Closed State Model Research Articles

Antagonistic Mechanism of α-Conotoxin BuIA toward the Human α3β2 Nicotinic Acetylcholine Receptor

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that are abundantly expressed in the central and peripheral nervous systems, playing an important role in mediating neurotransmitter release and inter-synaptic signaling. Dysfunctional nAChRs are associated with neurological disorders, and studying the structure and function of nAChRs is essential for development of drugs or strategies for treatment of related diseases. α-Conotoxins are selective antagonists of the nAChR and are an important class of drug leads. So far, the antagonistic mechanism of α-conotoxins toward the nAChRs is still unclear. In this study, we built an α3β2 nAChR homology model and investigated its conformational transition mechanism upon binding with a highly potent inhibitor, α-conotoxin BuIA, through μs molecular dynamic simulations and site-directed mutagenesis studies. The results suggested that the α3β2 nAChR underwent global conformational transitions and was stabilized into a closed state with three hydrophobic gates present in the transmembrane domain by BuIA. Finally, the probable antagonistic mechanism of BuIA was proposed. Overall, the closed-state model of the α3β2 nAChR bound with BuIA is not only essential for understanding the antagonistic mechanism of α-conotoxins but also particularly valuable for development of therapeutic inhibitors in future.

Engineered high-affinity zinc binding site reveals gating configurations of a human proton channel.

The voltage-gated proton channel (HV1) is a voltage sensor that also conducts protons. The singular ability of protons to penetrate proteins complicates distinguishing closed and open channels. When we replaced valine with histidine at position 116 in the external vestibule of hHV1, current was potently inhibited by externally applied Zn2+ in a construct lacking the two His that bind Zn2+ in WT channels. High-affinity binding with profound effects at 10 nM Zn2+ at pHo 7 suggests additional groups contribute. We hypothesized that Asp185, which faces position 116 in our closed-state model, contributes to Zn2+ chelation. Confirming this prediction, V116H/D185N abolished Zn2+ binding. Studied in a C-terminal truncated monomeric construct, V116H channels activated rapidly. Anomalously, Zn2+ slowed activation, producing a time constant independent of both voltage and Zn2+ concentration. We hypothesized that slow turn-on of H+ current in the presence of Zn2+ reflects the rate of Zn2+ unbinding from the channel, analogous to drug-receptor dissociation reactions. This behavior in turn suggests that the affinity for Zn2+ is greater in the closed state of hHV1. Supporting this hypothesis, pulse pairs revealed a rapid component of activation whose amplitude decreased after longer intervals at negative voltages as closed channels bound Zn2+. The lower affinity of Zn2+ in open channels is consistent with the idea that structural rearrangements within the transmembrane region bring Arg205 near position 116, electrostatically expelling Zn2+. This phenomenon provides direct evidence that Asp185 opposes position 116 in closed channels and that Arg205 moves between them when the channel opens.

Atomic Mechanisms of Timothy Syndrome-Associated Mutations in Calcium Channel Cav1.2.

Timothy syndrome (TS) is a very rare multisystem disorder almost exclusively associated with mutations G402S and G406R in helix IS6 of Cav1.2. Recently, mutations R518C/H in helix IIS0 of the voltage sensing domain II (VSD-II) were described as a cause of cardiac-only TS. The three mutations are known to decelerate voltage-dependent inactivation (VDI). Here, we report a case of cardiac-only TS caused by mutation R518C. To explore possible impact of the three mutations on interdomain contacts, we modeled channel Cav1.2 using as templates Class Ia and Class II cryo-EM structures of presumably inactivated channel Cav1.1. In both models, R518 and several other residues in VSD-II donated H-bonds to the IS6-linked α1-interaction domain (AID). We further employed steered Monte Carlo energy minimizations to move helices S4–S5, S5, and S6 from the inactivated-state positions to those seen in the X-ray structures of the open and closed NavAb channel. In the open-state models, positions of AID and VSD-II were similar to those in Cav1.1. In the closed-state models, AID moved along the β subunit (Cavβ) toward the pore axis and shifted AID-bound VSD-II. In all the models R518 retained strong contacts with AID. Our calculations suggest that conformational changes in VSD-II upon its deactivation would shift AID along Cavβ toward the pore axis. The AID-linked IS6 would bend at flexible G402 and G406, facilitating the activation gate closure. Mutations R518C/H weakened the IIS0-AID contacts and would retard the AID shift. Mutations G406R and G402S stabilized the open state and would resist the pore closure. Several Cav1.2 mutations associated with long QT syndromes are consistent with this proposition. Our results provide a mechanistic rationale for the VDI deceleration caused by TS-associated mutations and suggest targets for further studies of calcium channelopathies.

