G219S mutagenesis as a means of stabilizing conformational flexibility in the bacterial sodium channel NaChBac

The NaChBac sodium channel from Bacillus halodurans is a homologue of eukaryotic voltage-gated sodium channels. It can be solubilized in a range of detergents and consists of four identical subunits assembled as a tetramer. Sodium channels are relatively flexible molecules, adopting different conformations in their closed, open and inactivated states. This study aimed to design and construct a mutant version of the NaChBac protein that would insert into membranes and retain its folded conformation, but which would have enhanced stability when subjected to thermal stress. Modelling studies suggested a G219S mutant would have decreased conformational flexibility due to the removal of the glycine hinge around the proposed gating region, thereby imparting increased resistance to unfolding. The mutant expressed in Escherichia coli and purified in the detergent dodecyl maltoside was compared to wildtype NaChBac prepared in a similar manner. The mutant was incorporated into the membrane fraction and had a nearly identical secondary structure to the wildtype protein. When the thermal unfolding of the G219S mutant was examined by circular dichroism spectroscopy, it was shown to not only have a Tm ～10°C higher than the wildtype, but also in its unfolded state it retained more ordered helical structure than did the wildtype protein. Hence the G219S mutant was shown to be, as designed, more thermally stable.     

A gating hinge in Na+ channels; a molecular switch for electrical signaling

Voltage-gated sodium channels are members of a large family with similar pore structures. The mechanism of opening and closing is unknown, but structural studies suggest gating via bending of the inner pore helix at a glycine hinge. Here we provide functional evidence for this gating model for the bacterial sodium channel NaChBac. Mutation of glycine 219 to proline, which would strongly favor bending of the alpha helix, greatly enhances activation by shifting its voltage dependence -51 mV and slowing deactivation by 2000-fold. The mutation also slows voltage-dependent inactivation by 1200-fold. The effects are specific because substitutions of proline at neighboring positions and substitutions of other amino acids at position 219 have much smaller functional effects. Our results fit a model in which concerted bending at glycine 219 in the S6 segments of NaChBac serves as a gating hinge. This gating motion may be conserved in most members of this large ion channel protein family.     

Tetrameric bacterial sodium channels: characterization of structure, stability, and drug binding

NaChBac from Bacillus halodurans is a bacterial homologue of mammalian voltage-gated sodium channels. It has been proposed that a NaChBac monomer corresponds to a single domain of the mammalian sodium channel and that, like potassium channels, four monomers form a tetrameric channel. However, to date, although NaChBac has been well-characterized for functional properties by electrophysiological measurements on protein expressed in tissue culture, little information about its structural properties exists because of the difficulties in expressing the protein in large quantities. In this study, we present studies on the overexpression of NaChBac in Escherichia coli, purification of the functional detergent-solubilized channel, its identification as a tetramer, and characterization of its secondary structure, drug binding, and thermal stability. These studies are correlated with a model produced for the protein and provide new insights into the structure-function relationships of this sodium channel.     

The C-terminal coiled-coil of the bacterial voltage-gated sodium channel NaChBac is not essential for tetramer formation, but stabilizes subunit-to-subunit interactions

The NaChBac is a prokaryotic homologue of the voltage-gated sodium channel found in the genome of the alkalophilic bacterium Bacillus halodurans C-125. Like a repeating cassette of mammalian sodium channel, the NaChBac possesses hydrophobic domains corresponding to six putative transmembrane segments and a pore loop, and exerts channel function by forming a tetramer although detailed mechanisms of subunit assembly remain unclear. We generated truncated mutants from NaChBac, and investigated their ability to form tetramers in relation to their channel functions. A mutant that deletes almost all of the C-terminal coiled-coil structure lost its voltage-dependent ion permeability, although it was properly translocated to the cell surface. The mutant protein was purified as a tetramer using a reduced concentration of detergent, but the association between the subunits was shown to be much weaker than the wild type. The chemical cross-linking, blue native PAGE, sedimentation velocity experiments, size exclusion chromatography, immunoprecipitation, and electron microscopy all supported its tetrameric assembly. We further purified the C-terminal cytoplasmic domain alone and confirmed its self-oligomerization. These data suggest that the C-terminal coiled-coil structure stabilizes subunit-to-subunit interactions of NaChBac, but is not critical for their tetramer formation.     

