Molecular architecture of the voltage-dependent Na channel: Functional evidence for α helices in the pore

Toshio Yamagishi, Ronald A. Li, Kate Hsu, Eduardo Marbán, Gordon F. Tomaselli

Research output: Contribution to journalArticle

31 Citations (Scopus)

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 NH2-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 NH2-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 Cd2+ 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 NH4+ 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 α 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.

Original languageEnglish (US)
Pages (from-to)171-181
Number of pages11
JournalJournal of General Physiology
Volume118
Issue number2
DOIs
StatePublished - Aug 23 2001
Externally publishedYes

Fingerprint

Bacterial Structures
Amino Acids
Tetrodotoxin
Cysteine
Mouth
Skeletal Muscle
Ions
methanethiosulfonate
Protein Domains

Keywords

  • Cadmium
  • Cysteine mutagenesis
  • Methanethiosulfonates
  • Pore helix
  • Tetrodotoxin

ASJC Scopus subject areas

  • Physiology

Cite this

Molecular architecture of the voltage-dependent Na channel : Functional evidence for α helices in the pore. / Yamagishi, Toshio; Li, Ronald A.; Hsu, Kate; Marbán, Eduardo; Tomaselli, Gordon F.

In: Journal of General Physiology, Vol. 118, No. 2, 23.08.2001, p. 171-181.

Research output: Contribution to journalArticle

Yamagishi, Toshio ; Li, Ronald A. ; Hsu, Kate ; Marbán, Eduardo ; Tomaselli, Gordon F. / Molecular architecture of the voltage-dependent Na channel : Functional evidence for α helices in the pore. In: Journal of General Physiology. 2001 ; Vol. 118, No. 2. pp. 171-181.
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