title: Multiple saxitoxin-binding sites in bullfrog muscle: tetrodotoxin-sensitive sodium channels and tetrodotoxin-insensitive sites of unknown function.
abstract: The possible presence of multiple sodium channel subtypes in bullfrog skeletal muscle was investigated in binding experiments with [3H]saxitoxin and in single-channel studies using planar lipid bilayers. Two classes of [3H]saxitoxin-binding sites were identified in membrane preparations. One class displayed a toxin specificity characteristic of voltage-dependent sodium channels: high affinity for saxitoxin (KD approximately equal to 0.5 nM), neosaxitoxin (KD approximately equal to 0.1 nM), and tetrodotoxin (KD approximately equal to 1.3 nM). A second class of membrane-associated binding sites exhibited high affinity for saxitoxin (KD approximately equal to 0.1 nM), lower affinity for neosaxitoxin (KD approximately equal to 25 nM), and complete insensitivity to tetrodotoxin at concentrations up to 32 microM. The first class corresponded to functional tetrodotoxin-sensitive sodium channels that could be incorporated and observed in planar bilayers in the presence of batrachotoxin. Similar attempts to incorporate tetrodotoxin-insensitive sodium channels from bullfrog muscle and heart membranes were unsuccessful. The unusual, tetrodotoxin-insensitive binding activity for [3H]saxitoxin was also found at nM levels in the high speed supernatant of homogenized skeletal muscle without the addition of detergents. This soluble class of sites exhibited low affinity for neosaxitoxin (KD approximately equal to 60 nM) and a very slow dissociation rate of [3H]saxitoxin (t0.5 approximately equal to 90 min), properties nearly identical to those of the tetrodotoxin-insensitive sites in membranes. The soluble saxitoxin-binding activity is also characterized by a more basic pH dependence and a complete lack of binding competition between saxitoxin and alkali cations. Bullfrog muscle appears to be a good tissue source for the purification of this soluble saxitoxin-binding protein.
mesh_terms: D029845:Amphibian Proteins; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D008051:Lipid Bilayers / Q000032:analysis; D009132:Muscles / Q000032:analysis*; D011892:Rana catesbeiana; D012964:Sodium / Q000378:metabolism*; D013779:Tetrodotoxin / Q000494:pharmacology*; D014316:Tritium
chemical_list: D029845:Amphibian Proteins; D002352:Carrier Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; C027525:saxitoxin-binding protein, Rana catesbeiana; D014316:Tritium; D013779:Tetrodotoxin; D012964:Sodium