric field, thus generating the conformational change that prompts channel opening. This sliding helix mechanism is aided by the transient formation of ion-pair interactions with countercharges located in the S2 and S3 helices within the VSDs. Recently, we identified a domain-specific ion-pair partner of R1 and R2 in VSD IV of Ca_V1.1 that stabilizes the activated state of this VSD and regulates the voltage dependence of current activation in a splicing-dependent manner. Structure modeling of the entire Ca_V1.1 in a membrane environment now revealed the participation in this process of an additional putative ion-pair partner (E216) located outside VSD IV, in the pore domain of the first repeat (IS5). This interdomain interaction is specific for Ca_V1.1 and Ca_V1.2 L-type calcium channels. Moreover, in Ca_V1.1 it is sensitive to insertion of the 19 amino acid peptide encoded by exon 29. Whole-cell patch-clamp recordings in dysgenic myotubes reconstituted with wild-type or E216 mutants of GFP-Ca_V1.1e (lacking exon 29) showed that charge neutralization (E216Q) or removal of the side chain (E216A) significantly shifted the voltage dependence of activation (V_1/2) to more positive potentials, suggesting that E216 stabilizes the activated state. Insertion of exon 29 in the GFP-Ca_V1.1a splice variant strongly reduced the ionic interactions with R1 and R2 and caused a substantial right shift of V_1/2, whereas no further shift of V_1/2 was observed on substitution of E216 with A or Q. Together with our previous findings, these results demonstrate that inter- and intradomain ion-pair interactions cooperate in the molecular mechanism regulating VSD function and channel gating in Ca_V1.1.     

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.     

The Cytosolic GH Loop Regulates the Phosphatidylinositol 4,5-Bisphosphate-induced Gating Kinetics of Kir2 Channels

Inwardly rectifying K⁺ (Kir) channels set the resting membrane potential and regulate cellular excitability. The activity of Kir channels depends critically on the phospholipid PIP₂. The molecular mechanism by which PIP₂ regulates Kir channel gating is poorly understood. Here, we utilized a combination of computational and electrophysiological approaches to discern structural elements involved in regulating the PIP₂-induced gating kinetics of Kir2 channels. We identify a novel role for the cytosolic GH loop. Mutations that directly or indirectly affect GH loop flexibility (e.g. V223L, E272G, D292G) increase both the on- and especially the off-gating kinetics. These effects are consistent with a model in which competing interactions between the CD and GH loops for the N terminus regulate the gating of the intracellular G loop gate.     

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.     

Control of pH and PIP2 Gating in Heteromeric Kir4.1/Kir5.1 Channels by H-Bonding at the Helix-Bundle Crossing

Inhibition by intracellular H⁺ (pH gating) and activation by phosphoinositides such as PIP₂ (PIP₂ gating) are key regulatory mechanisms in the physiology of inwardly-rectifying potassium (Kir) channels. Our recent findings suggest that PIP₂ gating and pH gating are controlled by an intrasubunit H-bond at the helix-bundle crossing between a lysine in TM1 and a backbone carbonyl group in TM2. This interaction only occurs in the closed state and channel opening requires this H-bond to be broken, thereby influencing the kinetics of PIP₂- and pH-gating in Kir channels. In this addendum, we explore the role of H-bonding in heteromeric Kir4.1/Kir5.1 channels. Kir5.1 subunits do not possess a TM1 lysine. However, homology modelling and molecular dynamics simulations demonstrate that the TM1 lysine in Kir4.1 is capable of H-bonding at the helix-bundle crossing. Consistent with this, the rates of pH and PIP₂ gating in Kir4.1/Kir5.1 channels (two H-bonds) were intermediate between those of wild-type homomeric Kir4.1 (four H-bonds) and Kir4.1(K67M) channels (no H-bonds) suggesting that the number of H-bonds in the tetrameric channel complex determines the gating kinetics. Furthermore, in heteromeric Kir4.1(K67M)/Kir5.1 channels, where the two remaining H-bonds are disrupted, we found that the gating kinetics were similar to Kir4.1(K67M) homomeric channels despite the fact that these two channels differ considerably in their PIP₂ affinities. This indicates that Kir channel PIP₂ affinity has little impact on either the PIP₂- or pH-gating kinetics.     

