Substituted hydrophobic and hydrophilic residues at methionine-68 influence the chaperone-like function of αB-crystallin

Amino acid residues 57–69 in B-crystallin have been implicated as a target protein binding site. Moreover, a direct correlation between the extent of -crystallin hydrophobicity and chaperone-like activity has been demonstrated. The purpose of this study was to mutate a moderately hydrophobic residue Met-68 (M-68) in the above region to strongly hydrophobic and hydrophilic residues and show whether chaperoning ability is affected with or without structural changes. Mutation of M-68 to Val, Ile or Thr did not result in significant changes in molecular mass and secondary and tertiary structures. However, the Val and Ile mutants showed significant improvement and the Thr mutant showed substantial loss in chaperone activity. Differences in chaperone function in the absence of any structural changes confirmed that the hydrophobicity or hydrophilicity of the substituted amino acid in the putative target protein binding site was the only contributing factor.     

Significance of the hydrophobic residues 225–230 of apoA-I for the biogenesis of HDL

We studied the significance of four hydrophobic residues within the 225–230 region of apoA-I on its structure and functions and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of an apoA-I[F225A/V227A/F229A/L230A] mutant in apoA-I^−/− mice decreased plasma cholesterol, HDL cholesterol, and apoA-I levels. When expressed in apoA-I^−/− × apoE^−/− mice, approximately 40% of the mutant apoA-I as well as mouse apoA-IV and apoB-48 appeared in the VLDL/IDL/LDL. In both mouse models, the apoA-I mutant generated small spherical particles of pre-β- and α4-HDL mobility. Coexpression of the apoA-I mutant and LCAT increased and shifted the-HDL cholesterol peak toward lower densities, created normal αHDL subpopulations, and generated spherical-HDL particles. Biophysical analyses suggested that the apoA-I[225–230] mutations led to a more compact folding that may limit the conformational flexibility of the protein. The mutations also reduced the ability of apoA-I to promote ABCA1-mediated cholesterol efflux and to activate LCAT to 31% and 66%, respectively, of the WT control. Overall, the apoA-I[225–230] mutations inhibited the biogenesis of-HDL and led to the accumulation of immature pre-β- and α4-HDL particles, a phenotype that could be corrected by administration of LCAT.     

Structure of the UreD–UreF–UreG–UreE complex in Helicobacter pylori: a model study

The molecular details of the protein complex formed by UreD, UreF, UreG, and UreE, accessory proteins for urease activation in the carcinogenic bacterium Helicobacter pylori, have been elucidated using computational modeling. The calculated structure of the complex supports the hypothesis of UreF acting as a GTPase activation protein that facilitates GTP hydrolysis by UreG during urease maturation, and provides a rationale for the design of new drugs against infections by ureolytic bacterial pathogens.     

White-noise stimulation of the Hodgkin–Huxley model

 We studied the combined influence of noise and constant current stimulations on the Hodgkin–Huxley neuron model through time and frequency analysis of the membrane-potential dynamics. We observed that, in agreement with experimental data (Guttman et al. 1974), at low noise and low constant current stimulation the behavior of the model is well approximated by that of the linearized Hodgkin–Huxley system. Conversely, nonlinearities due to firing dominate at large noise or current stimulations. The transition between the two regimes is abrupt, and takes place in the same range of noise and current intensities as the noise-induced transition characterized by the qualitative change in the stationary distribution of the membrane potential (Tanabe and Pakdaman 2001a). The implications of these results are discussed. Received: 27 July 2001 / Accepted in revised form: 18 December 2001     

Structure optimization of a new class of PPARγ antagonists

Peroxisome proliferator-activated receptor gamma (PPARγ) modulators have found wide application for the treatment of cancers, metabolic disorders and inflammatory diseases. Contrary to PPARγ agonists, PPARγ antagonists have been much less studied and although they have shown immunomodulatory effects, there is still no therapeutically useful PPARγ antagonist on the market. In contrast to non-competitive, irreversible inhibition caused by 2-chloro-5-nitrobenzanilide (GW9662), the recently described (E)-2-(5-((4-methoxy-2-(trifluoromethyl)quinolin-6-yl)methoxy)-2-((4-(trifluoromethyl)benzyl)oxy)-benzylidene)-hexanoic acid (MTTB, T-10017) is a promising prototype for a new class of PPARγ antagonists. It exhibits competitive antagonism against rosiglitazone mediated activation of PPARγ ligand binding domain (PPARγLBD) in a transactivation assay in HEK293T cells with an IC₅₀ of 4.3 µM against 1 µM rosiglitazone. The aim of this study was to investigate the structure-activity relationships (SAR) of the MTTB scaffold focusing on improving its physicochemical properties. Through this optimization, 34 new derivatives were prepared and characterized. Two new potent compounds (T-10075 and T-10106) with much improved drug-like properties and promising pharmacokinetic profile were identified.     

Determination of material parameters of the two-dimensional Holzapfel–Weizsäcker type model based on uniaxial extension data of arterial walls

Soft tissues are anisotropic materials yet a majority of mechanical property tests have been uniaxial, which often failed to recapitulate the tensile response in other directions. This paper aims to study the feasibility of determining material parameters of anisotropic tissues by uniaxial extension with a minimal loss of anisotropic information. We assumed that by preselecting a certain constitutive model, we could give the constitutive parameters based on uniaxial extension data from orthogonal strip samples. In our study, the Holzapfel–Weizsäcker type strain energy density function (H–W model) was used to determine the material parameters of arterial walls from two fresh donation bodies. The key points we applied were the relationships between strain components in uniaxial tensile tests and the methods of stochastic optimisation. Further numerical experiments were taken. The estimate–effect ratio, defined by the number of data with the precision of estimation less than 0.5% over whole size of data, was calculated to demonstrate the feasibility of our method. The material parameters for Chinese aorta and pulmonary artery were given with the maximum root mean square (RMS) errors 0.042, and the minimal estimate–effect ratio in numerical experiments was 90.79%. Our results suggest that the constitutive parameters of arterial walls can be determined from uniaxial extension data, given the passive mechanical behaviour governed by H–W model. This method may apply to other tissues using different constitutive models.     

Up-and-down topological mode of amyloid β-peptide lying on hydrophilic/hydrophobic interface of ganglioside clusters

Growing evidence has indicated that GM1 ganglioside specifically interacts with Amyloid β-peptide (Aβ) and thereby promotes Alzheimer’s disease-associated Aβ assembly. To characterize the conformation of Aβ bound to the ganglioside, we performed 920 MHz ultra-high field NMR analyses using isotopically labeled Aβ(1–40) in association with GM1 and lyso-GM1 micelles. Our NMR data revealed that (1) Aβ(1–40) forms discontinuous α-helices at the segments His¹⁴-Val²⁴ and Ile³¹-Val³⁶ upon binding to the gangliosidic micelles, leaving the remaining regions disordered, and (2) Aβ(1–40) lies on hydrophobic/hydrophilic interface of the ganglioside cluster exhibiting an up-and-down topological mode in which the two α-helices and the C-terminal dipeptide segment are in contact with the hydrophobic interior, whereas the remaining regions are exposed to the aqueous environment. These findings suggest that the ganglioside clusters serve as a unique platform for binding coupled with conformational transition of Aβ molecules, rendering their spatial rearrangements restricted to promote specific intermolecular interactions.     

