Use-dependent block with tetrodotoxin and saxitoxin at frog Ranvier nodes I. Intrinsic channel and toxin parameters

The use-dependent phasic blockage of sodium channels by tetrodotoxin (TTX) and saxitoxin (STX) was examined in frog nodes of Ranvier using trains of depolarizing pulses. The decline of the peak Na⁺ current from its initial value (I ₀) before the train to a stationary value (I ) after the train was more pronounced at more negative holding potentials. The relationship betweenI /I ₀ and holding potential was fitted by a sigmoid function which yielded values for the steepness of the voltage dependencies of around –15 mV for TTX and – 8 mV for STX. Similar values were obtained at toxin concentrations of 4 and 8 nM. The higher voltage sensitivity of STX versus TTX is interpreted in terms of the higher charge and the faster binding kinetics of STX. These differences also explain the frequency dependence of the decline of Na⁺ currents with STX (between 0.5 and 2 Hz) and the frequency independence with TTX. Variation of the pulse amplitude in a train of conditioning pulses revealed that the magnitude of the use-dependent actions of STX parallels the steady-state Na⁺ inactivation curveh . Inhibition of inactivation, by pre-treatment with chloramine-T, did not, however, abolish the use dependence. Instead, it introduced a change in the time constants of the decline of the Na⁺ currents and the magnitude became independent of the holding potential.     

Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule.

The marine guanidinium toxins, saxitoxin (STX) and tetrodotoxin (TTX), have played crucial roles in the study of voltage-gated Na+ channels. Because they have similar actions, sizes, and functional groups, they have been thought to associate with the channel in the same manner, and early mutational studies supported this idea. Recent experiments by. Biophys. J. 67:2305-2315) have suggested that the toxins bind differently to the isoform-specific domain I Phe/Tyr/Cys location. In the adult skeletal muscle Na+ channel isoform (microliter), we compared the effects on both TTX and STX affinities of mutations in eight positions known to influence toxin binding. The results permitted the assignment of energies contributed by each amino acid to the binding reaction. For neutralizing mutations of Asp400, Glu755, and Lys1237, all thought to be part of the selectivity filter of the channel, the loss of binding energy was identical for the two toxins. However, the loss of binding energy was quite different for vestibule residues considered to be more superficial. Specifically, STX affinity was reduced much more by neutralizations of Glu758 and Asp1532. On the other hand, mutation of Tyr401 to Cys reduced TTX binding energy twice as much as it reduced STX binding energy. Kinetic analysis suggested that all outer vestibule residues tested interacted with both toxins early in the binding reaction (consistent with larger changes in the binding than unbinding rates) before the transition state and formation of the final bound complex. We propose a revised model of TTX and STX binding in the Na+ channel outer vestibule in which the toxins have similar interactions at the selectivity filter, TTX has a stronger interaction with Tyr401, and STX interacts more strongly with the more extracellular residues.     

Use-dependent block of sodium channels in frog myelinated nerve by tetrodotoxin and saxitoxin at negative holding potentials

Na+ currents were measured in myelinated frog nerve fibres in the presence of nanomolar concentrations of tetrodotoxin (TTX) or saxitoxin (STX) in the extracellular solution. The Na+ currents declined during a train of depolarizing pulses if the fibre was held at hyperpolarizing potentials between the pulses. At a pulse frequency of 0.8 Hz, the peak Na+ currents were reduced to 70 or 60% of the initial value in 9.3 nM TTX and 3.5 nM STX solutions, respectively. A decline of Na+ currents was also observed in two-pulse experiments. The peak Na+ current during a second test pulse did not depend on the duration (0.2 to 12 ms) of the first pulse. It decreased with increasing interval between the pulses, reached a minimum and increased again. The results are interpreted with a use-dependent blockage of Na+ channels by TTX or STX at negative holding potentials. The effects were described quantitatively, assuming a fast affinity increase of toxin receptors at Na+ channels triggered by Na+ activation followed by slow toxin binding to channels and relaxation of the receptor affinity.     