Timothy Syndrome-Associated Mutations Affect State-Dependent Contacts in L-Type Calcium Channel

Timothy syndrome (TS) is a very rare disorder almost exclusively associated with mutations G402S and G406R in IS6 of Cav1.2. Recently, mutations R518C/H in IIS0 of the voltage sensing domain II (VSD-II) were described as a cause of cardiac-only Timothy syndrome. The three mutations decelerate the voltage-dependent inactivation (VDI). We describe a new case of cardiac-only Timothy syndrome caused by mutation R518C. We used Class Ia and Class II cryo-EM structures of presumably inactivated-state channel Cav1.1 to model Cav1.2 and explore possible impact of the three mutations on interdomain contacts. In both models, R518 and several other residues in VSD-II donated H-bonds to the IS6-linked α1-interaction domain (AID). We further employed steered Monte Carlo energy minimizations (MCM) to move helices S4-S5, S5 and S6 to positions seen in the X-ray structures of open and closed sodium channel NavAb. In the open-state models, positions of AID and VSD-II were similar to those in Cav1.1. In the closed-state models, AID moved along the β subunit (Cavβ) towards the pore axis and shifted AID-bound VSD-II. In all the models, R518 retained H-bonds with AID. Our models suggest that VSD-II rearrangement upon its activation would shift AID along Cavβ towards the pore axis. The AID-linked IS6 would bend at flexible G402 and G406, facilitating closure of the activation gate. Mutations R518C/H, which weakened the IIS0-AID contacts in our models, would retard the AID shift. Mutations G406R and G402S stabilized the open state and would resist the pore closure. Several Cav1.2 mutations associated with long QT syndromes are consistent with this scenario. Our results provide a mechanistic rationale for the VDI deceleration caused by TS-associated mutations. They also suggest targets for further studies of calcium channelopathies. Supported by grants to BSZ from RSF and NSERC.

A Lack of Significant Lipid Interactions in the Open State of MSCS Implies a Jack-In-The-Box Type Channel Gating Mechanism

Bacterial mechanosensitive channels are important for cell survival in changing osmotic environments. For the mechanosensitive channel of small conductance from Escherichia coli (Ec-MscS), seven residues have previously been shown to form important lipid contacts in the closed state of the channel. Based on open state crystal structures and closed state models, these residues interact with lipids in the closed state of the channel. Decreasing the hydrophobicity of these residues reduces bacterial survival upon osmotic downshock. Since the closed state appears to be stabilized by lipid interactions, we hypothesized that similar stabilizing lipid interactions could be identified in the open state. Using a computational model of open state Ec-MscS embedded in a lipid bilayer, eleven residues were determined to be lipid exposed with ten of these residues being unique to the open state of the channel. To identify the role of lipid interactions these residues were mutated to alanine and leucine to alter their interaction with the hydrophobic lipids. The effects of these mutations on channel function were assayed using osmotic downshock lysis assays, growth assays, and patch clamp electrophysiology. Mutations of the ten residues that are exposed to the lipid bilayer only in the open state of the channel effected a wildtype phenotype in all of these assays. The lack of phenotypic changes, for residues that interact with the lipid bilayer solely in the open state, suggests that these interactions are not critical for channel gating. This has led us to propose a Jack-In-The-Box model of gating for MscS, in which intrinsic lipid bilayer pressure holds the channel in the closed state. Upon relief of the intrinsic bilayer pressure by application of opposing extrinsic tension, MscS springs into the open state.