Models of the structure and gating mechanisms of the pore domain of the NaChBac ion channel

The NaChBac prokaryotic sodium channel appears to be a descendent of an evolutionary link between voltage-gated K_V and Ca_V channels. Like K_V channels, four identical six-transmembrane subunits comprise the NaChBac channel, but its selectivity filter possesses a signature sequence of eukaryotic Ca_V channels. We developed structural models of the NaChBac channel in closed and open conformations, using K⁺-channel crystal structures as initial templates. Our models were also consistent with numerous experimental results and modeling criteria. This study concerns the pore domain. The major differences between our models and K⁺ crystal structures involve the latter portion of the selectivity filter and the bend region in S6 of the open conformation. These NaChBac models may serve as a stepping stone between K⁺ channels of known structure and Na_V, Ca_V, and TRP channels of unknown structure.     

Transmembrane and extramembrane contributions to membrane protein thermal stability: studies with the NaChBac sodium channel

The thermal stabilities of the extramembranous and transmembranous regions of the bacterial voltage-gated sodium channel NaChBac have been characterised using thermal-melt synchrotron radiation circular dichroism (SRCD) spectroscopy. A series of constructs, ranging from the full-length protein containing both the C-terminal cytoplasmic and the transmembranous domains, to proteins with decreasing amounts of the cytoplasmic domain, were examined in order to separately define the roles of these two types of domains in the stability and processes of unfolding of a membrane protein. The sensitivity of the SRCD measurements over a wide range of wavelengths and temperatures has meant that subtle but reproducible conformational changes could be detected with accuracy. The residues in the C-terminal extramembranous domain were highly susceptible to thermal denaturation, but for the most part the transmembrane residues were not thermally-labile and retained their helical character even at very elevated temperatures. The process of thermal unfolding involved an initial irreversible unfolding of the highly labile distal extramembranous C-terminal helical region, which was accompanied by a reversible unfolding of a small number of helical residues in the transmembrane domain. This was then followed by the irreversible unfolding of a limited number of additional transmembrane helical residues at greatly elevated temperatures. Hence this study has been able to determine the different contributions and roles of the transmembrane and extramembrane residues in the processes of thermal denaturation of this multipass integral membrane protein.     

Molecular and functional determinants of local anesthetic inhibition of NaChBac

In our recent publication, we describe the local anesthetic (LA) inhibition of the prokaryotic voltage gated sodium channel NaChBac. Despite the numerous functional and putative structural differences with the mammalian sodium channels, the data show that LA compounds effectively and reversibly inhibit NaChBac channels in a concentration range similar to resting blockade on eukaryotic Navs. In addition to current reduction, LA application accelerated channel inactivation kinetics of NaChBac which could be accounted for in a simple state-model whereby local anesthetics increase the probability of entering the inactivated state. We have further explored what state (or states) local anesthetic blockade of NaChBac could pertain to eukaryotic sodium channels, and what molecular similarities exist between these disparate channel families. Here we show that the rate of recovery from inactivation remains unaffected in the presence of local anesthetics. Further, we show that two sites that support use-dependent inhibition in eukaryotic channels, do not affect block to the same extent when mutated in NaChBac channels. The data indicate that the molecular determinants and the inherent mechanisms for LA block are likely to be divergent between bacterial and eukaryotic Navs, but future experiments will help define possible similarities.     

Molecular and functional determinants of local anesthetic inhibition of NaChBac

In our recent publication, we describe the local anesthetic (LA) inhibition of the prokaryotic voltage gated sodium channel NaChBac. Despite the numerous functional and putative structural differences with the mammalian sodium channels, the data show that LA compounds effectively and reversibly inhibit NaChBac channels in a concentration range similar to resting blockade on eukaryotic Navs. In addition to current reduction, LA application accelerated channel inactivation kinetics of NaChBac which could be accounted for in a simple state-model whereby local anesthetics increase the probability of entering the inactivated state. We have further explored what state (or states) local anesthetic blockade of NaChBac could pertain to eukaryotic sodium channels, and what molecular similarities exist between these disparate channel families. Here we show that the rate of recovery from inactivation remains unaffected in the presence of local anesthetics. Further, we show that two sites that support use-dependent inhibition in eukaryotic channels, do not affect block to the same extent when mutated in NaChBac channels. The data indicate that the molecular determinants and the inherent mechanisms for LA block are likely to be divergent between bacterial and eukaryotic Navs, but future experiments will help define possible similarities.     