A structural model of the tetrodotoxin and saxitoxin binding site of the Na+ channel.

Biophysical evidence has placed the binding site for the naturally occurring marine toxins tetrodotoxin (TTX) and saxitoxin (STX) in the external mouth of the Na+ channel ion permeation pathway. We developed a molecular model of the binding pocket for TTX and STX, composed of antiparallel beta-hairpins formed from peptide segments of the four S5-S6 loops of the voltage-gated Na+ channel. For TTX the guanidinium moiety formed salt bridges with three carboxyls, while two toxin hydroxyls (C9-OH and C10-OH) interacted with a fourth carboxyl on repeats I and II. This alignment also resulted in a hydrophobic interaction with an aromatic ring of phenylalanine or tyrosine residues for the brainII and skeletal Na+ channel isoforms, but not with the cysteine found in the cardiac isoform. In comparison to TTX, there was an additional interaction site for STX through its second guanidinium group with a carboxyl on repeat IV. This model satisfactorily reproduced the effects of mutations in the S5-S6 regions and the differences in affinity by various toxin analogs. However, this model differed in important ways from previously published models for the outer vestibule and the selectivity region of the Na+ channel pore. Removal of the toxins from the pocket formed by the four beta-hairpins revealed a structure resembling a funnel that terminated in a narrowed region suitable as a candidate for the selectivity filter of the channel. This region contained two carboxyls (Asp384 and Glu942) that substituted for molecules of water from the hydrated Na+ ion. Simulation of mutations in this region that have produced Ca2+ permeation of the Na+ channel created a site with three carboxyls (Asp384, Glu942, and Glu1714) in proximity.     

Use-dependent block with tetrodotoxin and saxitoxin at frog Ranvier nodes II. Extrinsic influence of cations

Use-dependent declines of Na⁺ currents in myelinated frog nerve fibres were measured during a train of depolarizing pulses in solutions containing tetrodotoxin (TTX) or saxitoxin (STX). The following effects of external monovalent (Na⁺), divalent (Ca²⁺, Mg²⁺) and trivalent (La²⁺) cations on use dependence were found: Increasing the Ca²⁺ concentration from 2 to 8 mM shifts its voltage dependence by 20 mV whereas no significant use-dependent decline occurred at 0.2 mM Ca²⁺. Doubling the external Na⁺ concentration in 0.2 mM Ca²⁺ solutions did not initiate phasic block. External Mg²⁺ ions induced a smaller, and La²⁺ ions a larger, use dependence. The time constants of the current decline were 4-fold greater in 1.08 mM La²⁺. The static block of Na⁺ currents by La³⁺ could be directly demonstrated by the relief of block during a train of pulses. The results are qualitatively explained by a toxin binding site at the Na⁺ channel whose affinity for TTX or STX depends oni) the gating conformation of the channel, probably the inactivation andii) the occupancy of a blocking site by di- or trivalent external cations.     

On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel.

Voltage-sensitive sodium channels and calcium channels are homologous proteins with distinctly different selectivity for permeation of inorganic cations. This difference in function is specified by amino acid residues located within P-region segments that link presumed transmembrane elements S5 and S6 in each of four repetitive Domains I, II, III, and IV. By analyzing the selective permeability of Na+, K+, and Ca2+ in various mutants of the mu 1 rat muscle sodium channel, the results in this paper support the concept that a conserved motif of four residues contributed by each of the Domains I-IV, termed the DEKA locus in sodium channels and the EEEE locus in calcium channels, determines the ionic selectivity of these channels. Furthermore, the results indicate that the Lys residue in Domain III of the sodium channel is the critical determinant that specifies both the impermeability of Ca2+ and the selective permeability of Na+ over K+. We propose that the alkylammonium ion of the Lys(III) residue acts as an endogenous cation within the ion binding site/selectivity filter of the sodium channel to tune the kinetics and affinity of inorganic cation binding within the pore in a manner analogous to ion-ion interactions that occur in the process of multi-ion channel conduction.     