On the Location of Binding Sites of 2-Guanidinobenzimidazole in the Voltage-Gated Proton Channel

Voltage-gated proton channels (Hv1) play important roles in the respiratory burst, in pH regulation, in spermatozoa, in apoptosis, and in cancer metastasis. The ability to block them selectively is an important target for drug development and for biophysical studies of channel function. Recently, 2-guanidobenzimidazole (2-GBI), a selective and state-dependent blocker of Hv1 channels was identified. However, the exact location of the binding site for a blocker as well as its apparent state-dependence has yet to be established. Recently, we have investigated the structure of the closed and open states of the voltage-gated proton channel through a combination of modeling and experimental analysis. To understand the mode binding and blocking and to hopefully identify the binding pocket, an initial docking study was performed for all of the ligands reported by Hong et al (25) as having significant binding affinity with the open channel monomer.We tested whether our open- and closed-state models could explain the state-dependence of the binding of the guanidine analogue 2-guanidobenzimidazole (2-GBI) from the cytosolic side of the channel. A combination of free-energy simulations and molecular docking established the architecture of the cytosolic binding site for 2-GBI. The site is centered near F198, consistent with the experimental data allowing for interactions between the positively-charged guanidine moiety of 2-GBI and the negatively-charged residues E201, D222, and E219. The aromatic ring of 2-GBI is stabilized by residues in the proximity of F198. In the closed state model, E201, D222, and E219, are occupied by the guanidine moieties of R255 and R258 from S4, thereby preventing 2-GBI access to its binding site. Additional binding modes were investigated by a combination of MD and Free Energy simulations.

Molecular dynamics investigation of Cl− transport through the closed and open states of the 2α12β2γ2 GABAA receptor

The α1β2γ2 gamma-aminobutyric type A receptor (GABAAR) is one of the most widely expressed GABAAR subtypes in the mammalian brain. GABAARs belonging to the Cys-loop superfamily of ligand-gated ion channels have been identified as key targets for many clinical drugs, and the motions that govern the gating mechanism are still not well understood. In this study, an open-state GABAAR was constructed using the structure of the glutamate-gated chloride channel (GluCl), which has a high sequence identity to GABAAR. A closed-state model was constructed using the structure of the nicotinic acetylcholine receptor (nAChR). Molecular dynamics simulations of the open-state and closed-state GABAAR were performed. We calculated the electrostatic potential of the two conformations, the pore radius of the two ion channels and the root-mean-square fluctuation. We observed the presence of two positively charged girdles around the ion channel and found flexible regions in the GABAAR. Then, the free-energy of chloride ion permeations through the closed-state and open-state GABAAR has been estimated using adaptive biasing force (ABF) simulation. For the closed-state GABAAR, we observed two major energy barriers for chloride ion translocation in the transmembrane domain (TMD). For the open-state GABAAR, there was only one energy barrier formed by two Thr261 (α1), two Thr255 (β2) and one Thr271 (γ2). By using ABF simulation, the overall free-energy profile is obtained for Cl− transporting through GABAAR, which gives a complete map of the ion channel of Cl− permeation.

