Specific inhibition of [3H] saxitoxin binding to skeletal muscle sodium channels by geographutoxin II, a polypeptide channel blocker.
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.
Y
Y
Ohizumi
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Nakamura
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Kobayashi
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WA
Catterall
D000595:Amino Acid Sequence; D029845:Amphibian Proteins; D000818:Animals; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D005724:Ganglia / Q000378:metabolism; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008978:Mollusk Venoms / Q000494:pharmacology*; D009132:Muscles / Q000378:metabolism*; D010456:Peptides, Cyclic; D051381:Rats; D011919:Rats, Inbred Strains; D012530:Saxitoxin / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism
D029845:Amphibian Proteins; D002352:Carrier Proteins; D007473:Ion Channels; D008978:Mollusk Venoms; D010456:Peptides, Cyclic; C027525:saxitoxin-binding protein, Rana catesbeiana; D012530:Saxitoxin; C038094:geographutoxin II; D012964:Sodium