Reconstitution of neurotoxin-modulated ion transport by the voltage-regulated sodium channel isolated from the electroplax of Electrophorus electricus

R. L. Rosenberg, S. A. Tomiko, W. S. Agnew

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Abstract

The functional reconstitution of the voltage-regulated Na channel purified from the electroplax of Electrophorus electricus is described. Reconstitution was achieved by removing detergent with Bio-Beads SM-2 followed by freeze-thaw-sonication in the presence of added liposomes. This preparation displayed heat-stable binding of 3H-labeled tetrodotoxin (TTX) (K(d) = 33 nM). 22Na+ influx was stimulated 2- to 5-fold by alkaloid neurotoxins and blocked by TTX. Veratridine activated Na+ influx with a K 1/2 of 18 μM, and this activation was blocked by TTX precisely in parallel with specific [3H]TTX binding. Batrachotoxin stimulated 22Na+ flux more effectively than did veratridine. No effect of the peptide anemone toxin II was found. Insertion of the Na channel into membranes resulted in 60-70% of the TTX-binding sites facing the vesicle exterior. Thus, external TTX partially inhibited flux, whereas blockade was complete when TTX was also equilibrated with the vesicle interior. The lipid-soluble local anesthetics tetracaine and dibucaine inhibited flux completely. QX-222, a charged derivative of lidocaine, blocked only a fraction of the channels, apparently those oriented inside-out. Purified samples were predominantly composed of the M(r) 260,000-300,000 glycopeptide but contained variable quantities of smaller peptides. Veratridine-dependent flux and peptide compositions were determined on fractions across a gel filtration column profile. Stimulated flux codistributed only with the large peptide.

Original languageEnglish (US)
Pages (from-to)1239-1243
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume81
Issue number4 I
DOIs
StatePublished - 1984

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