Modeling of Open, Closed, and Open-Inactivated States of the hERG1 Channel: Structural Mechanisms of the State-Dependent Drug Binding

The human ether-a-go-go related gene 1 (hERG1) K ion channel is a key element for the rapid component of the delayed rectified potassium current in cardiac myocytes. Since there are no crystal structures for hERG channels, creation and validation of its reliable atomistic models have been key targets in molecular cardiology for the past decade. In this study, we developed and vigorously validated models for open, closed, and open-inactivated states of hERG1 using a multistep protocol. The conserved elements were derived using multiple-template homology modeling utilizing available structures for Kv1.2, Kv1.2/2.1 chimera, and KcsA channels. Then missing elements were modeled with the ROSETTA De Novo protein-designing suite and further refined with all-atom molecular dynamics simulations. The final ensemble of models was evaluated for consistency to the reported experimental data from biochemical, biophysical, and electrophysiological studies. The closed state models were cross-validated against available experimental data on toxin footprinting with protein-protein docking using hERG state-selective toxin BeKm-1. Poisson-Boltzmann calculations were performed to determine gating charge and compare it to electrophysiological measurements. The validated structures offered us a unique chance to assess molecular mechanisms of state-dependent drug binding in three different states of the channel.

A Limited 4 Å Radial Displacement of the S4-S5 Linker Is Sufficient for Internal Gate Closing in Kv Channels

Voltage-gated ion channels are responsible for the generation of action potentials in our nervous system. Conformational rearrangements in their voltage sensor domains in response to changes of the membrane potential control pore opening and thus ion conduction. Crystal structures of the open channel in combination with a wealth of biophysical data and molecular dynamics simulations led to a consensus on the voltage sensor movement. However, the coupling between voltage sensor movement and pore opening, the electromechanical coupling, occurs at the cytosolic face of the channel, from where no structural information is available yet. In particular, the question how far the cytosolic pore gate has to close to prevent ion conduction remains controversial. In cells, spectroscopic methods are hindered because labeling of internal sites remains difficult, whereas liposomes or detergent solutions containing purified ion channels lack voltage control. Here, to overcome these problems, we controlled the state of the channel by varying the lipid environment. This way, we directly measured the position of the S4-S5 linker in both the open and the closed state of a prokaryotic Kv channel (KvAP) in a lipid environment using Lanthanide-based resonance energy transfer. We were able to reconstruct the movement of the covalent link between the voltage sensor and the pore domain and used this information as restraints for molecular dynamics simulations of the closed state structure. We found that a small decrease of the pore radius of about 3-4 Å is sufficient to prevent ion permeation through the pore.

Initial steps in the opening of a Shaker potassium channel

The structural model of a K(V) (K(+)-selective, voltage-gated) channel in the open state is known (Protein Data Bank ID code 2R9R). Each subunit of the channel has four negatively charged residues distributed in the transmembrane segments S1, S2, and S3 that bind to and facilitate the movement within the membrane of the positively charged, voltage-sensing residues of S4. When extrapolated to the closed state, the two outermost negatively charged residues are exposed to extracellular fluid and not bound to S4 residues, all of which have theoretically been driven inward by voltage. If this closed state model is correct, these residues are available to bind external cations. We examined the effects of La(3+) on voltage-gated Shaker K(+) channels. Addition of the trivalent cation La(3+) (50 μM) extracellularly markedly prolongs the lag that precedes channel opening and slows the subsequent rise of K(+) current (I(K)) at all voltages. Decay kinetics of I(K) at negative voltages are unaltered. Gating current (I(g)) recorded from a nonconducting mutant shows that La(3+) reduces the initial amplitude of I(g) nearly twofold. We postulate that, in the resting state, La(3+) binds to the unoccupied, outermost negative residues, hindering outward S4 motion, thus increasing the lag on activation and slowing the rise of I(K). In the activated state, La(3+) is displaced by outward movement of arginine residues in S4; La(3+), therefore, is not present to affect channel closing. The results give strong support to the closed state model of the K(V) channel and a clear explanation of the effect of multivalent cations on cellular excitability.