Structure–Function Relationships of the Vasopressin Prohormone Domains

1. In this review the structure–function relationships of the different vasopressin prohormone domains are dated and discussed, with special reference to the neurophysin and glycopeptide domains.2. The primary structures of the currently known neurophysins and glycopeptide sequences are compared and discussed.3. The hormone-binding and aggregational properties of neurophysin are reviewed and related to a possible function within the regulated secretory pathway.4. It is proposed, based on the properties reviewed here as well as our own data shown here, that the sorting of the vasopressin prohormone is initiated by hormone binding, which triggers aggregation of the prohormone into the characteristic dense cores of the regulated secretory pathway.5. This may suggest that prohormone sorting into the regulated secretory pathway is, in general, determined by noncovalent, intramolecular interactions that promote aggregation.     

Role of the hydrophobic and charged residues in the 218–226 region of apoA-I in the biogenesis of HDL

We investigated the significance of hydrophobic and charged residues 218–226 on the structure and functions of apoA-I and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of apoA-I[L218A/L219A/V221A/L222A] in apoA-I^−/− mice decreased plasma cholesterol and apoA-I levels to 15% of wild-type (WT) control mice and generated pre-β- and α4-HDL particles. In apoA-I^−/− × apoE^−/− mice, the same mutant formed few discoidal and pre-β-HDL particles that could not be converted to mature α-HDL particles by excess LCAT. Expression of the apoA-I[E223A/K226A] mutant in apoA-I^−/− mice caused lesser but discrete alterations in the HDL phenotype. The apoA-I[218–222] and apoA-I[E223A/K226A] mutants had 20% and normal capacity, respectively, to promote ABCA1-mediated cholesterol efflux. Both mutants had ∼65% of normal capacity to activate LCAT in vitro. Biophysical analyses suggested that both mutants affected in a distinct manner the structural integrity and plasticity of apoA-I that is necessary for normal functions. We conclude that the alteration of the hydrophobic 218–222 residues of apoA-I disrupts apoA-I/ABCA1 interactions and promotes the generation of defective pre-β particles that fail to mature into α-HDL subpopulations, thus resulting in low plasma apoA-I and HDL. Alterations of the charged 223, 226 residues caused milder but discrete changes in HDL phenotype.     

Are the interactions between recombinant prion proteins and polymeric surfaces related to the hydrophilic/hydrophobic balance?

New non-fouling tubes are developed and their influence on the adhesion of neuroproteins is studied. Recombinant prion proteins are considered as a single component representative of hydrophobic proteins. Samples are stored for 24?h at 4?°C in tubes coated with two different coatings: poly(N-isopropylacrylamide) as a hydrophilic surface and a plasma-fluorinated coating as a hydrophobic one. The protein adhesion is monitored by ELISA tests, XPS and confocal microscopy. It appears that the highest recovery of recombinant prion protein in the liquid phase is obtained with the hydrophilic surface while the hydrophobic character of the storage tube induces an important amount of biological loss. However, the recovery is not complete even for tubes coated with poly(N-isopropylacrylamide).     

The minimal model of the hypothalamic–pituitary–adrenal axis

This paper concerns ODE modeling of the hypothalamic–pituitary– adrenal axis (HPA axis) using an analytical and numerical approach, combined with biological knowledge regarding physiological mechanisms and parameters. The three hormones, CRH, ACTH, and cortisol, which interact in the HPA axis are modeled as a system of three coupled, nonlinear differential equations. Experimental data shows the circadian as well as the ultradian rhythm. This paper focuses on the ultradian rhythm. The ultradian rhythm can mathematically be explained by oscillating solutions. Oscillating solutions to an ODE emerges from an unstable fixed point with complex eigenvalues with a positive real parts and a non-zero imaginary parts. The first part of the paper describes the general considerations to be obeyed for a mathematical model of the HPA axis. In this paper we only include the most widely accepted mechanisms that influence the dynamics of the HPA axis, i.e. a negative feedback from cortisol on CRH and ACTH. Therefore we term our model the minimal model. The minimal model, encompasses a wide class of different realizations, obeying only a few physiologically reasonable demands. The results include the existence of a trapping region guaranteeing that concentrations do not become negative or tend to infinity. Furthermore, this treatment guarantees the existence of a unique fixed point. A change in local stability of the fixed point, from stable to unstable, implies a Hopf bifurcation; thereby, oscillating solutions may emerge from the model. Sufficient criteria for local stability of the fixed point, and an easily applicable sufficient criteria guaranteeing global stability of the fixed point, is formulated. If the latter is fulfilled, ultradian rhythm is an impossible outcome of the minimal model and all realizations thereof. The second part of the paper concerns a specific realization of the minimal model in which feedback functions are built explicitly using receptor dynamics. Using physiologically reasonable parameter values, along with the results of the general case, it is demonstrated that un-physiological values of the parameters are needed in order to achieve local instability of the fixed point. Small changes in physiologically relevant parameters cause the system to be globally stable using the analytical criteria. All simulations show a globally stable fixed point, ruling out periodic solutions even when an investigation of the ‘worst case parameters’ is performed.     

Parametric analysis of the ratio-dependent predator–prey model

We present a complete parametric analysis of stability properties and dynamic regimes of an ODE model in which the functional response is a function of the ratio of prey and predator abundances. We show the existence of eight qualitatively different types of system behaviors realized for various parameter values. In particular, there exist areas of coexistence (which may be steady or oscillating), areas in which both populations become extinct, and areas of "conditional coexistence" depending on the initial values. One of the main mathematical features of ratio-dependent models, distinguishing this class from other predator-prey models, is that the Origin is a complicated equilibrium point, whose characteristics crucially determine the main properties of the model. This is the first demonstration of this phenomenon in an ecological model. The model is investigated with methods of the qualitative theory of ODEs and the theory of bifurcations. The biological relevance of the mathematical results is discussed both regarding conservation issues (for which coexistence is desired) and biological control (for which extinction is desired).     

Parameter non-identifiability of the Gyllenberg–Webb ODE model

An ODE model introduced by Gyllenberg and Webb (Growth Develop Aging 53:25–33, 1989) describes tumour growth in terms of the dynamics between proliferating and quiescent cell states. The passage from one state to another and vice versa is modelled by two functions $r_o$ and $r_i$ depending on the total tumour size. As these functions do not represent any observable quantities, they have to be identified from the observations. In this paper we show that there is an infinite number of pairs ( $r_o, r_i$ ) corresponding to the same solution of the ODE system and the functions ( $r_o, r_i$ ) will be classified in terms of this equivalence. Surprisingly, the technique used for this classification permits a uniqueness proof of the solution of the ODE model in a non-Lipschitz case. The reasoning can be widened to a more general setting including an extension of the Gyllenberg–Webb model with a nonlinear birth rate. The relevance of this result is discussed in a preclinical application scenario.     

