Identification and affinity labeling of very high affinity binding sites for the phenylalkylamine series of Ca+ channel blockers in the Drosophila nervous system

Abstract

The interaction of putative Ca2+ channels of Drosophila head membranes with molecules of the phenylalkylamine series was studied from binding experiments using (-)-[3H]D888 and (+/-)-[3H]verapamil. These ligands recognize a single class (Kd = 0.1-0.4 nM; Bmax = 1600-1800 fmol/mg of protein) of very high affinity binding sites. The most potent molecule in the phenylalkylamine series was (-)-verapamil with a Kd value as exceptionally low as 4.7 pM. Molecules in the benzothiazepine and diphenylbutylpiperidine series of Ca2+ channel blockers as well as bepridil inhibited (-)-[3H]D888 binding in a competitive way with Kd values between 12 and 190 nM, suggesting a close correlation, as in the mammalian system, between these receptor sites and those recognizing phenylalkylamines. A tritiated (arylazido)phenylalkylamine with high affinity for the Drosophila head membranes, phenylalkylamine receptor Kd = 0.24 nM), was used in photoaffinity experiments. A protein of Mr 135,000 +/- 5,000 was specifically labeled after ultraviolet irradiation.