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

Yamagishi T, Li RA, Hsu K, Marbán E, Tomaselli GF - J. Gen. Physiol. (2001)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Cardiology, Department of Medicine, The Johns Hopkins University, Baltimore, MD 21205, USA.

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.

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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+ (Gx) compared with that in 140 mM Na+(GNa). The selectivity of the wild-type channel and the selectivity filter mutant K1237C are shown for comparison.
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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+ (Gx) compared with that in 140 mM Na+(GNa). The selectivity of the wild-type channel and the selectivity filter mutant K1237C are shown for comparison.

Mentions: Several residues in both the third (K1237) and fourth domain (G1530, W1531, and D1532) P segments of the Na channel influence ionic selectivity (Heinemann et al. 1992; Chiamvimonvat et al. 1996; Favre et al. 1996; Perez-Garcia et al. 1997; Tsushima et al. 1997b). We examined all mutants for changes in ion selectivity; only R395C and R750C altered the selectivity of the channel, and R395C altered selectivity most dramatically. We compared the whole-cell conductances in external solutions of different monovalent and divalent cation composition to the Na+ conductance of the mutants. Fig. 5 (A and B) shows representative R395C currents and current-voltage relationships. Fig. 5 C summarizes the ion-specific conductances compared with Na+. For comparison, the previously reported domain III DEKA mutant, K1237C, is shown (Chiamvimonvat et al. 1996; Perez-Garcia et al. 1997). R395C is more permeable to NH4+, K+, and Ca2+ than the wild-type channel. R750C exhibits enhanced permeability to NH4+ and modest K+ permeability, but does not support divalent cation flux. The selectivity phenotype of F745C is similar to the wild-type.


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

Yamagishi T, Li RA, Hsu K, Marbán E, Tomaselli GF - J. Gen. Physiol. (2001)

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+ (Gx) compared with that in 140 mM Na+(GNa). The selectivity of the wild-type channel and the selectivity filter mutant K1237C are shown for comparison.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233827&req=5

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+ (Gx) compared with that in 140 mM Na+(GNa). The selectivity of the wild-type channel and the selectivity filter mutant K1237C are shown for comparison.
Mentions: Several residues in both the third (K1237) and fourth domain (G1530, W1531, and D1532) P segments of the Na channel influence ionic selectivity (Heinemann et al. 1992; Chiamvimonvat et al. 1996; Favre et al. 1996; Perez-Garcia et al. 1997; Tsushima et al. 1997b). We examined all mutants for changes in ion selectivity; only R395C and R750C altered the selectivity of the channel, and R395C altered selectivity most dramatically. We compared the whole-cell conductances in external solutions of different monovalent and divalent cation composition to the Na+ conductance of the mutants. Fig. 5 (A and B) shows representative R395C currents and current-voltage relationships. Fig. 5 C summarizes the ion-specific conductances compared with Na+. For comparison, the previously reported domain III DEKA mutant, K1237C, is shown (Chiamvimonvat et al. 1996; Perez-Garcia et al. 1997). R395C is more permeable to NH4+, K+, and Ca2+ than the wild-type channel. R750C exhibits enhanced permeability to NH4+ and modest K+ permeability, but does not support divalent cation flux. The selectivity phenotype of F745C is similar to the wild-type.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Cardiology, Department of Medicine, The Johns Hopkins University, Baltimore, MD 21205, USA.

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.

Show MeSH
Related in: MedlinePlus