Local anesthetic inhibition of a bacterial sodium channel

Recent structural breakthroughs with the voltage-gated sodium channel from Arcobacter butzleri suggest that such bacterial channels may provide a structural platform to advance the understanding of eukaryotic sodium channel gating and pharmacology. We therefore set out to determine whether compounds known to interact with eukaryotic Na(V)s could also inhibit the bacterial channel from Bacillus halodurans and NaChBac and whether they did so through similar mechanisms as in their eukaryotic homologues. The data show that the archetypal local anesthetic (LA) lidocaine inhibits resting NaChBac channels with a dissociation constant (K(d)) of 260 μM, and channels displayed a left-shifted steady-state inactivation gating relationship in the presence of the drug. Extracellular application of QX-314 to expressed NaChBac channels had no effect on sodium current, whereas internal exposure via injection of a bolus of the quaternary derivative rapidly reduced sodium conductance, consistent with a hydrophilic cytoplasmic access pathway to an internal binding site. However, the neutral derivative benzocaine applied externally inhibited NaChBac channels, suggesting that hydrophobic pathways can also provide drug access to inhibit channels. Alternatively, ranolazine, a putative preopen state blocker of eukaryotic Na(V)s, displayed a K(d) of 60 μM and left-shifted the NaChBac activation-voltage relationship. In each case, block enhanced entry into the inactivated state of the channel, an effect that is well described by a simple kinetic scheme. The data suggest that although significant differences exist, LA block of eukaryotic Na(V)s also occurs in bacterial sodium channels and that NaChBac shares pharmacological homology to the resting state of vertebrate Na(V) homologues.     

Detecting rearrangements of shaker and NaChBac in real-time with fluorescence spectroscopy in patch-clamped mammalian cells

Time-resolved fluorescence detection of site-directed probes is a major tool in the investigation of structure-function relationships of voltage-dependent ion channels. However, the technique has been limited so far to the Xenopus-oocyte system making it difficult to study proteins, like, e.g., the prokaryotic sodium channel NaChBac, whose expression in oocytes is insufficient or whose physiological functions are distorted in oocytes. To expand the application of site-directed fluorescence detection to these proteins, we used two techniques-semiconfocal epifluorescence and total internal reflection fluorescence-to detect time-resolved fluorescence changes from site-directed labeled proteins expressed in mammalian cells under patch-clamp conditions, and investigated the characteristics and limitations of the techniques. The voltage-sensitive dye, di-8-ANEPPS, was used to monitor control of the membrane voltage in epifluorescence and total internal reflection fluorescence. Fluorescence changes in patch-clamped cells were recorded from a Shaker channel mutant (M356C) labeled in the S3-S4 linker using semiconfocal epifluorescence. The gating kinetics and fluorescence changes were in accordance with previous studies using fluorescence spectroscopy in Xenopus-oocyte systems. We applied our technique to the prokaryotic sodium channel NaChBac. Voltage-dependent protein-rearrangements of S4 could be detected that are independent of inactivation. Comparison of the S3-S4 linker regions revealed structural differences to the KvAP voltage sensor. The results from the NaChBac channel point to structural requirements for the S3-S4 loop to generate a fluorescence signal.     

The pore, not cytoplasmic domains, underlies inactivation in a prokaryotic sodium channel

Kinetics and voltage dependence of inactivation of a prokaryotic voltage-gated sodium channel (NaChBac) were investigated in an effort to understand its molecular mechanism. NaChBac inactivation kinetics show strong, bell-shaped voltage dependence with characteristic time constants ranging from approximately 50 ms at depolarized voltages to a maximum of approximately 100 s at the inactivation midpoint. Activation and inactivation parameters for four different covalently linked tandem dimer or tandem tetramer constructs were indistinguishable from those of the wild-type channel. Point mutations in the outer part of the pore revealed an important influence of the S195 residue on the process of inactivation. For two mutants (S195D and S195E), the maximal and minimal rates of inactivation observed were increased by approximately 2.5-fold, and the midpoint of the steady-state inactivation curve was shifted approximately 20 mV in the hyperpolarizing direction, compared to the wild-type channel. Our data suggest that pore vestibule structure is an important determinant of NaChBac inactivation, whereas the inactivation mechanism is independent of the number of free cytoplasmic N- and C-termini in the functional channel. In these respects, NaChBac inactivation resembles C-type or slow inactivation modes observed in other voltage-gated K and Na channels.     

