The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics. However, the structural basis of the dynamic process from the closed to open states in MscL is not fully understood. In this study, we performed molecular dynamics (MD) simulations on the initial process of MscL opening in response to a tension increase in the lipid bilayer. To identify the tension-sensing site(s) in the channel protein, we calculated interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids. We found that Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL. Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78, and the force was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices in the inner leaflet of the bilayer. The radial dragging force induced radial sliding of the crossing portions, leading to a gate expansion. Calculated energy for this expansion is comparable to an experimentally estimated energy difference between the closed and the first subconductance state, suggesting that our model simulates the initial step toward the full opening of MscL. The model also successfully mimicked the behaviors of a gain of function mutant (G22N) and a loss of function mutant (F78N), strongly supporting that our MD model did simulate some essential biophysical aspects of the mechano-gating in MscL.     

From membrane tension to channel gating: A principal energy transfer mechanism for mechanosensitive channels

Mechanosensitive (MS) channels are evolutionarily conserved membrane proteins that play essential roles in multiple cellular processes, including sensing mechanical forces and regulating osmotic pressure. Bacterial MscL and MscS are two prototypes of MS channels. Numerous structural studies, in combination with biochemical and cellular data, provide valuable insights into the mechanism of energy transfer from membrane tension to gating of the channel. We discuss these data in a unified two‐state model of thermodynamics. In addition, we propose a lipid diffusion‐mediated mechanism to explain the adaptation phenomenon of MscS.     

The dynamics of protein-protein interactions between domains of MscL at the cytoplasmic-lipid interface

The bacterial mechanosensitive channel of large conductance, MscL, is one of the best characterized mechanosensitive channels serving as a paradigm for how proteins can sense and transduce mechanical forces. The physiological role of MscL is that of an emergency release valve that opens a large pore upon a sudden drop in the osmolarity of the environment. A crystal structure of a closed state of MscL shows it as a homopentamer, with each subunit consisting of two transmembrane domains (TM). There is consensus that the TM helices move in an iris like manner tilting in the plane of the membrane while gating. An N-terminal amphipathic helix that lies along the cytoplasmic membrane (S1), and the portion of TM2 near the cytoplasmic interface (TM2_ci), are relatively close in the crystal structure, yet predicted to be dynamic upon gating. Here we determine how these two regions interact in the channel complex, and study how these interactions change as the channel opens. We have screened 143 double-cysteine mutants of E. coli MscL for their efficiency in disulfide bridging and generated a map of protein-protein interactions between these two regions. Interesting candidates have been further studied by patch clamp and show differences in channel activity under different redox potentials; the results suggest a model for the dynamics of these two domains during MscL gating.     

The dynamics of protein-protein interactions between domains of MscL at the cytoplasmic-lipid interface

The bacterial mechanosensitive channel of large conductance, MscL, is one of the best characterized mechanosensitive channels serving as a paradigm for how proteins can sense and transduce mechanical forces. The physiological role of MscL is that of an emergency release valve that opens a large pore upon a sudden drop in the osmolarity of the environment. A crystal structure of a closed state of MscL shows it as a homopentamer, with each subunit consisting of two transmembrane domains (TM). There is consensus that the TM helices move in an iris like manner tilting in the plane of the membrane while gating. An N-terminal amphipathic helix that lies along the cytoplasmic membrane (S1), and the portion of TM2 near the cytoplasmic interface (TM2_ci), are relatively close in the crystal structure, yet predicted to be dynamic upon gating. Here we determine how these two regions interact in the channel complex, and study how these interactions change as the channel opens. We have screened 143 double-cysteine mutants of E. coli MscL for their efficiency in disulfide bridging and generated a map of protein-protein interactions between these two regions. Interesting candidates have been further studied by patch clamp and show differences in channel activity under different redox potentials; the results suggest a model for the dynamics of these two domains during MscL gating.     

Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL

MscL is multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by tension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified MscL reconstituted in liposomes, we recorded single channel currents and varied the pressure gradient (P) to vary the tension (T). The tension was calculated from P and the radius of curvature was obtained using video microscopy of the patch. The probability of being open (Po) has a steep sigmoidal dependence on T, with a midpoint (T1/2) of 11.8 dyn/cm. The maximal slope sensitivity of Po/Pc was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distribution, the energy difference between the closed and fully open states in the unstressed membrane was DeltaE = 18.6 kBT. If the mechanosensitivity arises from tension acting on a change of in-plane area (DeltaA), the free energy, TDeltaA, would correspond to DeltaA = 6.5 nm2. MscL is not a binary channel, but has four conducting states and a closed state. Most transition rates are independent of tension, but the rate-limiting step to opening is the transition between the closed state and the lowest conductance substate. This transition thus involves the greatest DeltaA. When summed over all transitions, the in-plane area change from closed to fully open was 6 nm2, agreeing with the value obtained in the two-state analysis. Assuming a cylindrical channel, the dimensions of the (fully open) pore were comparable to DeltaA. Thus, the tension dependence of channel gating is primarily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to involve conformational changes internal to the channel that don't involve changes in area.     

On the conformation of the COOH-terminal domain of the large mechanosensitive channel MscL

COOH-terminal (S3) domains are conserved within the MscL family of bacterial mechanosensitive channels, but their function remains unclear. The X-ray structure of MscL from Mycobacterium tuberculosis (TbMscL) revealed cytoplasmic domains forming a pentameric bundle (Chang, G., R.H. Spencer, A.T. Lee, M.T. Barclay, and D.C. Rees. 1998. SCIENCE: 282:2220-2226). The helices, however, have an unusual orientation in which hydrophobic sidechains face outside while charged residues face inside, possibly due to specific crystallization conditions. Based on the structure of pentameric cartilage protein, we modeled the COOH-terminal region of E. coli MscL to better satisfy the hydrophobicity criteria, with sidechains of conserved aliphatic residues all inside the bundle. Molecular dynamic simulations predicted higher stability for this conformation compared with one modeled after the crystal structure of TbMscL, and suggested distances for disulfide trapping experiments. The single cysteine mutants L121C and I125C formed dimers under ambient conditions and more so in the presence of an oxidant. The double-cysteine mutants, L121C/L122C and L128C/L129C, often cross-link into tetrameric and pentameric structures, consistent with the new model. Patch-clamp examination of these double mutants under moderately oxidizing or reducing conditions indicated that the bundle cross-linking neither prevents the channel from opening nor changes thermodynamic parameters of gating. Destabilization of the bundle by replacing conservative leucines with small polar residues, or complete removal of COOH-terminal domain (Delta110-136 mutation), increased the occupancy of subconducting states but did not change gating parameters substantially. The Delta110-136 truncation mutant was functional in in vivo osmotic shock assays; however, the amount of ATP released into the shock medium was considerably larger than in controls. The data strongly suggest that in contrast to previous gating models (Sukharev, S., M. Betanzos, C.S. Chiang, and H.R. Guy. 2001a. NATURE: 409:720-724.), S3 domains are stably associated in both closed and open conformations. The bundle-like assembly of cytoplasmic helices provides stability to the open conformation, and may function as a size-exclusion filter at the cytoplasmic entrance to the MscL pore, preventing loss of essential metabolites.     

Toward elucidating the heat activation mechanism of the TRPV1 channel gating by molecular dynamics simulation