Structure–activity relationships of the thujaplicins for inhibition of human tyrosinase

Tyrosinase inhibitors have become increasingly critical agents in cosmetic, agricultural, and medicinal products. Although a large number of tyrosinase inhibitors have been reported, almost all the inhibitors were unfortunately evaluated by using commercial available mushroom tyrosinase. Here, we examined the inhibitory effects of three isomers of thujaplicin (α, β, and γ) on human tyrosinase and analyzed their binding modes using homology model and docking studies. As the results, γ-thujaplicin was found to strongly inhibit human tyrosinase with the IC₅₀ of 1.15 μM, extremely superior to a well-known tyrosinase inhibitor kojic acid (IC₅₀ = 571.17 μM). MM-GB/SA binding free energy decomposition analyses suggested that the potent inhibitory activity of γ-thujaplicin may be due to the interactions with His367, Ile368, and Val377 (hot spot amino acid residues) in human tyrosinase. Furthermore, the binding mode of α-thujaplicin indicated that Val377 and Ser380 may cause van der Waals clashes with the isopropyl group of α-thujaplicin. These results provide a novel structural insight into the hot spot of human tyrosinase for the specific binding of γ-thujaplicin and a way to optimize not only thujaplicins but also other lead compounds as specific inhibitors for human tyrosinase in a rational manner.     

The dynamics of the lynx–hare system: an application of the Lotka–Volterra model

The Lotka–Volterra model of predator–prey dynamics was used for approximation of the wellknown empirical time series on the lynx–hare system in Canada that was collected by the Hudson Bay Company in 1845–1935. The model was assumed to demonstrate satisfactory data approximation if the sets of deviations of the model and empirical data for both time series satisfied a number of statistical criteria (for the selected significance level). The frequency distributions of deviations between the theoretical (model) trajectories and empirical datasets were tested for symmetry (with respect to the Y-axis; the Kolmogorov–Smirnov and Lehmann–Rosenblatt tests) and the presence or absence of serial correlation (the Swed–Eisenhart and “jumps up–jumps down” tests). The numerical calculations show that the set of points of the space of model parameters, when the deviations satisfy the statistical criteria, is not empty and, consequently, the model is suitable for describing empirical data.     

The Chemical Structure of the Intermediate Metabolites of Catechin I–IV

Examinations were made on two intermediate metabolites excreted after administration of (+)-catechin to the rabbit. From the infrared and ultraviolet spectra of these two substances (C₁₁H₁₂O₃ and C₁₁H₁₂O₄) and chemical properties of their various derivatives, they were identified as 3-hydroxyphenyl lactone and 3, 4-dihydroxyphenyl lactone. However, it still remains unknown whether they are δ- or γ-lactone.     

Stability and flexibility of marginally hydrophobic–segment stalling at the endoplasmic reticulum translocon

Many membrane proteins are integrated into the endoplasmic reticulum membrane through the protein-conducting channel, the translocon. Transmembrane segments with insufficient hydrophobicity for membrane integration are frequently found in multispanning membrane proteins, and such marginally hydrophobic (mH) segments should be accommodated, at least transiently, at the membrane. Here we investigated how mH-segments stall at the membrane and their stability. Our findings show that mH-segments can be retained at the membrane without moving into the lipid phase and that such segments flank Sec61α, the core channel of the translocon, in the translational intermediate state. The mH-segments are gradually transferred from the Sec61 channel to the lipid environment in a hydrophobicity-dependent manner, and this lateral movement may be affected by the ribosome. In addition, stalling mH-segments allow for insertion of the following transmembrane segment, forming an N_cytosol/C_lumen orientation, suggesting that mH-segments can move laterally to accommodate the next transmembrane segment. These findings suggest that mH-segments may be accommodated at the ER membrane with lateral fluctuation between the Sec61 channel and the lipid phase.     

Investigation of Turing instability for the Gierer–Meinhardt model

The dependence of the emergence of Turing instability for a distributed system of nonlinear differential equations that describe hydra morphogenesis based on the oscillatory properties of the corresponding trajectories of the system was investigated. The limits in the parameter space that provide diffusive instability were obtained. The frequency and amplitude dependences of the resulting spatial self oscillations on the values of the main parameters were investigated. Comparative analysis of the properties of the distributed system and corresponding trajectories of the system was carried out and the analytical conclusions were confirmed by the solutions of the system that were found using MATLAB.

     

Adsorption of bacterial capsular exopolymers to hydrophilic and hydrophobic surfaces: A 2D‐FTIR analysis

Analysis of the adsorption of capsular exopolymers (EPS) from Pseudomonas sp. NCIMB 2021 to hydrophilic and hydrophobic gold surfaces was examined, in situ, using Fourier transform infrared spectroscopy. The molecular sequence of events occurring upon EPS adsorption to hydrophilic and hydrophobic surfaces has been elucidated using dynamic 2D‐FTIR correlation spectroscopy. This method of analysis enables the enhancement of the resolution of overlapping spectral features and the elucidation of time‐dependent changes. The data reveal the existence of surface dependent adsorption mechanisms. At both surfaces, the aromatic tyrosyl side chains of the protein moiety displace water. This is followed by an adsorption step dominated by carboxylate groups. However, at the hydrophobic surface, the two steps are interrupted by the ingress of water back to the surface. Furthermore, the amount of neutral exopolymer present was greater at the hydrophilic surface than the hydrophobic surface.     

Structural model of the open–closed–inactivated cycle of prokaryotic voltage-gated sodium channels

