Biochemical characterization of insect neuronal sodium channels

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Abstract
The pharmacological uniqueness of the insect sodium channels is indicated by their ability to bind the excitatory and depressant insect selective neurotoxins derived from scorpion venom. The latter were shown to bind and modify sodium conductance exclusively in insect neuronal membranes. The insect sodium channel polypeptides were identified by immunoprecipitation using site‐directed antibody, anti SP19 (corresponding to a highly conserved segment in the sodium channel α subunits), followed by radiophosphorylation and SDS‐PAGE autoradiography. Sodium channel polypeptide in the central nervous system (CNS) of insects belonging to four distinct orders (Orthoptera, Dyctioptera, Diptera, and Lepidoptera) were shown to (1) serve as substrates for phosphorylation by cAMP‐dependent protein kinase; (2) be devoid of disulfide linkage to smaller subunits unlike sodium channels in vertebrate CNS; (3) be glycoproteins as demonstrated by endoglycosidase F treatment and binding to lectins; and (4) reveal a diversity with regard to their apparent molecular mass (Mr 240,000–280,000) and partial peptide maps. The locust sodium channels were functionally solubilized (monitored by [3H]saxitoxin (STX) binding) by 1% cholate, 0.2% Triton X‐100, and 0.22% phosphatidylcholine. About 40% of STX binding activity was recovered in the solubilized fraction without affecting affinity (Kd = 0.5 nM). The time and temperature dependent lability of STX binding activity, in the solubilized fraction, was prevented by 20 nM STX. Partial purification of the insect sodium channel by an anion exchanger yielded 20% recovery and a 3.5 times increase in specific STX binding activity. The presence of a radiophosphorylated 245,000 α‐subunit band coincided with the STX binding activity during purification. In sum, the above information concerning the solubilization and characterization of the insect sodium channel will pave the way to the molecular identification of the receptor sites of the insect selective neurotoxins.

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