As a key cellular sensor, the TRPV1 cation channel undergoes a gating transition from a closed state to an open state in response to various physical and chemical stimuli including noxious heat. Despite years of study, the heat activation mechanism of TRPV1 gating remains enigmatic at the molecular level. Toward elucidating the structural and energetic basis of TRPV1 gating, we have performed molecular dynamics (MD) simulations (with cumulative simulation time of 3 μs), starting from the high‐resolution closed and open structures of TRPV1 solved by cryo‐electron microscopy. In the closed‐state simulations at 30°C, we observed a stably closed channel constricted at the lower gate (near residue I679), while the upper gate (near residues G643 and M644) is dynamic and undergoes flickery opening/closing. In the open‐state simulations at 60°C, we found higher conformational variation consistent with a large entropy increase required for the heat activation, and both the lower and upper gates are dynamic with transient opening/closing. Through ensemble‐based structural analyses of the closed state versus the open state, we revealed pronounced closed‐to‐open conformational changes involving the membrane proximal domain (MPD) linker, the outer pore, and the TRP helix, which are accompanied by breaking/forming of a network of closed/open‐state specific hydrogen bonds. By comparing the closed‐state simulations at 30°C and 60°C, we observed heat‐activated conformational changes in the MPD linker, the outer pore, and the TRP helix that resemble the closed‐to‐open conformational changes, along with partial formation of the open‐state specific hydrogen bonds. Some of the residues involved in the above key hydrogen bonds were validated by previous mutational studies. Taken together, our MD simulations have offered rich structural and dynamic details beyond the static structures of TRPV1, and promising targets for future mutagenesis and functional studies of the TRPV1 channel. Proteins 2016; 84:1938–1949. © 2016 Wiley Periodicals, Inc.     

Conversion of a mechanosensitive channel protein from a membrane-embedded to a water-soluble form by covalent modification with amphiphiles

Covalent modification of integral membrane proteins with amphiphiles may provide a general approach to the conversion of membrane proteins into water-soluble forms for biophysical and high-resolution structural studies. To test this approach, we mutated four surface residues of the pentameric Mycobacterium tuberculosis mechanosensitive channel of large conductance (MscL) to cysteine residues as anchors for amphiphile attachment. A series of modified ion channels with four amphiphile groups attached per channel subunit was prepared. One construct showed the highest water solubility to a concentration of up to 4mg/ml in the absence of detergent. This analog also formed native-like, alpha-helical homo-pentamers in the absence of detergent as judged by circular dichroism spectroscopy, size-exclusion chromatography and various light-scattering techniques. Proteins with longer, or shorter polymers attached, or proteins modified exclusively with polar cysteine-reactive small molecules, exhibited reduced to no solubility and higher-order aggregation. Electron microscopy revealed a homogeneous population of particles consistent with a pentameric channel. Solubilization of membrane proteins by covalent attachment of amphiphiles results in homogeneous particles that may prove useful for crystallization, solution NMR spectroscopy, and electron microscopy.     

Fundamental gating mechanism of nicotinic receptor channel revealed by mutation causing a congenital myasthenic syndrome

We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation epsilonA411P in the amphipathic helix of the acetylcholine receptor (AChR) epsilon subunit. Myasthenic patients from three unrelated families are either homozygous for epsilonA411P or are heterozygous and harbor a null mutation in the second epsilon allele, indicating that epsilonA411P is recessive. We expressed human AChRs containing wild-type or A411P epsilon subunits in 293HEK cells, recorded single channel currents at high bandwidth, and determined microscopic rate constants for individual channels using hidden Markov modeling. For individual wild-type and mutant channels, each rate constant distributes as a Gaussian function, but the spread in the distributions for channel opening and closing rate constants is greatly expanded by epsilonA411P. Prolines engineered into positions flanking residue 411 of the epsilon subunit greatly increase the range of activation kinetics similar to epsilonA411P, whereas prolines engineered into positions equivalent to epsilonA411 in beta and delta subunits are without effect. Thus, the amphipathic helix of the epsilon subunit stabilizes the channel, minimizing the number and range of kinetic modes accessible to individual AChRs. The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes. The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well.     

Essential function of the N‐termini tails of the proteasome for the gating mechanism revealed by molecular dynamics simulations