In excitable cells, the initiation of the action potential results from the opening of voltage-gated sodium channels. These channels undergo a series of conformational changes between open, closed, and inactivated states. Many models have been proposed for the structural transitions that result in these different functional states. Here, we compare the crystal structures of prokaryotic sodium channels captured in the different conformational forms and use them as the basis for examining molecular models for the activation, slow inactivation, and recovery processes. We compare structural similarities and differences in the pore domains, specifically in the transmembrane helices, the constrictions within the pore cavity, the activation gate at the cytoplasmic end of the last transmembrane helix, the C-terminal domain, and the selectivity filter. We discuss the observed differences in the context of previous models for opening, closing, and inactivation, and present a new structure-based model for the functional transitions. Our proposed prokaryotic channel activation mechanism is then compared with the activation transition in eukaryotic sodium channels.Eukaryotic sodium channels are complex, multidomain proteins that assemble into pseudotetrameric structures. Sodium channels are also present in some prokaryotes (Ren et al., 2001), where they are homologous but simpler, single-domain proteins that assemble to form functional homotetramers (Nurani et al., 2008). Both eukaryotic and prokaryotic sodium channels consist of voltage-sensor (VS) and pore regions that are physically separated in three dimensions. Pore-only channels can be constructed, which are capable of undergoing opening and closing transitions and support ion flux in a manner similar to that of intact channels (McCusker et al., 2011; Shaya et al., 2011). Although the kinetics of their processes of conversion between states are different, both eukaryotic and prokaryotic channels exhibit activation, recovery, and slow inactivation transitions (Koishi et al., 2004; Charalambous and Wallace, 2011); in addition, eukaryotic channels undergo a fast inactivation process that is not observed in prokaryotic channels (Pavlov et al., 2005). Prokaryotic sodium channels have also been shown to be blocked by eukaryotic sodium channel antagonists (Bagnéris et al., 2014).Many models have been proposed for the structural changes that result in the open, closed, and slow inactivated states of prokaryotic sodium channels (e.g., Kuzmenkin et al., 2004; Zhao et al., 2004; Pavlov et al., 2005; Shafrir et al., 2008). Although there are, as yet, no crystal structures of eukaryotic sodium channels, the structures of several prokaryotic sodium channel orthologues have recently been determined by x-ray crystallography (Payandeh et al., 2011, 2012; McCusker et al., 2012; Zhang et al., 2012; Bagnéris et al., 2013; Shaya et al., 2014). These include the voltage-gated sodium channels from Magnetococcus marinus, formerly known as Magnetococcus spirillium (NavMs); from Arcobacter butzler (NavAb); from Rickettsiales sp. HIMB114 (NavRh); and from Alkalilimnicola ehrlichei (NavAe). These structures now make it possible to observe various states of prokaryotic sodium channels because they have apparently captured the pore regions in partially and fully open (NavMs), closed (NavAb and NavAe), and inactivated (NavAb and NavRh) conformations, thereby enabling structural comparisons that provide new insights into the transition processes.Because the transmembrane pore regions of the NavMs and NavAb pores have very high sequence identities (∼69%; McCusker et al., 2012; Fig. 1), our comparisons will primarily be confined to these structures because with this level of similarity, differences seen are likely to be state dependent rather than a consequence of sequence dissimilarities. They enable comparisons of structures that we designate as the following: partially (pOpen_Ms; Protein Data Bank [PDB] accession no. 4F4L; 3.5-Å resolution; McCusker et al., 2012) and fully open (Open_Ms; PDB accession no. 3ZJZ, 2.9-Å resolution; Bagnéris et al., 2013), closed (Closed_Ab; PBD accession no. 3RVY, an I217C mutant; 2.7-Å resolution; Payandeh et al., 2011), and inactivated (Inactivated_Ab; PDB accession no. 4EKW, 3.2-Å resolution; Payandeh et al., 2012) forms. The other inactivated form for which there is a crystal structure, NavRh (PDB accession no. 4DXW; 3.0-Å resolution; Zhang et al., 2012), and the other closed form, NavAe (PDB accession no. 4LTO; 3.5-Å resolution; Shaya et al., 2014), are more distant homologues (only 42 and 45% identity, respectively, in the pore region; Fig. 1). In addition, although both the NavAb and NavMs homologues have been shown to support sodium flux (Payandeh et al., 2011; D’Avanzo et al., 2013), neither NavRh nor NavAe has yet been shown to exhibit functional activity; therefore, we considered them to be less suitable for detailed comparisons. Although the resolutions of the structures are modest, all are sufficient to define the types and magnitudes of the conformational differences described in the comparisons made in this Review. In addition to these crystal structures, however, two structures of another orthologue, NavCt (PDB accession no. 4BGN, ∼9-Å resolution; Tsai et al., 2013), have been determined by electron crystallography, but those structures are at too low a resolution to make viable comparisons.Open in a separate windowFigure 1.Sequence alignment, using Clustal Omega (Sievers et al., 2011), of the prokaryotic sodium channel orthologues: NavMs from Magnetococcus marinus MC-1 (UniProt accession no. A0L5S6), NavAb from Arcobacter butzleri RM4018 (UniProt accession no. A8EVM5), NavAe from Alkalilimnicola ehrlichii MLHE-1 (UniProt accession no. {"type":"entrez-protein","attrs":{"text":"Q0ABW0","term_id":"122312570","term_text":"Q0ABW0"}}Q0ABW0), NavCt from Caldalkalibacillus thermarum TA2.A1 (UniProt accession no. F5L478), NavRh from Rickettsiales sp. HIMB114 (UniProt accession no. D0RMU8), and domain IV (DIV) of the human Nav1.4 sodium channel (UniProt accession no. {"type":"entrez-protein","attrs":{"text":"P35499","term_id":"292495096","term_text":"P35499"}}P35499). The identities of the helical regions (transmembrane helices S1–S6), the N-terminal intracellular helix S1N, the S4–S5 linker helix, the P1 and P2 pore helices, and the intracellular C-terminal coiled-coil (CC) helix are indicated in the horizontal colored tubes above the sequences and are based on the crystal structures of NavAb for the VS region (cyan), on the crystal structure of NavMs for the pore region (green), and on the site-directed electron paramagnetic resonance spectroscopy of NavMs and circular dichroism truncation studies of NaChBac for the CTD (gray). The vertical magenta bar indicates the extracellular negatively charged (ENC) region formed by D49 (in S2), and the red bars are the intracellular negatively charged (INC) region formed by E59 (in S2) and D81 (in S3) involved in the gating charge transfer across the membrane through sequential interactions with four arginine residues in S4 (indicated by the cyan vertical bars); in the “up” conformation, which corresponds to the activated state, residues E59 and D81 form the salt-bridged pairs. The residues comprising the SF are highlighted in light green vertical bars. The residues in purple are proposed to be the start of the twist in S6 that is implicated in activation gate opening (light purple from the partially open structure and dark purple in the fully open structure). The residue in yellow indicates the location of the final hydrophobic constriction region (HC3) in the closed structure, which effectively corresponds to the location of the activation gate.This Review focuses on comparisons of the Closed_Ab–pOpen_Ms–Open_Ms–Inactivated_Ab crystal structures, developing a structure-based model for the functional transitions. Although these crystal structures provide an important new structural context for understanding the nature of the transitions, it must be remembered that any models developed from them will ultimately require verification by functional studies under voltage-clamp conditions that probe native structures in real time and in the context of cell membranes.