Differential effects of sulfhydryl reagents on saxitoxin and tetrodotoxin block of voltage-dependent Na channels.

We have probed a cysteine residue that confers resistance to tetrodotoxin (TTX) block in heart Na channels, with membrane-impermeant, cysteine-specific, methanethiosulfonate (MTS) analogs. Covalent addition of a positively charged group to the cysteinyl sulfhydryl reduced pore conductance by 87%. The effect was selectively prevented by treatment with TTX, but not saxitoxin (STX). Addition of a negatively charged group selectively inhibited STX block without affecting TTX block. These results agree with models that place an exposed cysteinyl sulfhydryl in the TTX site adjacent to the mouth of the pore, but do not support the contention that STX and TTX are interchangeable. The surprising differences between the two toxins are consistent with the hypothesis that the toxin-receptor complex can assume different conformations when STX or TTX bound.     

电压门控Na^＋通道亚型及其功能——新的研究热点

依靠现代分子生物学技术及电生理的记录,探讨各种Ｎａ＾＋通道亚型在中枢与周边神经系统以及一些非兴奋性组织细胞中的分布,表达,突变及其对信息调控的功能特征,已成为当今神经生物学等学科发展中的一个研究新热点,本文将侧重对有关哺乳动物Ｎａ＾＋通道亚型的分类,在不同组织细胞中的分布及其表达调控的功能机制等一些研究进展做一简要的回眸。     

Structural basis for the voltage-gated Na+ channel selectivity of the scorpion alpha-like toxin BmK M1

Scorpion alpha-like toxins are proteins that act on mammalian and insect voltage-gated Na+ channels. Therefore, these toxins constitute an excellent target for examining the foundations that underlie their target specificity. With this motive we dissected the role of six critical amino acids located in the five-residue reverse turn (RT) and C-tail (CT) of the scorpion alpha-like toxin BmK M1. These residues were individually substituted resulting in 11 mutants and were subjected to a bioassay on mice, an electrophysiological characterization on three cloned voltage-gated Na+ channels (Nav1.2, Nav1.5 and para), a CD analysis and X-ray crystallography. The results reveal two molecular sites, a couplet of residues (8-9) in the RT and a hydrophobic surface consisting of residues 57 and 59-61 in the CT, where the substitution with specific residues can redirect the alpha-like characteristics of BmK M1 to either total insect or much higher mammal specificity. Crystal structures reveal that the pharmacological ramification of these mutants is accompanied by the reshaping of the 3D structure surrounding position 8. Furthermore, our results also reveal that residues 57 and 59-61, located at the CT, enclose the critical residue 58 in order to form a hydrophobic "gasket". Mutants of BmK M1 that interrupt this hydrophobic surface significantly gain insect selectivity.     

Batrachotoxin-activated Na+ channels in planar lipid bilayers. Competition of tetrodotoxin block by Na+

Single Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers formed from neutral phospholipids and were observed in the presence of batrachotoxin. The batrachotoxin-modified channel activates in the voltage range -120 to - 80 mV and remains open almost all the time at voltages positive to -60 mV. Low levels of tetrodotoxin (TTX) induce slow fluctuations of channel current, which represent the binding and dissociation of single TTX molecules to single channels. The rates of association and dissociation of TTX are both voltage dependent, and the association rate is competitively inhibited by Na+. This inhibition is observed only when Na+ is increased on the TTX binding side of the channel. The results suggest that the TTX receptor site is located at the channel's outer mouth, and that the Na+ competition site is not located deeply within the channel's conduction pathway.     

