Analysis of pyrethroid binding by use of a photoreactive analogue: Possible role for GTP-binding proteins in pyrethroid activity

Abstract

Neurotoxicity of pyrethroids has been attributed to their activity on a variety of enzymes, membrane receptors, and ion channels—especially voltage-dependent sodium channels. To study pyrethroid binding, I have synthesized decyanoazidofenvalerate (DeCAF), a photoactivatable arylazide derivative of fenvalerate. DeCAF is insecticidal and induces repetitive firing in invertebrate nerve axons. SDS-PAGE analysis of photoderivatized proteins from rat brain membranes indicated that [ 3H]DeCAF covalently labeled a 36-kDa membrane-bound protein. This labeling was completely abolished by fenvalerate and unlabeled DeCAF, and partially inhibited by other pyrethroids. While labeling was not inhibited by alkaloid toxins or polypeptide venoms that modify voltage-dependent sodium channels, photolabeling was stimulated by tetrodotoxin and saxitoxin, specific blockers of voltage-dependent sodium channels. The [ 3H]DeCAF-labeled protein could not be identified as a known sodium channel subunit or as the 36-kDa subunit of the peripheral benzodiazepine receptor (another target of pyrethroids). [ 3H]DeCAF labeling was modified by addition of either GTPγS or pretreatment of membranes with cholera toxin. These results indicate that pyrethroids interact with a 36-kDa protein that is associated with voltage-sensitive sodium channels and is affected by modifiers of GTP-binding proteins.