Defining the conformational states represented in the crystal structures

The full-length NavAb channel has its VS in the “up” conformation (with residues E59 and D81 forming salt-bridged pairs to arginines in S4; Fig. 1) (Payandeh et al., 2011). This conformation of the VS is considered to be associated with the activated or fully open state as defined by disulfide cross-linking experiments (DeCaen et al., 2008, 2009). In addition, because in crystals there is no transmembrane potential, we expected that the structure would include an open form of the pore, as would be seen when the channel gate opens in response to membrane depolarization. However, in these crystals, the activation gate, and thus the pore region, appears to be in the closed conformation: the pore has no continuous transmembrane pathway that would enable the passage of sodium ions from the extracellular to the intracellular surface (McCusker et al., 2012; Fig. 2, A and B). Although the selectivity filter (SF) is sufficiently open to enable ions to enter the pore, the bottom of its cavity (at the activation gate) is blocked (Payandeh et al., 2011) so ions cannot exit. This structure has thus been described as being in a “pre-open” state, in other words, with a VS conformation that has primed the gate for opening but in which the gate is actually closed. This demonstrates that the structures of the VS and pore regions can be uncoupled, and may indicate the presence of an additional conformation intermediate between the “open” and “closed” states. In the comparisons in this Review, which focuses on the pore region responsible for the opening and closing of the transmembrane pathway, we have designated this structure as “closed” (Closed_Ab) based on the state of the pore region. It is very similar in dimensions and features to the NavAe closed pore (Shaya et al., 2014), a structure that does not contain a VS region, another indication that the pore region state is not solely determined by the VS state.Open in a separate windowFigure 2.Accessible surface differences between the open and closed structures. (A; middle) Accessible surface plots (made using CAVER software; Chovancova et al., 2012) showing radius versus distance through the central axis of the pore for Closed_Ab-I217C (slate blue), Closed_Ae (red), Inactivated_Ab (gray), Open_Ms (light green), and pOpen_Ms (turquoise). (Left and right) Slab surface mode/cartoon depictions of the pores, sliced along the transmembrane direction for the Closed_Ab (left, slate blue) and Open_Ms (right, light green) pores, highlighting the sites of the first minor constriction (blue underlay: present for both forms with the responsible residues, V213 in the closed form and I215 in the open form), the second minor construction (orange underlay: present only in the closed form, residue I217C), and the third major constriction (yellow underlay: present only for the closed form, residue M221) at the intracellular end of the cavity. I217C is the mutation in the closed structure that enabled crystallization at higher resolution. (B) Detailed view of the three “hydrophobic constriction” (HC) regions noted above shown in cartoon and stick mode for the Closed_Ab (slated blue) and Open_Ms (light green) structures. The distances shown were measured between two diagonally opposite residues. It is clear that the narrow constrictions at HC2 and HC3 (6.62 and 4.81 Å) seen in the closed structure are not present in the open structure (where the equivalent distances are 14.84 and 17.18 Å).One of the two NavMs pore structures (Bagnéris et al., 2013) has been designated to be in the fully open state (Open_Ms) by virtue of its transmembrane pathway being of sufficient diameter along the full length of the pore, from the extracellular surface to the intracellular surface (Fig. 2, A and B), to enable the translocation of sodium ions across the cell membrane (Ulmschneider et al., 2013). It has all four monomers with a splayed conformation at their intracellular ends. In contrast, in the partially open (pOpen_Ms) structure (McCusker et al., 2012) (which lacks the last 37 residues of its C-terminal domain [CTD]), only one of the four monomers of the tetramer adopts an open splayed conformation, with the result that the activation gate is only partially open; it is not sufficient to permit ion translocation but is more widely open than the closed structure (Fig. 2 A). The other three monomers in this structure are effectively equivalent to the closed-state pore conformations. A model “open” structure was constructed (McCusker et al., 2012) based on using the most open of the four monomers to produce a symmetric tetramer, which was very similar to the structure later determined of the fully open state (Bagnéris et al., 2013). In the partially open structure, the distal end of the CTD, which has been proposed to form a stabilizing four-helix coiled-coil (Powl et al., 2010; Irie et al., 2012; Bagnéris et al., 2013; Shaya et al., 2014), is absent, although it is present in the fully open structure; this may have enabled the uncoupled opening of the four S6 helices, and this asymmetry suggests there may be an asynchrony in the functional transition between states.The two inactivated forms of NavAb (Inactivated_Ab; Payandeh et al., 2012) and NavRh (Inactivated_Rh; Zhang et al., 2012) have been so designated based on their closed pores and collapsed SFs (although the authors of the NavAb structure were careful to describe it as being in a “potentially” inactivated state, largely because there was no confirming functional data). Both of these structures have closed activation gates, which are very similar to the Closed_Ab structure, but their SFs are unlike any of the other structures, and would be too narrow to enable ions to enter the pore (Figs. 2 A, middle, and 4, C and D, and Table S1).Open in a separate windowFigure 4.Comparisons of the SF regions. (Left and middle columns) Views from the extracellular surface showing the size of the SF central holes, defined as the white areas in the middle of each structure. (Right column) Side view superpositions of the SFs in cartoon and stick depictions, in each case comparing the Open_Ms structure (light green) with the corresponding structure in that row. The identities of the SF residues (TLES) are indicated. Only two monomers are shown for clarity. (A; left) Open_Ms (pale green) and (middle) pOpen_Ms (turquoise) structures. (B; middle) Closed_Ab (slate) and overlay (right) comparison of Closed_Ab and Open_Ms. (C) As in B for Inactivated_Ab (AB tetramer; gray). (D) As in B for Inactivated_Ab (CD tetramer; gray). (E) As in B for Closed_Ae (raspberry). The distances between the narrowest parts of the SFs in each case are given in Table S1.