Development of Atomistic Models for Closed, Open and Open-Inactivated States of hERG1 Channel using Rosetta Protein Modeling Suite and Molecular Dynamics Simulations

The human ether-a-go-go related gene 1 (hERG1) K ion channel is a key element for the rapid component of the delayed rectified potassium current in cardiac myocytes. It is essential for the normal repolarization phase of the cardiac action potential. Loss of function mutations in hERG1 cause increased duration of ventricular repolarization which leads to prolongation of the time interval between Q and T waves of the body surface electrocardiogram (long QT syndrome-LQTS). hERG1 K+ channel is a homotetramer composed of four identical homologous subunits each containing six transmembrane (TM) helices S1-S6. Single hERG channels are either closed, open or inactivated conformations depending on TM voltage. Although creation and validation of reliable 3D atomistic models of the hERG channel has been a key target in molecular cardiology for last decade, there are no reported S1-S6 TM homology models of the channel in different states yet in literature. In this study, we derived open, closed and open-inactivated states of hERG1 using ROSETTA protein modeling suite and side-chain placements are optimized by molecular dynamics simulations. Although backbone templates are modeled on available crystal structures of Kv1.2 and KcsA channels, the missing parts are modeled de novo. Final models are evaluated for consistency to the reported structural elements discovered mainly on the basis of mutagenesis and electrophysiology. Closed state models are further validated by protein-protein docking using selective peptide toxin and available experimental data on toxin foot-printing. Derived models of hERG1 in different states offer an indispensable template for rational drug design as well as better understanding of molecular mechanisms of hERG channel upon binding of openers or blockers.

A Specific Two-pore Domain Potassium Channel Blocker Defines the Structure of the TASK-1 Open Pore

Two-pore domain potassium (K2P) channels play a key role in setting the membrane potential of excitable cells. Despite their role as putative targets for drugs and general anesthetics, little is known about the structure and the drug binding site of K2P channels. We describe A1899 as a potent and highly selective blocker of the K2P channel TASK-1. As A1899 acts as an open-channel blocker and binds to residues forming the wall of the central cavity, the drug was used to further our understanding of the channel pore. Using alanine mutagenesis screens, we have identified residues in both pore loops, the M2 and M4 segments, and the halothane response element to form the drug binding site of TASK-1. Our experimental data were used to validate a K2P open-pore homology model of TASK-1, providing structural insights for future rational design of drugs targeting K2P channels.

Structure of HsdS Subunit from Thermoanaerobacter tengcongensis Sheds Lights on Mechanism of Dynamic Opening and Closing of Type I Methyltransferase

Type I DNA methyltransferases contain one specificity subunit (HsdS) and two modification subunits (HsdM). The electron microscopy model of M.EcoKI-M2S1 methyltransferase shows a reasonable closed state of this clamp-like enzyme, but the structure of the open state is still unclear. The 1.95 Å crystal structure of the specificity subunit from Thermoanaerobacter tengcongensis (TTE-HsdS) shows an unreported open form inter-domain orientation of this subunit. Based on the crystal structure of TTE-HsdS and the closed state model of M.EcoKI-M2S1, we constructed a potential open state model of type I methyltransferase. Mutational studies indicated that two α-helices (aa30-59 and aa466-495) of the TTE-HsdM subunit are important inter-subunit interaction sites in the TTE-M2S1 complex. DNA binding assays also highlighted the importance of the C-terminal region of TTE-HsdM for DNA binding by the TTE-M2S1 complex. On the basis of structural analysis, biochemical experiments and previous studies, we propose a dynamic opening and closing mechanism for type I methyltransferase.

PIP2-Binding to an Open State Model of Kir1.1 Probed by Multiscale Biomolecular Simulations

Phosphatidylinositol bisphosphate (PIP2) is an activator of mammalian inwardly rectifying potassium (Kir) channels. We have used multiscale simulations, via a sequential combination of coarse-grained and atomistic molecular dynamics to explore the interactions of PIP2 molecules within the inner leaflet of a lipid bilayer membrane with possible binding sites on both open and closed state models of the Kir1.1 (ROMK) channel. Coarse-grained simulations of the channels in PIP2-containing lipid bilayers identified the PIP2-binding site on each channel. These models of the PIP2-channel complexes were refined by conversion to an atomistic representation followed by molecular dynamics simulation in a lipid bilayer. The binding site in the closed state agrees with previous mutagenesis data of Kir1.1 as well as with previous modeling studies of related Kir channels. Intriguingly, analysis of the open state model reveals a differential interaction of PIP2 with key residues thought to be involved in PIP2 activation of the channel. These models will serve as a framework for the functional validation of PIP2 interactions with Kir1.1 and provide a fresh insight into how PIP2 stabilizes the open state of the Kir channel.

