A model of sodium channels

We have explored the permeation and blockage of ions in sodium channels, relating the channel structure to function using electrostatic profiles and Brownian dynamics simulations. The model used resembles the KcsA potassium channel with an added external vestibule and a shorter selectivity filter. The electrostatic energy landscape seen by permeating ions is determined by solving Poisson's equation. The two charged amino acid rings of Glu-Glu-Asp-Asp (EEDD) and Asp-Glu-Lys-Ala (DEKA) around the selectivity filter region are seen to play a crucial role in making the channel sodium selective, and strongly binding calcium ions such that they block the channel. Our model closely reproduces a range of experimental data including the current-voltage curves, current-concentration curves and blockage of monovalent ions by divalent ions.     

Electrostatic basis of valence selectivity in cationic channels

We examine how a variety of cationic channels discriminate between ions of differing charge. We construct models of the KcsA potassium channel, voltage gated sodium channel and L-type calcium channel, and show that they all conduct monovalent cations, but that only the calcium channel conducts divalent cations. In the KcsA and sodium channels divalent ions block the channel and prevent any further conduction. We demonstrate that in each case, this discrimination and some of the more complex conductance properties of the channels is a consequence of the electrostatic interaction of the ions with the charges in the channel protein. The KcsA and sodium channels bind divalent ions strongly enough that they cannot be displaced by other ions and thereby block the channel. On the other hand, the calcium channel binds them less strongly such that they can be destabilized by the repulsion of another incoming divalent ion, but not by the lesser repulsion from monovalent ions.     

Simulations of calcium channel block by trivalent cations: Gd competes with permeant ions for the selectivity filter

Current through L-type calcium channels (Ca_V1.2 or dihydropyridine receptor) can be blocked by micromolar concentrations of trivalent cations like the lanthanide gadolinium (Gd³⁺). It has been proposed that trivalent block is due to ions competing for a binding site in both the open and closed configuration, but possibly with different trivalent affinities. Here, we corroborate this general view of trivalent block by computing conductance of a model L-type calcium channel. The model qualitatively reproduces the Gd³⁺ concentration dependence and the effect that substantially more Gd³⁺ is required to produce similar block in the presence of Sr²⁺ (compared to Ba²⁺) and even more in the presence of Ca²⁺. Trivalent block is explained in this model by cations binding in the selectivity filter with the charge/space competition mechanism. This is the same mechanism that in the model channel governs other selectivity properties. Specifically, selectivity is determined by the combination of ions that most effectively screen the negative glutamates of the protein while finding space in the midst of the closely packed carboxylate groups of the glutamate residues.     

Brownian dynamics study of flux ratios in sodium channels

Measurements of unidirectional fluxes in ion channels provide one of the experimental methods for studying the steps involved in ion permeation in biological pores. Conventionally, the number of ions in the pore is inferred by fitting the ratio of inward and outward currents to an exponential function with an adjustable parameter known as the flux ratio exponent. Here we investigate the relationship between the number of ions in the pore and the flux ratio exponent in a model sodium channel under a range of conditions. Brownian dynamics simulations enable us to count the precise number of ions in the channel and at the same time measure the currents flowing across the pore in both directions. We show here that the values of the flux ratio exponent n′ ranges between 1 and 3 and is highly dependent on the ionic concentrations in which measurements are made. This is a consequence of the fact that both inward and outward currents are susceptible to saturation with increasing concentration. These results indicate that measurements of the flux ratio exponent cannot be directly related to the number of ions in the pore and that interpretation of such experimental measurements requires careful consideration of the conditions in which the study is made.     

Contribution of residues in second transmembrane domain of ASIC1a protein to ion selectivity