Structural transitions associated with opening and closing

The extracellular turret and vestibule surfaces (comprised of the S5–S6 linker, including the P1, P2, and SF regions; Fig. 1) are very similar in the Open_Ms and Closed_Ab pores, suggesting that these are not substantially involved in the gating transition. In addition, the N-terminal ends of their S6 helices (Fig. 3 B) are virtually superimposable, as are the C-terminal ends of the S5 helices. However, the intracellular surface features differ substantially (Fig. 3 A) as a result of differences in the relative orientations and splaying of the C-terminal ends of their S6 helices away from the pore axis. Also, the N-terminal ends of the S5 helices appear to have moved slightly with respect to each other. Had the open pore structure had its VS attached, this latter movement would have impinged on the S4–S5 linker (Fig. 3 A, circle) of an adjacent monomer. We speculate that the reason the NavAb pore is closed is that the linker region is in the closed conformation, and that if the linker had been more closely coupled to the VS in its open conformation in the crystal, the NavAb pore structure would also have been of an open conformation.Open in a separate windowFigure 3.Differences between the open and closed structures. (A) Comparison of the Open_Ms (green) pore and the Closed_Ab (slate blue) crystal structures, depicted in cylindrical mode viewed from the intracellular surface. The equivalent residue numbers for the pore domain were G129 to M221 in NavMs and G130 to M222 in NavAb. Three-dimensional alignments (in all cases the least-squares superpositions were done using residues 145–198 [or their sequence equivalents] at the top of helices S5 and S6) and figures were made using PyMOL software (Schrödinger, LLC). The motions associated with the S5 and S6 helices are indicated by the small and large arrows, respectively. One of the Closed_Ab monomers is shown in gray, so that it can be seen that the region of the S4–S5 linker that the S5 helix in the open state would impinge on (magenta circle) is in the adjacent, not the same, monomer. (B) The Cα carbons of the S6 helixes (in stick motif) showing that the Closed_Ab (slate blue), Inactivated_Ab (gray), and Closed_Ae (red) structures overlay closely, but that the Open_Ms (green) deviates from the other structures starting at residue T206. (C) Plot of the delta phi (blue) and delta psi (red) angles in the S6 helix as a function of residue number. Values are those of Open_Ms structure (PDB accession no. 3ZJZ-A chain) minus those of the Closed_Ab structure (3RVY-A chain), demonstrating that the differences start after residue T206 in NavMs and continue to the end of the S6 helix. The single peak at around residue 155 is not related to the transition but simply arises from different interactions of the two proteins, with the different crystallization detergent molecules present adjacent to this site. (D) Secondary structure alignments compared using the 2Struc server (Klose et al., 2010). The position corresponding to the T206 residue in helix S6 is indicated by the black box in both parts. (Top) Open_Ms versus Closed_Ab. The locations of the S5 and S6 helices are indicated by the horizontal green bars. Both structures have essentially identical secondary structures, even around T206. (Bottom) Open_Ms versus Inactivated_Ab. The biggest differences are at the top of S5 (purple box) and in the turret loop (cyan box), not in helix S6 nor the region around T206.When the structures of the S6 pore helices of all the conformations are examined in detail (Fig. 3 B), the differences at the quaternary structural level between the open and closed (and inactivated) structures appear to arise from a twist in the middle of the S6 helix structures beginning at residue T206. The extracellular ends of all of the S6 helices in all of the closed and inactivated structures superimpose closely (Fig. 3 B), but the Open_Ms structure is an outlier. This results from a change in the backbone Ramachandran angles between the open and closed forms, which perpetrates along the C-terminal end of S6 starting at T206 (Fig. 3 C). The consequence of this deviation is that the C-terminal end of the S6 helix in the Open_Ms structure moves away from the axis of the top part of the helix, thereby opening the activation gate. In the lower resolution pOpen_Ms structure, residue T209 appeared to be the focal point of the bend, but in the higher resolution fully Open_Ms structure, it is clear that the beginning of the bend occurs one turn earlier in S6, at T206. Notably, the changes between open and closed structures are subtle enough so that they do not change the secondary structure (Fig. 3 D) of the S6 helix. What starts as a relatively small change in the middle of the helix is translated to a motion of >5 Å at the end of S6 that forms the activation gate. This suggests a simple (energetically inexpensive) mechanism for opening and closing: a twist in the backbone that does not disrupt the hydrogen bonding pattern is sufficient to open the gate. It does not require major rearrangements of the rest of the structure and is thus compatible with a rapid opening, such as that which gives rise to the initial phase of the action potential in excitable cells. The asynchrony of the motions of the four monomers, as suggested by the partially open structure, suggests that there may also be an asynchrony of the process of opening.The open and closed structures have very similar cavity regions (Fig. 2 A), with a minor hydrophobic constriction (designated HC1) near the bottom of the cavity (Fig. 2 A) near residue I215 in NavMs (equivalent to V213 in NavAb) that is not sufficient to prevent ions continuing their passage. However, the Closed_Ab form has two additional major hydrophobic constrictions (HC2 and HC3) further down toward the extracellular surface (Fig. 2 A) at approximately the level of residues M221 and I217 (numbering according to NavAb sequence; notably, the latter corresponds to the site of the mutation to a cysteine in the NavAb construct). Neither of these constrictions is seen in the open NavMs structure. HC3 (which obscures the exit) would exclude passage of even totally dehydrated sodium ions in the closed structure.Because the SFs of NavMs and NavAb have identical sequences (TLESWS; Fig. 1), they can be directly compared. The SFs of the Open_Ms (Fig. 4 A) and Closed_Ab (Fig. 4 B) structures are superimposable (Fig. 4 B, right), thus indicating that the closing of the activation gate, which prevents ion translocation, does not affect the potential entry of ions into the pore. In contrast, the SFs of the inactivated forms (Fig. 4, C and D) are very different from those of both the open and closed states, suggesting that they are altered during the inactivation process.

The structure and role of the CTD in opening and closing

All of the initial crystal structures determined (NavAb, NavMs, and NavRh) have elucidated the nature of the transmembrane domains (effectively to the end of the S6 helix) of the channels, but the structures of their CTDs (beyond the end of helix S6) were not interpretable, although these regions were present in the proteins used to produce the crystals. The structure of the CTD of the open conformation was first determined not by crystallography but by a combination of spectroscopic methods (Powl et al., 2010; Bagnéris et al., 2013), and shown to have a disordered region adjacent to the activation gate and a distal coiled-coil region (Figs. 1 and ​and5);5); molecular dynamics calculations (Bagnéris et al., 2013) suggested that flexibility of the region adjacent to the activation gate in the open state could account for the lack of defined structure in the crystal. The CTD in the closed NavAe pore (Shaya et al., 2014) is the only other such domain that has been defined structurally, this time by crystallographic means. Like the open CTD, its distal end formed a coiled-coil, but unlike the open CTD, the proximal end of the structure, nearest to the activation gate, was helical and relatively well ordered. These observations suggested that although the C-terminal end of the CTD remains unchanged in the two states, the end adjacent to the activation gate undergoes an ordered-to-disordered transition when the gate is opened (Fig. 5), thereby accommodating the separation movement at the end of the S6 helix that forms the gate, without uncoiling the distal coiled-coil that has been proposed to have a structural role in stabilizing the tetrameric structure in the membrane (Mio et al., 2010; Powl et al., 2010). Functional studies on the NavMs channel (Bagnéris et al., 2013), as well as on another orthologue (NavSulP from Sulfitobacer pontiacus; Irie et al., 2012), have indicated that this region (especially the EEE motif near the top of the CTD) plays a role in inactivation, recovery, and closing, perhaps by promotion of the conformational change of the S6 helix. In addition, in the NavAe pore structure, there is some density in the extracellular region beyond the end of the transmembrane segments that could be a calcium or other cation. Domain DIV of the human Nav1.4 sodium channel also possesses a similar negatively charged region just after its activation gate (Fig. 1), which appears to be a common feature in the prokaryotic sodium channels.Open in a separate windowFigure 5.Schematic diagram of (1) closed, (2) open, and (3) inactivated states of prokaryotic sodium channels based on crystallographic studies. Only two monomers are shown in each figure for clarity. The color scheme is as in Fig. 1. The VS S1–S4 helices are depicted as cyan bars, the S4–S5 linker is an orange bar, the S5–S6 helices of the pore region are green bars, and the coiled-coil region of the CTD is gray. The extracellular negatively charged region in S2 is in a magenta circle, and the intracellular negatively charged regions in S2 and S3 are in red circles. The four arginines in S4 involved in the gating charge transfer across the membrane through sequential interactions, with the extracellular negatively charged region and the intracellular negatively charged region represented by a “+.” The residues forming the SF are indicated by red boxes. The residue in purple is the start of the S6 twist that results in the open gate; the residue in yellow is the third hydrophobic constriction site in the closed form, which is not constricted in the open form, and indicates the position of the activation gate. The white bars represent the helical region present in the CTD in the closed and inactivated structures, and the corresponding dotted gray lines are the disordered CTD linker region in the open conformation.

What produces the open and closed states in the crystals?