Prediction and Verification of Critical Tension Sensing Residues in the E. coli Mechanosensitive Channel of Small Conductance (EC-MSCS)

We have previously used a combination of random mutagenesis and molecular dynamics simulations to identify and understand critical residues for tension sensation in MscL (mechanosensitive channel of large conductance) [Maurer et al JBC 2003, 278, 21076-21082; Elmore et. al Biophysical J., 2003, 85, 1512-1524]. In MscL, we were able to demonstrate a strong correlation between lipid-protein interaction energy, determined using a molecular dynamics simulation of the closed state structure, and mutant channel function. Upon mutation of amino acid residues in transmembrane domains that exhibited significant lipid interaction over the course of the simulation displayed were more likely than other transmembrane residues to display either a loss of function or a gain of function phenotype in bacterial assays. Here, we have employed a similar computational analysis to identify potentially critical lipid-binding residues for tension sensation in MscS. Molecular dynamics simulations of a closed state model of MscS were carried out in the presence of an explicit lipid membrane. Energetic analysis of lipid-protein interactions in the first transmembrane domain (TM1) and the second transmembrane domain (TM2) indicated ten residues (TM1: L35, I39, L42, I43, R46, N50, R54; and TM2: R74, L78, I82) with increased lipid interactions. Using site directed mutagenesis these residues were mutated to alanine and their ability to rescue MscL/MscS/MscK null E. coli from osmotic downshock was determined. Generally, mutating lipid interacting residues to alanine resulted in MscS channels that exhibited decreased ability to rescue bacteria from osmotic downshock, following a similar trend to our previous studies of MscL.

Identification of a protein–protein interaction between KCNE1 and the activation gate machinery of KCNQ1

KCNQ1 channels assemble with KCNE1 transmembrane (TM) peptides to form voltage-gated K+ channel complexes with slow activation gate opening. The cytoplasmic C-terminal domain that abuts the KCNE1 TM segment has been implicated in regulating KCNQ1 gating, yet its interaction with KCNQ1 has not been described. Here, we identified a protein–protein interaction between the KCNE1 C-terminal domain and the KCNQ1 S6 activation gate and S4–S5 linker. Using cysteine cross-linking, we biochemically screened over 300 cysteine pairs in the KCNQ1–KCNE1 complex and identified three residues in KCNQ1 (H363C, P369C, and I257C) that formed disulfide bonds with cysteine residues in the KCNE1 C-terminal domain. Statistical analysis of cross-link efficiency showed that H363C preferentially reacted with KCNE1 residues H73C, S74C, and D76C, whereas P369C showed preference for only D76C. Electrophysiological investigation of the mutant K+ channel complexes revealed that the KCNQ1 residue, H363C, formed cross-links not only with KCNE1 subunits, but also with neighboring KCNQ1 subunits in the complex. Cross-link formation involving the H363C residue was state dependent, primarily occurring when the KCNQ1–KCNE1 complex was closed. Based on these biochemical and electrophysiological data, we generated a closed-state model of the KCNQ1–KCNE1 cytoplasmic region where these protein–protein interactions are poised to slow activation gate opening.

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.