Acid-sensing ion channels (ASICs) are proton-gated cation-selective channels expressed in the peripheral and central nervous systems. The ion permeation pathway of ASIC1a is defined by residues 426–450 in the second transmembrane (TM2) segment. The gate, formed by the intersection of the TM2 segments, localizes near the extracellular boundary of the plasma membrane. We explored the contribution to ion permeation and selectivity of residues in the TM2 segment of ASIC1a. Studies of accessibility with positively charged methanethiosulfonate reagents suggest that the permeation pathway in the open state constricts below the gate, restricting the passage to large ions. Substitution of residues in the intracellular vestibule at positions 437, 438, 443, or 446 significantly increased the permeability to K⁺ versus Na⁺. ASIC1a shows a selectivity sequence for alkali metals of Na⁺>Li⁺>K⁺≫Rb⁺>Cs⁺. Alanine and cysteine substitutions at position 438 increased, to different extents, the relative permeability to Li⁺, K⁺, Rb⁺, and Cs⁺. For these mutants, ion permeation was not a function of the diameter of the nonhydrated ion, suggesting that Gly-438 encompasses an ion coordination site that is essential for ion selectivity. M437C and A443C mutants showed slightly increased permeability to K⁺, Rb⁺, and Cs⁺, suggesting that substitutions at these positions influence ion discrimination by altering molecular sieving. Our results indicate that ion selectivity is accomplished by the contribution of multiple sites in the pore of ASIC1a.     

Elucidation of the molecular basis of selective recognition uncovers the interaction site for the core domain of scorpion alpha-toxins on sodium channels

Neurotoxin receptor site-3 at voltage-gated Na(+) channels is recognized by various peptide toxin inhibitors of channel inactivation. Despite extensive studies of the effects of these toxins, their mode of interaction with the channel remained to be described at the molecular level. To identify channel constituents that interact with the toxins, we exploited the opposing preferences of LqhαIT and Lqh2 scorpion α-toxins for insect and mammalian brain Na(+) channels. Construction of the DIV/S1-S2, DIV/S3-S4, DI/S5-SS1, and DI/SS2-S6 external loops of the rat brain rNa(v)1.2a channel (highly sensitive to Lqh2) in the background of the Drosophila DmNa(v)1 channel (highly sensitive to LqhαIT), and examination of toxin activity on the channel chimera expressed in Xenopus oocytes revealed a substantial decrease in LqhαIT effect, whereas Lqh2 was as effective as at rNa(v)1.2a. Further substitutions of individual loops and specific residues followed by examination of gain or loss in Lqh2 and LqhαIT activities highlighted the importance of DI/S5-S6 (pore module) and the C-terminal region of DIV/S3 (gating module) of rNa(v)1.2a for Lqh2 action and selectivity. In contrast, a single substitution of Glu-1613 to Asp at DIV/S3-S4 converted rNa(v)1.2a to high sensitivity toward LqhαIT. Comparison of depolarization-driven dissociation of Lqh2 and mutant derivatives off their binding site at rNa(v)1.2a mutant channels has suggested that the toxin core domain interacts with the gating module of DIV. These results constitute the first step in better understanding of the way scorpion α-toxins interact with voltage-gated Na(+)-channels at the molecular level.     

Kinetic properties of single sodium channels in rat heart and rat brain

Single Na channel currents were compared in ventricular myocytes and cortical neurons of neonatal rats using the gigaseal patch-clamp method to determine whether tissue-specific differences in gating can be detected at the single-channel level. Single-channel currents were recorded in cell-attached and excised membrane patches at test potentials of -70 to -20 mV and at 9-11 degrees C. In both cell-attached and excised patches brain Na channel mean open time progressively increased from less than 1 ms at -70 mV to approximately 2 ms at -20 mV. Near threshold, single openings with dispersed latencies were observed. By contrast, in cell-attached patches, heart Na channel mean open time peaked near -50 mV, was three times brain Na channel mean open time, and declined continuously to approximately 2 ms at -20 mV. Near threshold, openings occurred frequently usually as brief bursts lasting several milliseconds and rarely as prolonged bursts lasting tens of milliseconds. Unlike what occurs in brain tissue where excision did not change gating, in excised heart patches both the frequency of prolonged bursting and the mean open time of single units increased markedly. Brain and cardiac Na channels can therefore be distinguished on the basis of their mean open times and bursting characteristics.     