Divalent Cations Slow Activation of EAG Family K Channels through Direct Binding to S4

Voltage-gated K⁺ channels share a common voltage sensor domain (VSD) consisting of four transmembrane helices, including a highly mobile S4 helix that contains the major gating charges. Activation of ether-a-go-go (EAG) family K⁺ channels is sensitive to external divalent cations. We show here that divalent cations slow the activation rate of two EAG family channels (Kv12.1 and Kv10.2) by forming a bridge between a residue in the S4 helix and acidic residues in S2. Histidine 328 in the S4 of Kv12.1 favors binding of Zn²⁺ and Cd²⁺, whereas the homologous residue Serine 321 in Kv10.2 contributes to effects of Mg²⁺ and Ni²⁺. This novel finding provides structural constraints for the position of transmembrane VSD helices in closed, ion-bound EAG family channels. Homology models of Kv12.1 and Kv10.2 VSD structures based on a closed-state model of the Shaker family K⁺ channel Kv1.2 match these constraints. Our results suggest close conformational conservation between closed EAG and Shaker family channels, despite large differences in voltage sensitivity, activation rates, and activation thresholds.     

A residue at the cytoplasmic entrance of BK-type channels regulating single-channel opening by its hydrophobicity

Single large-conductance calcium-activated K⁺ (BK) channels encoded by the mSlo gene usually have synchronous gating, but a Drosophila dSlo (A2/C2/E2/G5/10) splice variant (dSlo1A) exhibits very flickery openings. To probe this difference in gating, we constructed a mutant I323T. This channel exhibits four subconductance levels similar to those of dSlo1A. Rectification of the single-channel current-voltage relation of I323T decreased as [Ca²⁺ ]_in increased from 10 to 300 μM. Mutagenesis suggests that the hydrophobicity of the residue at the position is important for the wild-type gating; i.e., increasing hydrophobicity prolongs open duration. Molecular dynamics simulation suggests that four hydrophobic pore-lining residues at position 323 of mSlo act cooperatively in a “shutter-like” mechanism gating the permeation of K⁺ ions. Rate-equilibrium free energy relations analysis shows that the four I323 residues in an mSlo channel have a conformation 65% similar to the closed conformation during gating. Based on these observations, we suggest that the appearance of rectification and substates of BK-type channels arise from a reduction of the cooperativity among these four residues and a lower probability of being open.     

Multiscale modeling shows that dielectric differences make NaV channels faster than KV channels

The generation of action potentials in excitable cells requires different activation kinetics of voltage-gated Na (Na_V) and K (K_V) channels. Na_V channels activate much faster and allow the initial Na⁺ influx that generates the depolarizing phase and propagates the signal. Recent experimental results suggest that the molecular basis for this kinetic difference is an amino acid side chain located in the gating pore of the voltage sensor domain, which is a highly conserved isoleucine in K_V channels but an equally highly conserved threonine in Na_V channels. Mutagenesis suggests that the hydrophobicity of this side chain in Shaker K_V channels regulates the energetic barrier that gating charges cross as they move through the gating pore and control the rate of channel opening. We use a multiscale modeling approach to test this hypothesis. We use high-resolution molecular dynamics to study the effect of the mutation on polarization charge within the gating pore. We then incorporate these results in a lower-resolution model of voltage gating to predict the effect of the mutation on the movement of gating charges. The predictions of our hierarchical model are fully consistent with the tested hypothesis, thus suggesting that the faster activation kinetics of Na_V channels comes from a stronger dielectric polarization by threonine (Na_V channel) produced as the first gating charge enters the gating pore compared with isoleucine (K_V channel).