Molecular Dynamics Simulation of the Kv1.2 Potassium Channel

Voltage-gated ion channels are membrane proteins that open and close in response to voltage changes across the membrane. Voltage-gated potassium (Kv) channels are composed of two structurally and functionally separate domains, a voltage-sensing domain (VSD) and the ion conducting pore (PD). The voltage-sensing domain is comprised of four transmembrane segments that are highly charged and move in response to changes in the electric potential resulting in opening of the channel. We have developed complete models of the Kv1.2 channel in the open and closed state using the homology, de novo, and domain assembly methods of the structure prediction program ROSETTA. Molecular dynamics (MD) simulations were carried out to refine selected candidates of the open and closed state models of Kv1.2 in an explicit water-membrane environment. Each model was simulated for 100ns in the presence of a positive or negative voltage using the program NAMD. The magnitude of the gating charge that is transferred across the membrane upon opening of the channel is calculated based on 500ns of MD simulations of the full-channel and the isolated VSDs. In addition FEP simulations were performed to calculate the free energy of neutralizing (transmembrane) charged residues on the voltage-sensing helices, and to determine the fraction of the transmembrane voltage acting on each residue. The contribution of several charged residues (on the S4 and S2 segments) to the gating charge was then obtained by calculating the “electric” displacement of each amino acid side chain in the external field. The results show that the transmembrane electric field is focused inside the voltage sensor domains and that the gating charge arises mainly from the three positively charged residues located on the S4 segment.

Direct Binding Of Divalent Cations To The S4 Region In Eag Family K+ Channels Reveals A Closed Conformation Shared With Kv1 Channels

Voltage-gated K+ channels share a common voltage sensor domain (VSD) consisting of four transmembrane helices, including a highly mobile S4 domain. Nevertheless, it is an open question as to whether functionally diverse voltage-gated K+ channels, such as Shaker channels and the sub-threshold ether-a-go-go (EAG) family channels, will also share common conformational changes during voltage gating. EAG channels have a unique divalent binding site formed by two family-specific acidic charges in S2 and S3. However, the conformational similarity between this divalent-bound state and models of Shaker closed states has been difficult to assess in the absence of constraints for S4 position. We show here that divalent cations slow the activation rate of two EAG family channels (Kv12.1 and Kv10.2) by directly binding to the S4 domain and locking it to those acidic charges on neighboring helices. Histidine 328 in the S4 of Kv12.1 favors binding of Zn2+ and Cd2+, while the homologous residue Serine 321 in Kv10.2 contributes to high affinity binding of Mg2+ and Ni2+. This novel finding provides definitive constraints for the positions of S2, S3 and S4 in a deep closed state. Homology models of Kv12.1 and Kv10.2 VSD structures based on a closed state model of the Shaker family delayed rectifier K+ channel Kv1.2 match divalent binding constraints precisely. Thus our results suggest remarkable conformational conservation between EAG and Shaker family channels early in the activation pathway, despite large differences in voltage sensitivity, activation rates and activation thresholds.

Molecular Dynamic Simulation of the Kv1.2 Voltage-Gated Potassium Channel in Open and Closed State Conformations

We performed 45 ns atomistic force field based molecular dynamics (MD) simulations to explore the structure and dynamics of the Kvl.2 voltage-dependent potassium ion channel in both open and closed state conformations. The Kv1.2 crystal structure (PDB 2A79) based open state model and homologically derived closed state model are embedded in a hydrated lipid membrane. We present a detailed analysis of the protein stability, the environment of the gating-charge-carrying residues, the salt bridge within the voltage sensor, S4 segment helix movement, helix interaction between S4-S5 linker and S6, interaction between subunits, and interaction between protein and lipid membrane. The four subunits of the channel lost the symmetry from the starting structure during the simulation, especially the voltage sensors. According to our comparative analysis of the open and closed state conformations, S4 in our simulation behaves more near the screw helix model except for the tilt action. The S4 segment azimuthal rotation angle difference between two conformations shifts about 60 degrees after being embedded in a membrane environment. The S4-S5 linker helix remains stable. The contact area between the S4 and S5 from the adjacent subunit increases after transition to the closed state from the open state. The current investigation provided valuable information to our understanding of the structure and dynamics of membrane-associated helices and the gating mechanism of the voltage-dependent potassium ion channel of the Kv channel.