Differential Lipid Dependence of the Function of Bacterial Sodium Channels

The lipid bilayer is important for maintaining the integrity of cellular compartments and plays a vital role in providing the hydrophobic and charged interactions necessary for membrane protein structure, conformational flexibility and function. To directly assess the lipid dependence of activity for voltage-gated sodium channels, we compared the activity of three bacterial sodium channel homologues (NaChBac, NavMs, and NavSp) by cumulative ²²Na⁺ uptake into proteoliposomes containing a 3∶1 ratio of 1-palmitoyl 2-oleoyl phosphatidylethanolamine and different “guest” glycerophospholipids. We observed a unique lipid profile for each channel tested. NavMs and NavSp showed strong preference for different negatively-charged lipids (phosphatidylinositol and phosphatidylglycerol, respectively), whilst NaChBac exhibited a more modest variation with lipid type. To investigate the molecular bases of these differences we used synchrotron radiation circular dichroism spectroscopy to compare structures in liposomes of different composition, and molecular modeling and electrostatics calculations to rationalize the functional differences seen. We then examined pore-only constructs (with voltage sensor subdomains removed) and found that in these channels the lipid specificity was drastically reduced, suggesting that the specific lipid influences on voltage-gated sodium channels arise primarily from their abilities to interact with the voltage-sensing subdomains.     

Fluorescence and conformational stability studies of Staphylococcus nuclease and its mutants, including the less stable nuclease-concanavalin A hybrids

We report steady-state and time-resolved fluorescence studies with the single tryptophan protein, Staphylococcus aureus A, and several of its site-directed mutants. A couple of these mutants, nuclease-conA and nuclease-conA-S28G (which are hybrid proteins containing a six amino acid beta-turn substitute from concanavalin A), are found to have a much lower thermodynamic stability than the wild type. The thermal transition temperatures for nuclease-conA and S28G are 32.8 and 30.5 degrees C, which are about 20 degrees C lower than the Tm for wild-type nuclease A. These mutant proteins also are denatured by a much lower concentration of the denaturants urea and guanidine hydrochloride. We also show that an unfolding transition in the structure of the nuclease-conA hybrids can be induced by relatively low hydrostatic pressure (approximately 700 bar). The free energy for unfolding of nuclease-conA (and nuclease-conA-S28G) is found to be only 1.4 kcal/mol (and 1.2 kcal/mol) by thermal, urea, guanidine hydrochloride, and pressure unfolding. Time-resolved fluorescence intensity and anisotropy measurements with nuclease-conA-S28G show the temperature-, urea-, and pressure-perturbed states each to have a reduced average intensity decay time and to depolarize with a rotational correlation time of approximately 1.0 ns (as compared to a rotational correlation time of 11 ns for the native form of nuclease-conA-S28G at 20 degrees C).     

Probing the folding and unfolding of wild-type and mutant forms of bacteriorhodopsin in micellar solutions: evaluation of reversible unfolding conditions

Wild-type and mutant forms of bacteriorhodopsin (sbR) from Halobacterium salinarium, produced by Escherichia coli overexpression of a synthetic gene, were reversibly unfolded in 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 3-[(3-cholamidopropyl)dimethylamino]-2-hydroxyl-1-propane (CHAPSO), and sodium dodecyl sulfate (SDS) mixed micelles. To study the effect on protein stability by substitutions on the hydrophobic surface with polar residues, the unfolding behavior of a G113Q, G116Q mutant [sbR(Q2)] was compared to the wild-type sbR [sbR(WT)]. sbR(Q2) was more sensitive to SDS-induced unfolding than sbR(WT) under equilibrium conditions, and kinetic experiments showed that sbR(Q2) was more sensitive to acid-induced denaturation and thermal unfolding than sbR(WT). Since the mutations in sbR(Q2) were on the detergent-embedded hydrophobic surface of sbR, protein destabilization by these mutations supports the concept that the membrane-embedded segments are important for the stability of sbR. Our experiments provide the basis for studying the thermodynamic stability of sbR by evaluating reversible folding and unfolding conditions in DMPC/CHAPSO/SDS mixed micelles.     