Block of tetrodotoxin-resistant Na+ channel pore by multivalent cations: gating modification and Na+ flow dependence

Tetrodotoxin-resistant (TTX-R) Na(+) channels are much less susceptible to external TTX but more susceptible to external Cd(2+) block than tetrodotoxin-sensitive (TTX-S) Na(+) channels. Both TTX and Cd(2+) seem to block the channel near the "DEKA" ring, which is probably part of a multi-ion single-file region adjacent to the external pore mouth and is involved in the selectivity filter of the channel. In this study we demonstrate that other multivalent transitional metal ions such as La(3+), Zn(2+), Ni(2+), Co(2+), and Mn(2+) also block the TTX-R channels in dorsal root ganglion neurons. Just like Cd(2+), the blocking effect has little intrinsic voltage dependence, but is profoundly influenced by Na(+) flow. The apparent dissociation constants of the blocking ions are always significantly smaller in inward Na(+) currents than those in outward Na(+) current, signaling exit of the blocker along with the Na(+) flow and a high internal energy barrier for "permeation" of these multivalent blocking ions through the pore. Most interestingly, the activation and especially the inactivation kinetics are slowed by the blocking ions. Moreover, the gating changes induced by the same concentration of a blocking ion are evidently different in different directions of Na(+) current flow, but can always be correlated with the extent of pore block. Further quantitative analyses indicate that the apparent slowing of channel activation is chiefly ascribable to Na(+) flow-dependent unblocking of the bound La(3+) from the open Na(+) channel, whereas channel inactivation cannot happen with any discernible speed in the La(3+)-blocked channel. Thus, the selectivity filter of Na(+) channel is probably contiguous to a single-file multi-ion region at the external pore mouth, a region itself being nonselective in terms of significant binding of different multivalent cations. This region is "open" to the external solution even if the channel is "closed" ("deactivated"), but undergoes imperative conformational changes during the gating (especially the inactivation) process of the channel.     

Ion selectivity of the Kat1 K+ channel pore

Kat1 is a highly selective inward-rectifying K⁺ channel that opens for extended periods under conditions of extreme hyperpolarization. Over 200 point mutants in the pore region of the Kat1 K⁺ channel were generated and examined in the yeast Saccharomyces cerevisiae and Xenopus oocytes to assess the effect of the mutations on ion selectivity. Substitutions at the tyrosine of the signature sequence G-Y-G resulted in the most significant alterations in ion selectivity, consistent with its role in the selectivity filter. However, greater than 80% of the mutations throughout the greater pore region also conferred a defect in selectivity demonstrating that the entire pore of Kat1 contributes to the ion selectivity of this channel. Surprisingly, we identified a novel class of mutant channel that conferred enhanced selectivity of K⁺ over Na⁺. Mutants of this class frequently displayed sensitivity to the competing ion Cs⁺. This finding has led us to speculate that the Kat1 channel pore has evolved to balance not only K⁺/Na⁺ selectivity, but selectivity over Cs⁺, and possibly a wide spectrum of potential competing ions.     

Site-specific mutations in a minimal voltage-dependent K+ channel alter ion selectivity and open-channel block

MinK is a small membrane protein of 130 amino acids with a single potential membrane-spanning alpha-helical domain. Its expression in Xenopus oocytes induces voltage-dependent, K(+)-selective channels. Using site-directed mutagenesis of a synthetic gene, we have identified residues in the hydrophobic region of minK that influence both ion selectivity and open-channel block. Single amino acid changes increase the channel's relative permeability for NH4+ and Cs+ without affecting its ability to exclude Na+ and Li+. Blockade by two common K+ channel pore blockers, tetraethylammonium and Cs+, was also modified. These results suggest that an ion selectivity region and binding sites for the pore blockers within the conduction pathway have been modified. We conclude that the gene encoding minK is a structural gene for a K+ channel protein.     

Peregrination of the selectivity filter delineates the pore of the human voltage-gated proton channel hHV1

