A "minimal" sodium channel construct consisting of ligated S5-P-S6 segments forms a toxin-activatable ionophore

Zhenhui Chen, Carmen Alcayaga, Benjamin A. Suárez-Isla, Brian O'Rourke, Gordon Tomaselli, Eduardo Marbán

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

The large size (six membrane-spanning repeats in each of four domains) and asymmetric architecture of the voltage-dependent Na+ channel has hindered determination of its structure. With the goal of determining the minimum structure of the Na+ channel permeation pathway, we created two stable cell lines expressing the voltage-dependent rat skeletal muscle Na+ channel (μ1) with a polyhistidine tag on the C terminus (μHis) and pore-only μ1 (μPore) channels with S1-S4 in all domains removed. Both constructs were recognized by a Na+ channel-specific antibody on a Western blot. μHis channels exhibited the same functional properties as wild-type μ1. In contrast, μPore channels did not conduct Na+ currents nor did they bind [3H]saxitoxin. Veratridine caused 40 and 54% cell death in μHis- and μPore-expressing cells, respectively. However, veratridine-induced cell death could only be blocked by tetrodotoxin in cells expressing μHis, but not μPore. Furthermore, using a fluorescent Na+ indicator, we measured changes in intracellular Na+ induced by veratridine and a brevotoxin analogue, pumiliotoxin. When calibrated to the maximum signal after addition of gramicidin, the maximal percent increases in fluorescence (AF) were 35 and 31% in cells expressing μHis and μPore, respectively. Moreover, in the presence of 1 μM tetrodotoxin, ΔF decreased significantly to 10% in μHis- but not in μPore-expressing cells (43%). In conclusion, S5-P-S6 segments of μ1 channels form a toxin-activable ionophore but do not reconstitute the Na+ channel permeation pathway with full fidelity.

Original languageEnglish (US)
Pages (from-to)24653-24658
Number of pages6
JournalJournal of Biological Chemistry
Volume277
Issue number27
DOIs
StatePublished - Jul 5 2002

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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