Oligonucleotide directed mutagenesis of Escherichia coli 5S ribosomal RNA: construction of mutant and structural analysis.

The ribosomal 5S RNA gene from the rrnB operon of E. coli was mutagenised in vitro using a synthetic oligonucleotide hybridised to M13 ssDNA containing that gene. The oligonucleotide corresponded to the 5S RNA sequence positions 34 to 51 and changed the guanosine at position 41 to a cytidine. The DNA containing the desired mutation was identified by dot blot hybridisation and introduced back into the plasmid pKK 3535 which contains the total rrnB operon in pBR 322. Plasmid coded 5S rRNA was selectively labeled with 32p using a modified maxi-cell system, and the replacement of guanosine G41 by cytidine was confirmed by RNA sequencing. The growth of cells containing mutant 5S rRNA was not altered by the base change, and the 5S rRNA was processed and incorporated into 50S ribosomal subunits and 70S ribosomes. The structure of wildtype and mutant 5S rRNA was compared by chemical modification of accessible guanosines with kethoxal and limited enzymatic digestion using RNase T1 and nuclease S1. These results showed that the wildtype and mutant 5S rRNA do not differ significantly in their structure. Furthermore, the formation, interconversion and stability of the two 5S rRNA A- and B-conformers are unchanged.     

Unfolding of the cold shock protein studied with biased molecular dynamics

The cold shock protein from Bacillus caldolyticus is a small beta-barrel protein that folds in a two-state mechanism. For the native protein and for several mutants, a wealth of experimental data are available on stability and folding, so that it is an optimal system to study this process. We compare data from unfolding simulations (trajectories of 5 and up to 12 ns) obtained with a bias potential at room temperature and from unbiased thermal unfolding simulations with experimental data. The unfolding patterns derived from the trajectories starting from different native-like conformations and subject to different unfolding conditions agree. The transition state found in the simulations of unfolding is close to the native structure in agreement with experiment. Moreover, a lower value of the free energy barrier of unfolding was found for the mutant R3E than for the mutant E46A and the native protein, as indicated by experimental data. The first unfolding event involves the three-stranded beta-sheet whose decomposition corresponds to the transition state. In contrast to conclusions drawn from experiments, we found that the two-stranded beta-strand forms the most stable substructure, which decomposes very late in the unfolding process. However, assuming that this structure forms very early in the folding process, our findings would not contradict the experiments but require a different interpretation of them.     

Pivotal role of Gly 121 in dihydrofolate reductase from Escherichia coli: the altered structure of a mutant enzyme may form the basis of its diminished catalytic performance

The structure and folding of dihydrofolate reductase (DHFR) from Escherichia coli and the mutant G121V-DHFR, in which glycine 121 in the exterior FG loop was replaced with valine, were studied by molecular dynamics simulations and CD and fluorescence spectroscopy. The importance of residue 121 for the chemical step during DHFR catalysis had been demonstrated previously. High-temperature MD simulations indicated that while DHFR and G121V-DHFR followed similar unfolding pathways, the strong contacts between the M20 loop and the FG loop in DHFR were less stable in the mutant. These contacts have been proposed to be involved in a coupled network of interactions that influence the protein dynamics and promote catalysis [Benkovic, S. J., and Hammes-Schiffer, S. (2003) Science 301, 1196-1202]. CD spectroscopy of DHFR and G121V-DHFR indicated that the two proteins existed in different conformations at room temperature. While the thermally induced unfolding of DHFR was highly cooperative with a midpoint at 51.6 +/- 0.7 degrees C, G121V-DHFR exhibited a gradual decrease in its level of secondary structure without a clear melting temperature. Temperature-induced unfolding and renaturation from the urea-denatured state revealed that both proteins folded via highly fluorescent intermediates. The formation of these intermediates occurred with relaxation times of 149 +/- 4.5 and 256 +/- 13 ms for DHFR and G121V-DHFR, respectively. The fluorescence intensity for the intermediates formed during refolding of G121V-DHFR was approximately twice that of the wild-type. While the fluorescence intensity then slowly decayed for DHFR toward a state representing the native protein, G121V-DHFR appeared to be trapped in a highly fluorescent state. These results suggest that the reduced catalytic activity of G121V-DHFR is the consequence of nonlocal structural effects that may result in a perturbation of the network of promoting motions.     

