Fluorescence Assay for Neurotoxin-Modulated Ion Transport by the Reconstituted Voltage-Activated Sodium Channel Isolated from Eel Electric Organ

Sally A. Tomiko, Robert L. Rosenberg, Mark C. Emerick, William S. Agnew

Research output: Contribution to journalArticle

Abstract

A fluorescence assay for measuring Na channel activation in liposomes containing voltage-sensitive Na channels isolated from Electrophorus electricus is described. The assay is based on transport of a heavy-metal cation, Tl+, through the activated channel to quench fluorescence of an internalized, water-soluble chromophore. The channel is “locked” in a chronically opened configuration with alkaloid neurotoxins such as veratridine or batrachotoxin. Diffusion potentials are used to amplify the signal, and enlarged liposomes (>8000 Å) result in time courses extended to the range of seconds. Analysis of the kinetics of quenching yields parameters that behave as linear functions of channel activation and reflect vesicle size and channel abundance. The k1/2's for activation by veratridine and batrachotoxin were 5 μM and 169 nM, respectively, and that for tetrodotoxin blockade was 4 nM. Externally applied QX-222 and tetrodotoxin each acted to partially block the stimulated signal, as expected for compounds that act on oppositely oriented channels in the membrane. Single-channel conductances estimated with either veratridine or batrachotoxin ranged between 0.6 and 40.7 pS, corresponding to transport numbers of (1.2 X 105) to (8.1 X 106) ions s-1 channel-1under the conditions of assay. The assay is approximately 100-fold more sensitive than radiotracer influx assays, requiring 1 fmol of protein per time course.

Original languageEnglish (US)
Pages (from-to)2162-2174
Number of pages13
JournalBiochemistry
Volume25
Issue number8
DOIs
StatePublished - Apr 1986
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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