Proteasome is involved in the degradation of proteins. Proteasome activators bind to the proteasome core particle (CP) and facilitate opening a gate of the CP, where Tyr8 and Asp9 in the N‐termini tails of the CP form the ordered open gate. In a double mutant (Tyr8Gly/Asp9Gly), the N‐termini tails are disordered and the stabilized open‐gate conformation cannot be formed. To understand the gating mechanism of the CP for the translocation of the substrate, four different molecular dynamics simulations were carried out: ordered‐ and Tyr8Gly/Asp9Gly disordered‐gate models of the CP complexed with an ATP‐independent PA26 and ordered‐ and disordered‐gate models of the CP complexed with an ATP‐dependent PAN‐like activator. The free‐energies of the translocation of a polypeptide substrate moving through the gate were estimated. In the ordered‐gate models, the substrate in the activator was more stable than that in the CP. The conformational entropy of the N‐termini tails of the CP was larger when the substrate was in the activator than in the CP. In the disordered‐gate models, the substrate in the activator was more destabilized than in the ordered‐gate models. The mutated N‐termini tails became randomized and their increased conformational entropy could no longer increase further even when the substrate was in the activator, meaning the randomized N‐termini tails had lost the ability to stabilize the substrate in the activator. Thus, it was concluded that the dynamics of the N‐termini tails entropically play a key role in the translocation of the substrate. Proteins 2014; 82:1985–1999. © 2014 Wiley Periodicals, Inc.     

Computer simulation of the KvAP voltage-gated potassium channel: steered molecular dynamics of the voltage sensor

The recent crystal structures of the voltage-gated potassium channel KvAP and its isolated voltage-sensing 'paddle' (composed of segments S1-S4) challenge existing models of voltage gating and raise a number of questions about the structure of the physiologically relevant state. We investigate a possible gating mechanism based on the crystal structures in a 10 ns steered molecular dynamics simulation of KvAP in a membrane-mimetic octane layer. The structure of the full KvAP protein has been modified by restraining the S2-S4 domain to the conformation of the isolated high-resolution paddle structure. After an initial relaxation, the paddle tips are pulled through the membrane from the intracellular to the extracellular side, corresponding to a putative change from closed to open. We describe the effect of this large-scale motion on the central pore domain, which remains largely unchanged, on the protein hydrogen-bonding network and on solvent. We analyze the motion of the S3b-S4 portion of the protein and propose a possible coupling mechanism between the paddle motion and the opening of the channel. Interactions between the arginine residues in S4, solvent and chloride ions are likely to play a role in the gating charge.     

Homology modeling and molecular dynamics simulations of the alpha1 glycine receptor reveals different states of the channel

Homology modeling is used to build initial models of the transmembrane domain of the human alpha1 glycine receptor (GlyR) based on the most recently published refined structure of nAChR (PDB ID: 2BG9). Six preliminary GlyR models are constructed using two different approaches. In one approach, five different homopentamers are built by symmetric assembly of alpha1 GlyR subunits using only one of the five unique chains of nAChR as a template. In a second approach, each nAChR subunit serves as a template for an alpha1 GlyR subunit. All six initial GlyR constructs are then embedded into a hydrated POPC lipid bilayer and subjected to molecular dynamics simulation for at least six nanoseconds. Each model is stable throughout the simulation, and the final models fall into three distinct categories. Homopentameric GlyR bundles using a single alpha nAChR subunit as a template appear to be in an open conformation. Under an applied external potential, permeation of Cl(-) ions is observed within several ns in a channel built on an alpha chain. Model channels built on non-alpha chains have a constriction either near the intracellular mouth or more centrally located in the pore domain, both of which may be narrow enough to close the channel and whose locations correspond to putative gates observed in nicotinicoid receptors. The differences between these three general models suggest that channel closure may be effected by either rotation or tangential tilting of TM2.     

Mouse acetylcholinesterase unliganded and in complex with huperzine A: A comparison of molecular dynamics simulations

A 1 ns molecular dynamics simulation of unliganded mouse acetylcholinesterase (AChE) is compared to a previous simulation of mouse AChE complexed with huperzine A (HupA). Several common features are observed. In both simulations, the active site gorge fluctuates in size during the 1 ns trajectory and is completely pinched off several times. Many of the residues in the gorge that formed hydrogen bonds with HupA in the simulation of the complex now form hydrogen bonds with other protein residues and water molecules in the gorge. The opening of a “backdoor” entrance to the active site that was found in the simulation of the complex is also observed in the unliganded simulation. Differences between the two simulations include overall lower structural rms deviations for residues in the gorge in the unliganded simulation, a smaller diameter of the gorge in the absence of HupA, and the disappearance of a side channel that was frequently present in the liganded simulation. The differences between the two simulations can be attributed, in part, to the interaction of AChE with HupA. © 1999 John Wiley & Sons, Inc. Biopoly 50: 35–43, 1999     