An obvious suggestion as to what causes NavMs pore structure to be open is that it is missing the constraints provided by the VS and S4–S5 linker regions present in the full-length channel structures. However, this cannot be the only reason, as the two structures of pore-only constructs, NavMs and NavAe, are in different states (NavMs is open, whereas NavAe is closed). It is also not because of the presence of CTD structures because, again, the CTDs are present in both of these two structures, albeit in different conformations. Furthermore, the partially open NavMs construct is missing the distal end of its CTD, yet one of its monomers is in the open state. The CTDs are present but not visible in any of the other structures. It is possible that the different CTD structures have a role in driving the transition (for example because of the different conformations at their N-terminal ends), but this cannot be determined from the structures in hand. It could also be because of the differences in crystal packing that enable the NavMs pores to open: in the NavMs crystals, the transmembrane regions of different pores are aligned next to each other, as if in a membrane bilayer, with a large enough gap to fit the CTD between two separate bilayers and thus allow sufficient room for the bottom of the pore to open. In the NavAe crystals, the CTDs are sandwiched between other CTDs and pore regions of symmetrically related molecules, rigidifying the whole structure and potentially preventing the opening.The different structures in the crystals may be caused by (or alternatively, result in) the presence or absences of ions within the pore, as sodium ions are visible in the SF of only the NavMs open-state crystal forms. However, in a version of the NavAb orthologue engineered to be calcium selective (Tang et al., 2014), the presence of calcium ions in that pore did not produce an open state, so the mere presence of ions appears not to be sufficient to produce an open pore. The most likely candidate in vivo is the VS-pore linker between helices S4 and S5, which could act as a lever, either pushing or pulling the pore domain when the VS is activated to its “up” position. But the linker is not present in either the NavMs open or partially open structures, nor in the NavAe closed structures. And although it is present in the NavAb structures, it is in the “wrong” position to influence the opening of the pore, so it may be a contributing, but not the only, driving force. This is an important issue and will need to be resolved in the future to enable a full understanding of the opening and closing processes.

Structural transitions associated with inactivation

Comparisons of the structure of the Inactivated_Ab channel with both the Closed_Ab and Open_Ms structures indicate areas of the protein primarily involved in the inactivation transition. The Inactivated_Ab structure has a very similar upper cavity region to both the open and closed structures, and an asymmetric inactivation gate conformation that, like the symmetrical gate of the closed state, is occluded. The activation gate structures of the S6 helices of both the Inactivated_Ab and Inactivated_Rh channels closely overlay the Closed_Ab (Fig. 3 B) but not the Open_Ms structures, suggesting that the hinge necessary to open the gate has not been activated. Although their S6 conformations appear to be the same, the structures of the two types of Inactivated_Ab tetramers (“AB” and “CD”) and the Closed_Ab form do exhibit differences at their C-terminal ends: the inactivated forms have between two and eight fewer ordered residues visible in the crystals, suggesting that they exhibit more mobility or flexibility in this region. Because the CTD has not been resolved in any of the inactivated form crystals, we cannot make any conclusions about whether the regions adjacent to the transmembrane helices are disordered (as in the open state) or helical (as in the closed state). Nor can we discern even if they exhibit the same coiled-coil structures at their distal ends that are present in both the open and closed forms.In contrast, the SF region of the Inactivated_Ab structure (as well as that of the inactivated NavRh structure) is very different from either the open or closed states (Fig. 4). It has been described as “collapsed,” and indeed it is of insufficient diameter to enable a full dehydrated sodium ion to enter into the pore, much less exit it. Because in this case comparisons can be made for two crystal structures of the same orthologue (NavAb), the differences in the inactivated and closed structures cannot be attributed to SF sequence differences. The changes involve both the P1 and P2 helices as well as the SF itself, and involve modest differences in the secondary structures of the turret and top of S5 (Fig. 3 D). The observation that structural changes associated with the inactivated structure primarily involve the SF region corresponds well with earlier functional studies suggesting that the region close to the SF is responsible for inactivation (Pavlov et al., 2005). Interestingly, too, is the correspondence with studies done many years ago using circular dichroism spectroscopy (Cronin et al., 2003) on eukaryotic (electric eel) sodium channels that were induced to adopt open, closed, and inactivated states through the use of toxins and drugs. Those functional studies indicated that the secondary structures of the open and closed channels were surprisingly very similar but that the secondary structures of the inactivated form was considerably different. This also corresponds with the structural observations for the prokaryotic channels, where there are no apparent changes in secondary structure between open and closed states but significant differences between the open and the inactivated state.The structural comparisons thus suggest that inactivation closes not only the extracellular but also the intracellular ends of the transmembrane passageway, although the extracellular changes appear to be the defining ones for the inactivation state.

Structural models for sodium channel opening–closing–inactivation mechanisms

Early models (e.g., Armstrong and Bezanilla, 1973; Hille, 1975; Lehmann-Horn and Jurkat-Rott, 1999; see also Hille, 2001, and Armstrong, 2007) proposed for eukaryotic sodium channel charge movement and gating were based primarily on functional observations and were produced, for the most part, before either any sodium channel sequences were determined or any crystal structures of any members of the voltage-gated cation channel superfamily were available. Structural models included suggestions such as the constriction of the SF or the shutting of two flaps (Lehmann-Horn and Jurkat-Rott, 1999), one at the extracellular surface and one at the intracellular surface, as being responsible for inactivation and closing, respectively. With the sequencing of the first eukaryotic sodium channel (Noda et al., 1984), three-dimensional models were developed that proposed the involvement of specific regions for the activation and inactivation processes (Guy and Seetharamulu, 1986; Yu et al., 2005). Many other approaches, including mutational effects, interactions with toxins, drug and ligand binding, and various spectroscopic studies, have since contributed to structure–function models of sodium channels (Catterall, 2012). More recently, the crystal structures of potassium channels (Doyle et al., 1998; Long et al., 2007) provided structural templates and information for understanding the electromechanical coupling and mechanism of their opening and closing. The sequence homology of sodium channels to these other members of the voltage-gated ion channel superfamily suggested that they would share a common architecture, albeit with very different SF regions, and led to more detailed models for sodium channel structures and functions (Yarov-Yarovoy et al., 2001; Shafrir et al., 2008; Zarrabi et al., 2010).Soon after the prokaryotic sodium channels were identified as simpler target molecules for biophysical studies (Ren et al., 2001), and well before any of the prokaryotic orthologue crystal structures were determined, a hinge model for NaChBac (the first sodium channel orthologue whose sequence was determined) was proposed. This model for opening of the activation gate, like the models for the MthK potassium channel (Jiang et al., 2002), was based on the presence of a glycine residue in the middle of the S6 helix. Because glycines lack side chains, this type of residue could act as a flexible pivot for the top and bottom of the S6 helix, which could then move to open the gate. This model was supported by functional studies that showed that mutations of the glycine altered the rate of inactivation and recovery (Ito et al., 2004; Zhao et al., 2004), and spectroscopic studies that showed that changing the glycine to a conformationally less flexible serine produced a more thermally stable protein (O’Reilly et al., 2008); later molecular dynamics calculations (Barber et al., 2012) also supported a hinge-bending model involving this glycine. This model for the mechanism became less favored, however, after the determination of the sequences of other orthologues, as they did not have a glycine in the equivalent position.The solution of the crystal structure of the prokaryotic sodium channel NavAb, with sequence and structural homology (especially in the VS regions) to single-domain tetrameric prokaryotic and eukaryotic potassium channel structures, prompted the suggestion (Payandeh et al., 2011) that the mechanism behind the coupling and pore opening might be similar to another model proposed for K⁺ channels (Long et al., 2007). Indeed, the VS of the Closed_Ab structure aligns well with the VS of the open Kv1.2 structure, although they diverge in the pore, specifically at the beginning of the base of the S5 helix, and their SFs are completely different both in sequence and in structure (one using side chains and backbone carbonyls, and the other narrower one using only backbone carbonyls to create the ion-binding sites). That model had the VS and pore regions moving as modular units, with the S4–S5 linker movement causing the conformational change. In the sodium channel case, the proposed change was a rotation-like motion of the S5 and S6 helices relative to each other, opening the pore in an iris-like dilation motion. This bears semblance to the twisting mechanism proposed for the opening and closing of the nonspecific ion channel in nicotinic acetylcholine receptors (Unwin, 2003), where the transmembrane helices of adjacent subunits change positions relative to each other, primarily on one surface, producing a rotational squeezing and release mechanism that changes the size of the pore along its length.