Novel Residues Lining the CFTR Chloride Channel Pore Identified by Functional Modification of Introduced Cysteines

Substituted cysteine accessibility mutagenesis (SCAM) has been used widely to identify pore-lining amino acid side chains in ion channel proteins. However, functional effects on permeation and gating can be difficult to separate, leading to uncertainty concerning the location of reactive cysteine side chains. We have combined SCAM with investigation of the charge-dependent effects of methanethiosulfonate (MTS) reagents on the functional permeation properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channels. We find that cysteines substituted for seven out of 21 continuous amino acids in the eleventh and twelfth transmembrane (TM) regions can be modified by external application of positively charged [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium [2-sulfonatoethyl] MTS (MTSES). Modification of these cysteines leads to changes in the open channel current–voltage relationship at both the macroscopic and single-channel current levels that reflect specific, charge-dependent effects on the rate of Cl⁻ permeation through the channel from the external solution. This approach therefore identifies amino acid side chains that lie within the permeation pathway. Cysteine mutagenesis of pore-lining residues also affects intrapore anion binding and anion selectivity, giving more information regarding the roles of these residues. Our results demonstrate a straightforward method of screening for pore-lining amino acids in ion channels. We suggest that TM11 contributes to the CFTR pore and that the extracellular loop between TMs 11 and 12 lies close to the outer mouth of the pore.     

Inhibitory effect of the recombinant Phoneutria nigriventer Tx1 toxin on voltage-gated sodium channels

Phoneutria nigriventer toxin Tx1 (PnTx1, also referred to in the literature as Tx1) exerts inhibitory effect on neuronal (Na_V1.2) sodium channels in a way dependent on the holding potential, and competes with μ-conotoxins but not with tetrodotoxin for their binding sites. In the present study we investigated the electrophysiological properties of the recombinant toxin (rPnTx1), which has the complete amino acid sequence of the natural toxin with 3 additional residues: AM on the N-terminal and G on the C-terminal. At the concentration of 1.5 μM, the recombinant toxin inhibits Na⁺ currents of dorsal root ganglia neurons (38.4 ± 6.1% inhibition at −80 mV holding potential) and tetrodotoxin-resistant Na⁺ currents (26.2 ± 4.9% at the same holding potential). At −50 mV holding potential the inhibition of the total current reached 71.3 ± 2.3% with 1.5 μM rPnTx1. The selectivity of rPnTx1 was investigated on ten different isoforms of voltage-gated sodium channels expressed in Xenopus oocytes. The order of potency for rPnTx1 was: rNa_V1.2 > rNa_V1.7 ≈ rNa_V1.4 ≥ rNa_V1.3 > mNa_V1.6 ≥ hNa_V1.8. No effect was seen on hNa_V1.5 and on the arthropods isoforms (DmNa_V1, BGNa_V1.1a and VdNa_V1). The IC₅₀ for Na_V1.2 was 33.7 ± 2.9 nM with a maximum inhibition of 83.3 ± 1.9%. The toxin did not alter the voltage-dependence of channel gating and was effective on Na_V1.2 channels devoid of inactivation. It was ineffective on neuronal calcium channels. We conclude that rPnTx1 has a promising selectivity, and that it may be a valuable model to achieve pharmacological activities of interest for the treatment of channelopathies and neuropathic pain.     

Slow actions of hyperpolarization on sodium channels in the membrane of myelinated nerve

The mean sodium current, $\text{I}$, and the variance of sodium current fluctuations, var, were measured in myelinated nerve during a depolarization to $\text{V = 40}\text{mV}$ applied from the resting potential ($\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= 0}$) or from a hyperpolarizing holding potential $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= ?28}\text{mV}$. From $\text{I}$ and var the relative variations in the number $\text{N}$ and the conductance γ of sodium channels following changes of the holding potential were calculated. Hyperpolarizing the membrane from $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= 0}$ to ?28 mV increased $\text{N}$ by a factor of 3.7, whereas γ decreased by a factor of 0.53. These actions of holding potential on sodium channels develop slowly since 500 ms prepulses to 0 or ?28 mV do not alter the values of $\text{N}$ and γ.     

Sodium permeability and sensitivity induced by mutations in the selectivity filter of the KcsA channel towards Kir channels

