Molecular architecture of the voltage-dependent Na channel: functional evidence for alpha helices in the pore

Abstract

The permeation pathway of the Na channel is formed by asymmetric loops (P segments) contributed by each of the four domains of the protein. In contrast to the analogous region of K channels, previously we (Yamagishi, T., M. Janecki, E. Marban, and G. Tomaselli. 1997. Biophys. J. 73:195-204) have shown that the P segments do not span the selectivity region, that is, they are accessible only from the extracellular surface. The portion of the P-segment NH(2)-terminal to the selectivity region is referred to as SS1. To explore further the topology and functional role of the SS1 region, 40 amino acids NH(2)-terminal to the selectivity ring (10 in each of the P segments) of the rat skeletal muscle Na channel were substituted by cysteine and expressed in tsA-201 cells. Selected mutants in each domain could be blocked with high affinity by externally applied Cd(2)+ and were resistant to tetrodotoxin as compared with the wild-type channel. None of the externally applied sulfhydryl-specific methanethiosulfonate reagents modified the current through any of the mutant channels. Both R395C and R750C altered ionic selectivity, producing significant increases in K(+) and NH(4)(+) currents. The pattern of side chain accessibility is consistent with a pore helix like that observed in the crystal structure of the bacterial K channel, KcsA. Structure prediction of the Na channel using the program PHDhtm suggests an alpha helix in the SS1 region of each domain channel. We conclude that each of the P segments undergoes a hairpin turn in the permeation pathway, such that amino acids on both sides of the putative selectivity filter line the outer mouth of the pore. Evolutionary conservation of the pore helix motif from bacterial K channels to mammalian Na channels identifies this structure as a critical feature in the architecture of ion selective pores.

Figure 1

Single-letter amino acid alignment of the residues in the P segments of the Na channel with KcsA. The bars over the KcsA sequence identify the turret and pore helix with the selectivity signature sequence enclosed in the box. Y62 is the first residue of the pore helix in KcsA, the underlined residues have their side chains exposed to the channel pore based on the crystal structure (Doyle et al. 1998). The residues in bold in all of the sequences are those predicted to be helical by PHDhtm. The italicized residues in the dShaker sequence were not categorized as helical by PHDhtm but exhibited a probability of >0.4 of being helical by the algorithm. The dotted box identifies the residues mutated in the μ1 channel in this study. The hH1 sequence is shown for comparison. The underlined residues indicate that the cysteine mutant at this position is accessible (i.e., altered Cd²+ and/or TTX block sensitivity) from the extracellular pore. The jagged underlined residues have been shown previously to be accessible from the outside of the channel. The DEKA selectivity filter of the Na channel is enclosed in a solid box. The numbering in parentheses, is according to the μ1 sequence.

Figure 2

The effect of external Cd²+ and TTX on the cysteine substitution mutants on the NH₂-terminal side of putative selectivity filter in μ1. (A) Bar plot of the IC₅₀s for Cd²+ block of each of the mutants that express current. The mutants R395C, R750C, L746C, F745C, Y1227C, L1228C, and I1519C have IC₅₀s (mean ± SD) 999 ± 64, 201 ± 10, 813 ± 10, 123 ± 19, 650 ± 106, 292 ± 94, and 173 ± 19 μM (P < 0.05 versus WT, 1,955 ± 207 μM), respectively. (B) Bar plot of the IC₅₀s for TTX of each of the mutants that express current. The mutants R395C, F745C, R750C, and L1228C have IC₅₀s for TTX block of (mean ± SD) 112 ± 32, 1,660 ± 425, 4,431 ± 661, and 441 ± 268 nM (P < 0.05 versus WT, 13 ± 4 nM), respectively. In cases without standard error bars, full dose–response curves were obtained in individual cells, and the lack of deviation from wild-type was confirmed in several other experiments using single doses of Cd²+ (0.5 mM) and TTX (50 nM).

Figure 3

No effect of MTS reagents on R395C. (A) Representative experiment with repeated external application of 1 mM Cd²+ and saturating concentrations of MTS reagents. None of the Cd²+-sensitive mutants exhibited current reduction or a change in the IC₅₀ for Cd²+ block after MTS application.

Figure 4

Single-channel Cd²+ block of R395C and R750C. (top) Representative single-channel currents after modification by 20 μM fenvalerate, elicited by voltage steps to −50 and −80 mV from a holding potential of −120 mV with 140 mM Na⁺ in the pipette. The single-channel current is reduced in the presence of 400 or 800 μM Cd²+ (middle) Single-channel current-voltage relationships for each of the mutant channels determined from three patches in the presence and absence of Cd²+. Cd²+ blocks each of the mutants in a voltage-dependent fashion. (bottom) A plot of the logarithm of the ratio of the blocked and unblocked single-channel current amplitude versus voltage gives the fractional electrical distance (δ) for Cd²+ binding.

Figure 5

Selectivity of the cysteine mutants with altered Cd²+ and TTX affinities. Peak inward currents (A) and current-voltage relationship (B) through R395C in the presence of different cations. (C) Plot of the ratio of the whole-cell conductance in 140 mM Li⁺, 140 mM NH₄ ⁺, 140 mM K⁺, and 70 mM Ca²+ (G_x) compared with that in 140 mM Na⁺(G_Na). The selectivity of the wild-type channel and the selectivity filter mutant K1237C are shown for comparison.

Figure 6

Structure of Na channel SS1 region of the P segments. (top) Helical wheel representations of the residues mutated in this study. The residues in bold italics are accessible from the aqueous pore; those enclosed in a box do not express current and thus have an indeterminate phenotype. The cysteine mutants that alter Cd²+/ TTX block sensitivity or have an indeterminate phenotype are on the same side of the helix. (bottom) Molecular model of the putative pore helices in the Na channel pore, views from the top (left) and side (right) are shown. The domains are positioned to approximate the distances between selectivity filter residues. The pore helices are rendered in ribbon format, the DEKA ring in yellow stick format, and accessible (green) or nonfunctional (salmon) positions in ball-and-stick format.

Figure 6

Structure of Na channel SS1 region of the P segments. (top) Helical wheel representations of the residues mutated in this study. The residues in bold italics are accessible from the aqueous pore; those enclosed in a box do not express current and thus have an indeterminate phenotype. The cysteine mutants that alter Cd²+/ TTX block sensitivity or have an indeterminate phenotype are on the same side of the helix. (bottom) Molecular model of the putative pore helices in the Na channel pore, views from the top (left) and side (right) are shown. The domains are positioned to approximate the distances between selectivity filter residues. The pore helices are rendered in ribbon format, the DEKA ring in yellow stick format, and accessible (green) or nonfunctional (salmon) positions in ball-and-stick format.