Models of voltage-dependent conformational changes in NaChBac channels

Models of the transmembrane region of the NaChBac channel were developed in two open/inactivated and several closed conformations. Homology models of NaChBac were developed using crystal structures of Kv1.2 and a Kv1.2/2.1 chimera as templates for open conformations, and MlotiK and KcsA channels as templates for closed conformations. Multiple molecular-dynamic simulations were performed to refine and evaluate these models. A striking difference between the S4 structures of the Kv1.2-like open models and MlotiK-like closed models is the secondary structure. In the open model, the first part of S4 forms an α-helix, and the last part forms a 3₁₀ helix, whereas in the closed model, the first part of S4 forms a 3₁₀ helix, and the last part forms an α-helix. A conformational change that involves this type of transition in secondary structure should be voltage-dependent. However, this transition alone is not sufficient to account for the large gating charge movement reported for NaChBac channels and for experimental results in other voltage-gated channels. To increase the magnitude of the motion of S4, we developed another model of an open/inactivated conformation, in which S4 is displaced farther outward, and a number of closed models in which S4 is displaced farther inward. A helical screw motion for the α-helical part of S4 and a simple axial translation for the 3₁₀ portion were used to develop models of these additional conformations. In our models, four positively charged residues of S4 moved outwardly during activation, across a transition barrier formed by highly conserved hydrophobic residues on S1, S2, and S3. The S4 movement was coupled to an opening of the activation gate formed by S6 through interactions with the segment linking S4 to S5. Consistencies of our models with experimental studies of NaChBac and K_v channels are discussed.     

Effects of helix and fingertip mutations on the thermostability of xyn11A investigated by molecular dynamics simulations and enzyme activity assays

Local conformational changes and global unfolding pathways of wildtype xyn11A recombinant and its mutated structures were studied through a series of atomistic molecular dynamics (MD) simulations, along with enzyme activity assays at three incubation temperatures to investigate the effects of mutations at three different sites to the thermostability. The first mutation was to replace an unstable negatively charged residue at a surface beta turn near the active site (D32G) by a hydrophobic residue. The second mutation was to create a disulphide bond (S100C/N147C) establishing a strong connection between an alpha helix and a distal beta hairpin associated with the thermally sensitive Thumb loop, and the third mutation add an extra hydrogen bond (A155S) to the same alpha helix. From the MD simulations performed, MM/PBSA energy calculations of the unfolding energy were in a good agreement with the enzyme activities measured from the experiment, as all mutated structures demonstrated the improved thermostability, especially the S100C/N147C proved to be the most stable mutant both by the simulations and the experiment. Local conformational analysis at the catalytic sites and the xylan access region also suggested that mutated xyn11A structures could accommodate xylan binding. However, the analysis of global unfolding pathways showed that structural disruptions at the beta sheet regions near the N-terminal were still imminent. These findings could provide the insight on the molecular mechanisms underlying the enhanced thermostability due to mutagenesis and changes in the protein unfolding pathways for further protein engineering of the GH11 family xylanase enzymes.     

Coupling between Residues on S4 and S1 Defines the Voltage-Sensor Resting Conformation in NaChBac

The voltage sensor is a four-transmembrane helix bundle (S1-S4) that couples changes in membrane potential to conformational alterations in voltage-gated ion channels leading to pore opening and ion conductance. Although the structure of the voltage sensor in activated potassium channels is available, the conformation of the voltage sensor at rest is still obscure, limiting our understanding of the voltage-sensing mechanism. By employing a heterologously expressed Bacillus halodurans sodium channel (NaChBac), we defined constraints that affect the positioning and depolarization-induced outward motion of the S4 segment. We compared macroscopic currents mediated by NaChBac and mutants in which E43 on the S1 segment and the two outermost arginines (R1 and R2) on S4 were substituted. Neutralization of the negatively charged E43 (E43C) had a significant effect on channel gating. A double-mutant cycle analysis of E43 and R1 or R2 suggested changes in pairing during channel activation, implying that the interaction of E43 with R1 stabilizes the voltage sensor in its closed/available state, whereas interaction of E43 with R2 stabilizes the channel open/unavailable state. These constraints on S4 dynamics that define its stepwise movement upon channel activation and positioning at rest are novel, to the best of our knowledge, and compatible with the helical-screw and electrostatic models of S4 motion.