The bacterial potassium (K⁺) channel KcsA provides an attractive model system to study ion permeation behavior in a selective K⁺-channel. We changed residue at the N-terminal end of the selectivity filter of KcsA (T74V) to its counterpart in inwardly rectifying K⁺-channels (Kir). The tetramer was found to be stable as unmodified KcsA. Under symmetrical and asymmetrical conditions, Na⁺ increased the inward current in the virtual absence of K⁺ however outward currents were nearly abolished which could be recovered upon internal K⁺ addition. Na⁺ also drastically increased the channel open time either in the presence or virtual absence of K⁺. Furthermore, the T74V mutation decreased the internal Ba²⁺ affinity of the channel possibly by binding to a K⁺ site in the pore. In additional experiments, another point mutation V76I in T74V mutant was carried out thus the selectivity filter resembled more the selectivity filter of Kir channels. The mutant tetramer was converted into monomers as determined by conventional gel electrophoresis. However, native like gel electrophoresis, Trp fluorescence and acrylamide quenching experiments indicated that this mutant still formed a tetramer and apparently adopted similar folding properties as unmodified KcsA. Single-channel experiments further demonstrated that the channel was selective for K⁺ over Na⁺ as Na⁺ blocked channel currents. These data suggest that single point mutation T74V alters the selectivity filter and allows simultaneous occupancy and conduction of K⁺ and Na⁺ probably via ion–ion interaction in the pore. In contrast, both mutations (T74V and V76I) in the same molecule seem to reorganize the pore conformation which controls the overall stability of a selective K⁺-channel.     

The cation selectivity filter of the bacterial sodium channel,NaChBac

The Bacillus halodurans voltage-gated sodium-selective channel (NaChBac) (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001b. SCIENCE: 294:2372-2375), is an ideal candidate for high resolution structural studies because it can be expressed in mammalian cells and its functional properties studied in detail. It has the added advantage of being a single six transmembrane (6TM) orthologue of a single repeat of mammalian voltage-gated Ca(2+) (Ca(V)) and Na(+) (Na(V)) channels. Here we report that six amino acids in the pore domain (LESWAS) participate in the selectivity filter. Replacing the amino acid residues adjacent to glutamatic acid (E) by a negatively charged aspartate (D; LEDWAS) converted the Na(+)-selective NaChBac to a Ca(2+)- and Na(+)-permeant channel. When additional aspartates were incorporated (LDDWAD), the mutant channel resulted in a highly expressing voltage-gated Ca(2+)-selective conductance.     

Nav1.6 channels generate resurgent sodium currents in spinal sensory neurons

The Na(v)1.6 voltage-gated sodium channel has been implicated in the generation of resurgent currents in cerebellar Purkinje neurons. Our data show that resurgent sodium currents are produced by some large diameter dorsal root ganglion (DRG) neurons from wild-type mice, but not from Na(v)1.6-null mice; small DRG neurons do not produce resurgent currents. Many, but not all, DRG neurons transfected with Na(v)1.6 produce resurgent currents. These results demonstrate for the first time the intrinsic ability of Na(v)1.6 to produce a resurgent current, and also show that cell background is critical in permitting the generation of these currents.     

Permeability properties of ENaC selectivity filter mutants.

The epithelial Na(+) channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na(+) absorption. The amiloride-sensitive ENaC is highly selective for Na(+) and Li(+) ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the alphaS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K(+) ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted alphaS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position alpha589, indicating that alphaS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na(+) and Li(+). These findings demonstrate the critical role of the pore size at the alphaS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the alphaS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the alphaS589 side chain is oriented toward the subunit-subunit interface and that substitution of alphaS589 by larger residues increases the pore diameter by adding extra volume at the subunit-subunit interface.     

Quantal charge redistributions accompanying the structural transitions of sodium channels

Asymmetric displacement currents, I _g , associated with the gating of nerve sodium channels have been recorded in cell-attached macropatches of Xenopus laevis oocytes injected with exogenous mRNA coding for rat-brain-II sodium channels. The I _g properties were found to be similar to those of gating currents previously observed in native nerve preparations. I _g fluctuations were measured in order to ascertain the discreteness of the conformational changes which precede the channel opening. The autocorrelation of the fluctuations is consistent with a shot-like character of the elementary I _g contributions. The variance of the fluctuations indicates that most of the gating-charge movement that accompanies the activation of a single sodium channel occurs in 2 to 3 brief packets, each carrying an equivalent of about 2.3 electron charges.     