A new structure-based model for the opening–closing–inactivation mechanisms

The observed structural differences described above between Open_Ms and Closed_Ab do not support a model based on a modular S5–S6 iris-like motion. In the prokaryotic sodium channel crystal structures, the C-terminal end of the S5 helix exhibits very little change between the two different states, and nothing suggests its movement is coupled with that of the S6 helix in either the same or an adjacent subunit within the tetramer. Instead, the opening and closing appears to involve a twist/bend in the middle of the S6 helix, which moves the activation gate to occlude or open the intracellular surface, enabling ion egress from the cavity (McCusker et al., 2012; Bagnéris et al., 2013; Video 1). This is remarkably similar to the earlier hinge model, except that there is no glycine at the hinge position. Instead, a threonine (T206 in the NavMs numbering scheme) is located at the juncture, in an equivalent position in the sequence to the glycine found in the NaChBac orthologue. The structure of NavMs shows that there would be sufficient space to enable the backbone motion even with the threonine side chain, as the Cβ substituent does not impinge on the region that moves. However, the enhanced flexibility of a glycine residue in NaChBac could be responsible for its faster rate of opening (Ren et al., 2001) than NavMs (Ulmschneider et al., 2013).Because the S1–S4 helices of the VS structure are already in the open conformation in the Closed_Ab and Inactivated_Ab structures, it initially seemed likely that the S4–S5 linker in these structures would be in a conformation similar to that which it would adopt in the open pore state. However, comparisons of the Open_Ms and Closed_Ab structures indicate that the positions of especially the S5 helices in the open pore would mitigate against the linker in the Closed_Ab structure being completely in the activated state because the end of the open state S5 helix would impinge on the linker. It is therefore suggested that the linker is in a “primed” state but that it moves in concert with the S5 and S6 helices as the activation gate is opened.The structure-based model for the slow inactivation process, like several of the early models (Lehmann-Horn and Jurkat-Rott, 1999), appears to primarily involve a closing of the SF to ion entry on the extracellular surface (Fig. 5). Rather than a flap folding over the entry, however, it appears that the SF itself folds in toward the center of the ion pathway, blocking entry of any sodium ions into the pore. It also appears, contrary to the double-flap mechanism, that the activation gate in the inactivated form is closed rather than open, although this could be a consequence of the crystallization and packing conditions (Video 2).Flexibility at the C-terminal region of S6 thus appears to be the defining feature of the prokaryotic voltage-gated sodium channel. It may be at least partially responsible for channel opening and for the cascade of events that occur at the SF and P loops during inactivation. The presence of a flexible linking region to the CTD would enable the opening and closing to occur without uncoiling the distal coiled-coil region, which may play an important role in stabilizing the tetrameric quaternary structure of the prokaryotic sodium channels (Video 3). This feature is not present (nor necessary) in eukaryotic sodium channels, which are single polypeptide chains. Instead, they seem to have a CTD that plays an important role in fast inactivation, a function that prokaryotic sodium channels do not exhibit.

Similarities and differences in the activation mechanisms of prokaryotic and eukaryotic sodium channels

Prokaryotic and eukaryotic sodium channels exhibit considerable homologies in the sequences of their transmembrane regions (Fig. 1), especially in the C-terminal ends of their S6 helices, although their SFs and CTDs are entirely different. Likewise, both exhibit transitions between activation, recovery, and slow inactivation functional states, albeit with different kinetics (as well as the absence of fast inactivation in prokaryotic channels). Therefore, it is of interest to consider if they might involve similar structural features in a common mechanism for opening and closing.New studies (Bagnéris et al., 2014) on the structure and electrophysiological consequences of binding channel blocker drugs to prokaryotic and human Na_v1.1 channels have shown the parallel nature of their block by a range of ligands that bind in the hydrophobic cavity and prevent passage of ions from the extracellular to intracellular surfaces. This apparently occurs not by closing the activation gate but by the hydrophobic ligands creating a barrier to exit at the end of the SF region, and is entirely consistent with the mechanisms described above.Furthermore, a recent study was done to test whether a similar mechanism of opening and closing could be operating in human Nav1.4 and prokaryotic sodium channels (Oelstrom et al., 2014). Channels with their fast inactivation mechanism disabled could be functionally held in either the open or closed state. An accessibility labeling technique was used to probe sites in S6 of domain IV (Fig. 1). Residues C terminal to the proposed activation gate were accessible to labeling from the intracellular side in both the open and closed states, whereas residues above this were only accessible in the open state. In the open state, the channel could be labeled up to the ring of hydrophobic residues immediately preceding the equivalent to residue T206 in the prokaryotic channels, again consistent with a mechanism involving a hinge mechanism in the middle of S6. These results strongly support the type of mechanism for opening and closing presented above, with a flexible hinge region that opens the pore up to the intracellular compartment in the open state, but not in the closed state.However, it is important to note that significant differences in the activation/inactivation and selectivity mechanisms do exist between prokaryotic and eukaryotic channels (see, for example, Ahern, 2013; Goldschen-Ohm et al., 2013; Finol-Urdaneta et al., 2014, for detailed discussions). These arise in large part because of the asymmetry of the pseudotetrameric eukaryotic structures as opposed to the simpler homotetramers that comprise the prokaryotic channels. They result in asynchronous movements of VSs, asymmetric ion-binding sites, and novel loops between the different domains that have specialized roles, for example in fast inactivation, which are not seen in the prokaryotic channels. Nevertheless, the availability of several crystal forms of prokaryotic voltage-gated sodium channels in different conformational states has vastly increased our knowledge of the structure–function relationships for these channels, and provides new insight into their mechanisms of opening, closing, and inactivation (Fig. 5). These, in turn, may inform our understanding of both the structure and function of sodium channels in general. The new model proposed, based on the detailed structures of prokaryotic channels in crystal environments, will, however, await confirmation by further functional experiments on channels in their biological context, as well as structural studies of eukaryotic channels.