Driven to near-experimental accuracy by refinement via molecular dynamics simulations

Protein model refinement has been an essential part of successful protein structure prediction. Molecular dynamics simulation-based refinement methods have shown consistent improvement of protein models. There had been progress in the extent of refinement for a few years since the idea of ensemble averaging of sampled conformations emerged. There was little progress in CASP12 because conformational sampling was not sufficiently diverse due to harmonic restraints. During CASP13, a new refinement method was tested that achieved significant improvements over CASP12. The new method intended to address previous bottlenecks in the refinement problem by introducing new features. Flat-bottom harmonic restraints replaced harmonic restraints, sampling was performed iteratively, and a new scoring function and selection criteria were used. The new protocol expanded conformational sampling at reduced computational costs. In addition to overall improvements, some models were refined significantly to near-experimental accuracy.     

Insight into the key features for ligand binding in Y1230 mutated c-Met kinase domain by molecular dynamics simulations

c-Met kinase has been considered as an attractive target for developing antitumor agents. The strong interactions between Tyr1230 and the inhibitors emphasized its importance for ligand binding. The clinically related Tyr1230 mutations have made negative impacts on current c-Met kinase inhibitors, especially the exquisitely selective ones, like PF-04217903, while the multi-targeted inhibitors, like Crizotinib, were not affected so much. In this study, the protein–ligand interactions between c-Met kinase domain (wild, Y1230C and Y1230H) and these inhibitors were compared. The binding site was expanded and the post-mutated regions became solvent accessible. The heavy dependency of PF-04217903 on the interactions with Tyr1230 resulted in the steep decrease of its potency against the Y1230 mutants. It was found that the ligand entrance region contributed consistently to the binding of Crizotinib, but not PF-04217903. Additional groups substituted in the ligand entrance region with stable interactions should be beneficial for improving the inhibitory activity of PF-04217903 against the Y1230 mutants. These findings will facilitate the discovery of potent inhibitors against Y1230 mutated c-Met kinase.     

Study of the stability and unfolding mechanism of BBA1 by molecular dynamics simulations at different temperatures.

BBA1 is a designed protein that has only 23 residues. It is the smallest protein without disulfide bridges that has a well-defined tertiary structure in solution. We have performed unfolding molecular dynamics simulations on BBA1 and some of its mutants at 300, 330, 360, and 400 K to study their kinetic stability as well as the unfolding mechanism of BBA1. It was shown that the unfolding simulations can provide insights into the forces that stabilize the protein. Packing, hydrophobic interactions, and a salt bridge between Asp12 and Lys16 were found to be important to the protein's stability. The unfolding of BBA1 goes through two major steps: (1) disruption of the hydrophobic core and (2) unfolding of the helix. The beta-hairpin remains stable in the unfolding because of the high stability of the type II' turn connecting the two beta-strands.     

Molecular dynamics simulations investigate the mechanism of Psalmotoxin 1 regulating gating process of an acid-sensing ion channel 1a at pH 5.5

Acid-sensing ion channel 1a (ASIC1a) is a cation channel activated by protons and causes neuronal death through central nervous system. Psalmotoxin1 (PcTx1) is a gating modifier for ASIC1a. The process of PcTx1 regulating the channel gating from the extracellular domain to the transmembrane domain is unclear. Here we used molecular dynamics (MD) simulations method to investigate how PcTx1 regulates the gating of the ASIC1a. Our results indicated that PcTx1can mainly regulate ASIC1a gating process through hydrogen bonds, which can affect their relative positions of several key domains in ASIC1a, further, a long-range conformational changes path was determined, which is composed of β1, β2, β10, α6, α7, β11, and β12 in ASIC1a.     

