Specific inhibition of [3H] saxitoxin binding to skeletal muscle sodium channels by geographutoxin II, a polypeptide channel blocker.

Y Ohizumi,H Nakamura,J Kobayashi,W A Catterall

doi:10.1016/s0021-9258(19)84540-4

Abstract

Geographutoxin II (GTX II), a peptide toxin isolated from Conus geographus, inhibited [3H]saxitoxin binding to receptor sites associated with voltage-sensitive Na channels in rat skeletal muscle homogenates and rabbit T-tubular membranes with K0.5 values of 60 nM for homogenates and 35 nM for T-tubular membranes in close agreement with concentrations that block muscle contraction. Scatchard analysis of [3H]saxitoxin binding to T-tubular membranes gave values of KD = 9.3 nM and Bmax = 300 fmol/mg of protein and revealed a primarily competitive mode of inhibition of saxitoxin binding by GTX II. The calculated KD values for GTX II were 24 nM for T-tubules and 35 nM for homogenates, respectively. In rat brain synaptosomes, GTX II caused a similar inhibitory effect on [3H]saxitoxin binding at substantially higher concentrations (K0.5 = 2 microM). In contrast, binding of [3H]batrachotoxin A 20-alpha-benzoate and 125I-labeled scorpion toxin to receptor sites associated with Na channels in synaptosomes was not affected by GTX II at concentrations up to 10 microM. Furthermore, [3H]saxitoxin binding to membranes of rat superior cervical ganglion was only blocked 10% by GTX II at 10 microM. These results indicate that GTX II interacts competitively with saxitoxin in binding at neurotoxin receptor site 1 on the sodium channel in a highly tissue-specific manner. GTX II is the first polypeptide ligand for this receptor site and the first to discriminate between this site on nerve and adult muscle sodium channels.

Highlights

Na channels in rat skeletal muscle homogenates and Materials-Extraction and purification of GTX I1 were made as rabbit T-tubular membranewsith K o .v~alues of6 0 . n ~ described previously [8, 9]. 30 pairs of the venom glands of C
Sites associated with Na channels in synaptosomweas s not affected by GTX I1 at concentrationsup to 10 p ~
The presence of 3 hydroxyproline residues and 6 cysteine residues in disulfide bonded form within the 22-residue sequence distinguishes the structure of GTX I1 and its congeners [10,11,12] from other polypeptide toxins that have been characterized

Summary

Introduction

Na channels in rat skeletal muscle homogenates and Materials-Extraction and purification of GTX I1 were made as rabbit T-tubular membranewsith K o .v~alues of6 0 . n ~ described previously [8, 9]. 30 pairs of the venom glands of C. The resulting concentration-effect curves (Fig. 2) show that GTX I1 inhibits contraction in rat and rabbit muscle with Ko.5 values of 60 and 50 nM, respectively. Inhibition of batrachotoxin-activated sodium channels by GTX I1 is voltage-dependent [12] as for saxitoxin and tetrodotoxin.

Results

Conclusion