Extraordinary selectivity is crucial to all proton-conducting molecules, including the human voltage-gated proton channel (hH_V1), because the proton concentration is >10⁶ times lower than that of other cations. Here we use “selectivity filter scanning” to elucidate the molecular requirements for proton-specific conduction in hH_V1. Asp¹¹², in the middle of the S1 transmembrane helix, is an essential part of the selectivity filter in wild-type (WT) channels. After neutralizing Asp¹¹² by mutating it to Ala (D112A), we introduced Asp at each position along S1 from 108 to 118, searching for “second site suppressor” activity. Surprisingly, most mutants lacked even the anion conduction exhibited by D112A. Proton-specific conduction was restored only with Asp or Glu at position 116. The D112V/V116D channel strikingly resembled WT in selectivity, kinetics, and ΔpH-dependent gating. The S4 segment of this mutant has similar accessibility to WT in open channels, because R211H/D112V/V116D was inhibited by internally applied Zn²⁺. Asp at position 109 allowed anion permeation in combination with D112A but did not rescue function in the nonconducting D112V mutant, indicating that selectivity is established externally to the constriction at F150. The three positions that permitted conduction all line the pore in our homology model, clearly delineating the conduction pathway. Evidently, a carboxyl group must face the pore directly to enable conduction. Molecular dynamics simulations indicate reorganization of hydrogen bond networks in the external vestibule in D112V/V116D. At both positions where it produces proton selectivity, Asp frequently engages in salt linkage with one or more Arg residues from S4. Surprisingly, mean hydration profiles were similar in proton-selective, anion-permeable, and nonconducting constructs. That the selectivity filter functions in a new location helps to define local environmental features required to produce proton-selective conduction.     

Control of voltage-gated K+ channel permeability to NMDG+ by a residue at the outer pore

Crystal structures of potassium (K⁺) channels reveal that the selectivity filter, the narrow portion of the pore, is only ∼3-Å wide and buttressed from behind, so that its ability to expand is highly constrained, and the permeation of molecules larger than Rb⁺ (2.96 Å in diameter) is prevented. N-methyl-d-glucamine (NMDG⁺), an organic monovalent cation, is thought to be a blocker of Kv channels, as it is much larger (∼7.3 Å in mean diameter) than K⁺ (2.66 Å in diameter). However, in the absence of K⁺, significant NMDG⁺ currents could be recorded from human embryonic kidney cells expressing Kv3.1 or Kv3.2b channels and Kv1.5 R487Y/V, but not wild-type channels. Inward currents were much larger than outward currents due to the presence of intracellular Mg²⁺ (1 mM), which blocked the outward NMDG⁺ current, resulting in a strong inward rectification. The NMDG⁺ current was inhibited by extracellular 4-aminopyridine (5 mM) or tetraethylammonium (10 mM), and largely eliminated in Kv3.2b by an S6 mutation that prevents the channel from opening (P468W) and by a pore helix mutation in Kv1.5 R487Y (W472F) that inactivates the channel at rest. These data indicate that NMDG⁺ passes through the open ion-conducting pore and suggest a very flexible nature of the selectivity filter itself. 0.3 or 1 mM K⁺ added to the external NMDG⁺ solution positively shifted the reversal potential by ∼16 or 31 mV, respectively, giving a permeability ratio for K⁺ over NMDG⁺ (P_K⁺/P_NMDG⁺) of ∼240. Reversal potential shifts in mixtures of K⁺ and NMDG⁺ are in accordance with P_K⁺/P_NMDG⁺, indicating that the ions compete for permeation and suggesting that NMDG⁺ passes through the open state. Comparison of the outer pore regions of Kv3 and Kv1.5 channels identified an Arg residue in Kv1.5 that is replaced by a Tyr in Kv3 channels. Substituting R with Y or V allowed Kv1.5 channels to conduct NMDG⁺, suggesting a regulation by this outer pore residue of Kv channel flexibility and, as a result, permeability.     