Multiple conformational states and gate opening of outer membrane protein TolC revealed by molecular dynamics simulations

Outer membrane protein TolC serves as an exit duct for exporting substances out of cell. The occluded periplasmic entrance of TolC is required to open for substrate transport, although the opening mechanism remains elusive. In this study, systematic molecular dynamics (MD) simulations for wild type TolC and six mutants were performed to explore the conformational dynamics of TolC. The periplasmic gate was shown to sample multiple conformational states with various degrees of gating opening. The gate opening was facilitated by all mutations except Y362F, which adopts an even more closed state than wild type TolC. The interprotomer salt‐bridge R367–D153 is turned out to be crucial for periplasmic gate opening. The mutations that disrupt the interactions at the periplasmic tip may affect the stability of the trimeric assembly of TolC. Structural asymmetry of the periplasmic gate was observed to be opening size dependent. Asymmetric conformations are found in moderately opening states, while the most and the least opening states are often more symmetric. Finally, it is shown that lowering pH can remarkably stabilize the closed state of the periplasmic gate. Proteins 2014; 82:2169–2179. © 2014 Wiley Periodicals, Inc.     

The mechanism of high affinity pentasaccharide binding to antithrombin,insights from Gaussian accelerated molecular dynamics simulations

Abstract

The activity of antithrombin (AT), a serpin protease inhibitor, is enhanced by heparin and heparin analogs against its target proteases, mainly thrombin, factors Xa and IXa. Considerable amount of information is available on the multistep mechanism of the heparin pentasaccharide binding and conformational activation. However, much of the details were inferred from ‘static’ structures obtained by X-ray diffraction. Moreover, limited information is available for the early steps of binding mechanism other than kinetic studies with various ligands. To gain insights into these processes, we performed enhanced sampling molecular dynamics (MD) simulations using the Gaussian Accelerated Molecular Dynamics (GAMD) method, applied previously in drug binding studies. We were able to observe the binding of the pentasaccharide idraparinux to a ‘non-activated’ AT conformation in two separate trajectories with low root mean square deviation (RMSD) values compared to X-ray structures of the bound state. These trajectories along with further simulations of the AT-pentasaccharide complex provided insights into the mechanisms of multiple conformational transitions, including the expulsion of the hinge region, the extension of helix D and the conformational behavior of the reactive center loop (RCL). We could also confirm the high stability of helix P in non-activated AT conformations, such states might play an important role in heparin binding. ‘Generalized correlation’ matrices revealed possible paths of allosteric signal propagation to the binding sites for the target proteases, factors Xa and IXa. Enhanced MD simulations of ligand binding to AT may assist the design of new anticoagulant drugs.

Communicated by Ramaswamy H. Sarma     

Gating of the bacterial sodium channel, NaChBac: voltage-dependent charge movement and gating currents

The bacterial sodium channel, NaChBac, from Bacillus halodurans provides an excellent model to study structure-function relationships of voltage-gated ion channels. It can be expressed in mammalian cells for functional studies as well as in bacterial cultures as starting material for protein purification for fine biochemical and biophysical studies. Macroscopic functional properties of NaChBac have been described previously (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001. Science. 294:2372-2375). In this study, we report gating current properties of NaChBac expressed in COS-1 cells. Upon depolarization of the membrane, gating currents appeared as upward inflections preceding the ionic currents. Gating currents were detectable at -90 mV while holding at -150 mV. Charge-voltage (Q-V) curves showed sigmoidal dependence on voltage with gating charge saturating at -10 mV. Charge movement was shifted by -22 mV relative to the conductance-voltage curve, indicating the presence of more than one closed state. Consistent with this was the Cole-Moore shift of 533 micros observed for a change in preconditioning voltage from -160 to -80 mV. The total gating charge was estimated to be 16 elementary charges per channel. Charge immobilization caused by prolonged depolarization was also observed; Q-V curves were shifted by approximately -60 mV to hyperpolarized potentials when cells were held at 0 mV. The kinetic properties of NaChBac were simulated by simultaneous fit of sodium currents at various voltages to a sequential kinetic model. Gating current kinetics predicted from ionic current experiments resembled the experimental data, indicating that gating currents are coupled to activation of NaChBac and confirming the assertion that this channel undergoes several transitions between closed states before channel opening. The results indicate that NaChBac has several closed states with voltage-dependent transitions between them realized by translocation of gating charge that causes activation of the channel.