Detergent extraction of a presumptive gating component from the voltage-dependent sodium channel.

Abstract

A physiologically characterized radiolabeled neurotoxin complex obtained from venom of the scorpion Leiurus quinquestriatue has been used to identify -detergent-solubilized presumptive sodium channel components in sucrose gradients. This toxin-binding component is found in extracts prepared from three sources of excitable membrane but appears to Electrophysiological experiments have provided a quantitative description ofthe time and field dependence ofgating processes that regulate voltage-dependent sodium channels. In contrast there is, as yet, little information on the structure and organization of these channels. One approach to providing such information is that of using physiologically characterized animal neurotoxins as selective affinity ligands for the identification of sodium channel components. Two neurotoxins that have proven to be of particular value in this regard are tetrodotoxin (TIX) and saxitoxin (STX). Although structurally different, these molecules have very similar physiological activities, binding to voltage-dependent sodium channels with high affinity to produce a selective sodium conductance block (1). This interaction effectively inhibits ion transport through the channel but does not appear to alter either the kinetics or voltage dependence of the asymmetry currents associated with channel gating (2, 3). These toxins also appear to show considerable biochemical specificity, recognizing the sodium channel to the relative exclusion of other membrane proteins (4). STX and TTX have been used as affinity ligands for the identification and partial purification of a sodium channel component solubilized from eel electroplaque (5) and rat skeletal muscle (6). The solubilized toxin-binding component has a molecular weight of 200,000-300,000 and a -sedimentation coefficient near 9 S. A rapid loss of high-affinity toxin-binding activity occurs in the presence ofexcess detergent or during procedures that strip bound lipid from the solubilized channel component; thus purification of this component in an active form appears possible only if exogenous lipid is added to the solubilization and chromatography buffers (7).A second group ofneurotoxins that may be ofsignificant value in biochemical studies on the sodium channel are those obtained from scorpion venoms. Scorpion toxins selectively alter channel gating without substantially changing channel conductance. Venoms from two species are of particular interest. Centruroides sculpturatus neurotoxins selectively alter the voltage dependence of sodium activation, or "m" gating (8), and neurotoxins isolated from Leiurus quinquestriatus characteristically slow the kinetics of sodium inactivation (9, 10).Of the scorpion neurotoxins, those from Leiurus have been the most extensively employed in biochemical studies on the sodium channel. Work on this venom by Miranda et aL (11) led to the isolation of five neurotoxic components. These toxins appear to be structurally related in that they have molecular weights near 6600, lack methionine, and show ...

Tunicamycin inhibits the expression of surface Na+ channels in cultured muscle cells

“…Pharmacological studies indicate that Na + channels consist of at least three separate components that possess binding sites for specific neurotoxins (Catterall, 1980b). The Na+ channel component that binds the neurotoxins tetrodotoxin (TTX) and saxitoxin has recently been solubilized in detergent, purified (Agnew et al, 1978;Barchi et al, 1980;Culp and McKenzie, 1981;Moore et al, 1982; Weigele and Barchi, 19821, and shown to interact with lectins (Cohen and Barchi, 1981), indicating that sugar moieties are linked to channel polypeptides.In the present study, we have used tunicamycin (TM), an antibiotic that inhibits the synthesis and transfer of core oligosaccarides to asparaginyl residues of nascent polypeptides (Kuo and Lampen, 1974;Takatsuki et al, 1975;Tkacz and Lampen, 1975) to investigate the contribution of carbohydrate moieties to the properties of Na+ channels expressed in cultured muscle cells. Our findings indicate that surface Na+ channels elaborated under conditions of impaired glycosylation have altered metabolic properties, as reflected by an accelerated rate of degradation.…”

“…Pharmacological studies indicate that Na + channels consist of at least three separate components that possess binding sites for specific neurotoxins (Catterall, 1980b). The Na+ channel component that binds the neurotoxins tetrodotoxin (TTX) and saxitoxin has recently been solubilized in detergent, purified (Agnew et al, 1978;Barchi et al, 1980;Culp and McKenzie, 1981;Moore et al, 1982;Weigele and Barchi, 19821, and shown to interact with lectins (Cohen and Barchi, 1981), indicating that sugar moieties are linked to channel polypeptides.…”