Extent of the selectivity filter conferred by the sixth transmembrane region in the CFTR chloride channel pore

Point mutations within the pore region of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^? channel have previously been shown to alter the selectivity of the channel between different anions, suggesting that part of the pore may form an anion 'selectivity filter'. However, the full extent of this selectivity filter region and the location of anion binding sites in the pore are currently unclear. As a result, comparisons between CFTR and other classes of Cl^? channel of known structure are difficult. We compare here the effects of point mutations at each of eight consecutive amino acid residues (arginine 334-serine 341) in the crucial sixth transmembrane region (TM6) of CFTR. Anion selectivity was determined using patch-clamp recording from inside-out membrane patches excised from transiently transfected mammalian cell lines. The results suggest that selectivity is predominantly controlled by a single site involving adjacent residues phenylalanine 337 and threonine 338, and that the selectivity conferred by this 'filter' region is modified by anion binding to flanking sites involving the more extracellular arginine 334 and the more intracellular serine 341. Other residues within this part of the pore play only minor roles in controlling anion permeability and conductance. Our results support a model in which specific TM6 residues make important contributions to a single, localized anion selectivity filter in the CFTR pore, and also contribute to multiple anion binding sites both within and on either side of the filter region.     

A novel benzazepinone sodium channel blocker with oral efficacy in a rat model of neuropathic pain

A series of benzazepinones were synthesized and evaluated for block of Na_v1.7 sodium channels. Compound 30 from this series displayed potent channel block, good selectivity versus other targets, and dose-dependent oral efficacy in a rat model of neuropathic pain.     

The up-regulation of voltage-gated sodium channels subtypes coincides with an increased sodium current in hippocampal neuronal culture model

Voltage-gated sodium channels (VGSC) have been linked to inherited forms of epilepsy. The expression and biophysical properties of VGSC in the hippocampal neuronal culture model have not been clarified. In order to evaluate mechanisms of epileptogenesis that are related to VGSC, we examined the expression and function of VGSC in the hippocampal neuronal culture model in vitro and spontaneously epileptic rats (SER) in vivo. Our data showed that the peak amplitude of transient, rapidly-inactivating Na⁺ current (I_Na,T) in model neurons was significantly increased compared with control neurons, and the activation curve was shifted to the negative potentials in model neurons in whole cell recording by patch–clamp. In addition, channel activity of persistent, non-inactivating Na⁺ current (I_Na,P) was obviously increased in the hippocampal neuronal culture model as judged by single-channel patch–clamp recording. Furthermore, VGSC subtypes Na_V1.1, Na_V1.2 and Na_V1.3 were up-regulated at the protein expression level in model neurons and SER as assessed by Western blotting. Four subtypes of VGSC proteins in SER were clearly present throughout the hippocampus, including CA1, CA3 and dentate gyrus regions, and neurons expressing VGSC immunoreactivity were also detected in hippocampal neuronal culture model by immunofluorescence. These findings suggested that the up-regulation of voltage-gated sodium channels subtypes in neurons coincided with an increased sodium current in the hippocampal neuronal culture model, providing a possible explanation for the observed seizure discharge and enhanced excitability in epilepsy.     

Arrangement and mobility of the voltage sensor domain in prokaryotic voltage-gated sodium channels

Prokaryotic voltage-gated sodium channels (Na(V)s) form homotetramers with each subunit contributing six transmembrane α-helices (S1-S6). Helices S5 and S6 form the ion-conducting pore, and helices S1-S4 function as the voltage sensor with helix S4 thought to be the essential element for voltage-dependent activation. Although the crystal structures have provided insight into voltage-gated K channels (K(V)s), revealing a characteristic domain arrangement in which the voltage sensor domain of one subunit is close to the pore domain of an adjacent subunit in the tetramer, the structural and functional information on Na(V)s remains limited. Here, we show that the domain arrangement in NaChBac, a firstly cloned prokaryotic Na(V), is similar to that in K(V)s. Cysteine substitutions of three residues in helix S4, Q107C, T110C, and R113C, effectively induced intersubunit disulfide bond formation with a cysteine introduced in helix S5, M164C, of the adjacent subunit. In addition, substituting two acidic residues with lysine, E43K and D60K, shifted the activation of the channel to more positive membrane potentials and consistently shifted the preferentially formed disulfide bond from T110C/M164C to Q107C/M164C. Because Gln-107 is located closer to the extracellular side of helix S4 than Thr-110, this finding suggests that the functional shift in the voltage dependence of activation is related to a restriction of the position of helix S4 in the lipid bilayer. The domain arrangement and vertical mobility of helix S4 in NaChBac indicate that the structure and the mechanism of voltage-dependent activation in prokaryotic Na(V)s are similar to those in canonical K(V)s.