Functional architecture of the inner pore of a voltage-gated Ca2+ channel

The inner pore of voltage-gated Ca2+ channels (VGCCs) is functionally important, but little is known about the architecture of this region. In K+ channels, this part of the pore is formed by the S6/M2 transmembrane segments from four symmetrically arranged subunits. The Ca2+ channel pore, however, is formed by four asymmetric domains of the same (alpha1) subunit. Here we investigated the architecture of the inner pore of P/Q-type Ca2+ channels using the substituted-cysteine accessibility method. Many positions in the S6 segments of all four repeats of the alpha1 subunit (Ca(v)2.1) were modified by internal methanethiosulfonate ethyltrimethylammonium (MTSET). However, the pattern of modification does not fit any known sequence alignment with K+ channels. In IIS6, five consecutive positions showed clear modification, suggesting a likely aqueous crevice and a loose packing between S6 and S5 segments, a notion further supported by the observation that some S5 positions were also accessible to internal MTSET. These results indicate that the inner pore of VGCCs is indeed formed by the S6 segments but is different from that of K+ channels. Interestingly some residues in IIIS6 and IVS6 whose mutations in L-type Ca2+ channels affect the binding of dihydropyridines and phenylalkylamines and are thought to face the pore appeared not to react with internal MTSET. Probing with qBBr, a rigid thiol-reactive agent with a dimension of 12 angstroms x 10 angstroms x 6 angstroms suggests that the inner pore can open to >10 angstroms. This work provides an impetus for future studies on ion permeation, gating, and drug binding of VGCCs.     

Slow inactivation does not block the aqueous accessibility to the outer pore of voltage-gated Na channels

Slow inactivation of voltage-gated Na channels is kinetically and structurally distinct from fast inactivation. Whereas structures that participate in fast inactivation are well described and include the cytoplasmic III-IV linker, the nature and location of the slow inactivation gating mechanism remains poorly understood. Several lines of evidence suggest that the pore regions (P-regions) are important contributors to slow inactivation gating. This has led to the proposal that a collapse of the pore impedes Na current during slow inactivation. We sought to determine whether such a slow inactivation-coupled conformational change could be detected in the outer pore. To accomplish this, we used a rapid perfusion technique to measure reaction rates between cysteine-substituted side chains lining the aqueous pore and the charged sulfhydryl-modifying reagent MTS-ET. A pattern of incrementally slower reaction rates was observed at substituted sites at increasing depth in the pore. We found no state-dependent change in modification rates of P-region residues located in all four domains, and thus no change in aqueous accessibility, between slow- and nonslow-inactivated states. In domains I and IV, it was possible to measure modification rates at residues adjacent to the narrow DEKA selectivity filter (Y401C and G1530C), and yet no change was observed in accessibility in either slow- or nonslow-inactivated states. We interpret these results as evidence that the outer mouth of the Na pore remains open while the channel is slow inactivated.     

Tonic and phasic tetrodotoxin block of sodium channels with point mutations in the outer pore region.

Tonic and use-dependent block by tetrodotoxin (TTX) has been studied in cRNA-injected Xenopus oocytes expressing mutants W386Y, E945Q, D1426K, and D1717Q, of the outer-pore region of the rat brain IIA alpha-subunit of sodium channels. The various phenotypes are tonically half-blocked at TTX concentrations, IC50(t), that span a range of more than three orders of magnitude, from 4 nM in mutant D1426K to 11 microM in mutant D1717Q. When stimulated with repetitive depolarizing pulses at saturating frequencies, all channels showed a monoexponential increase in their TTX-binding affinity with time constants that span an equally wide range of values ([TTX] approximately IC50(t), from approximately 60 s for D1426K to approximately 30 ms for D1717Q) and are in most phenotypes roughly inversely proportional to IC50(t). In contrast, all phenotypes show the same approximately threefold increase in their TTX affinity under stimulation. The invariance of the free-energy difference between tonic and phasic configurations of the toxin-receptor complex, together with the extreme variability of phasic block kinetics, is fully consistent with the trapped-ion mechanism of use dependence suggested by and developed by. Using this model, we estimated for each phenotype both the second-order association rate constant, kon, and the first-order dissociation rate constant, koff, for TTX binding. Except for mutant E945Q, all phenotypes have roughly the same value of kon approximately 2 microM-1 s-1 and owe their large differences in IC50(t) to different koff values. However, a 60-fold reduction in kon is the main determinant of the low TTX sensitivity of mutant E945Q. This suggests that the carboxyl group of E945 occupies a much more external position in the pore vestibule than that of the homologous residue D1717.