Native and detergent-solubilized membrane extracts from Drosophila heads contain binding sites for phenylalkylamine calcium channel blockers

Abstract

Membrane extracts from Drosophila melanogaster heads contain binding sites for [ 3H]D-600, a verapamil-like calcium channel blocker. Scatchard analysis suggested a two binding site model with a dissociation constant ( K d) of 3.3 ± 0.28 nM and a B max of 1.3 ± 0.14 pmol/mg protein for the high affinity site and a K d of 150 ± 28 nM and a B max of 29 ± 10 pmol/mg protein for the low affinity site. The rank order of affinity of phenylalkylamine binding to the high affinity site was (−)-D-888 > (±)-verapamil > (±)-D-600. Displacement of (−)-[ 3H])D-888 binding by phenylalkylamines is stereoselective. The diphenylpiperazine cinnarizine is an effective inhibitor of [ 3H]D-600 binding. However, [ 3H]D-600 binding is only partially inhibited by diltiazem and bepridil at 10 −4M concentrations, and is not affected by dihydropyridine calcium channel drugs at concentrations < 10 −5M. Moreover, only extremely low specific [ 3H]nitrendipine, (+)-[ 3H]PN200-110, or d-(cis)- [ 3H]diltiazem binding was detectable. The phenylalkylamine photoaffinity probe, [N-methyl- 3H]LU 49888, labeled two Proteinase-K sensitive proteins of 136,000 and 27,000 Da. The high molecular weight, trypsin-sensitive band behaves like the high affinity phenylalkylamine receptor, while the low molecular weight, trypsin-insensitive band has properties of the low affinity receptor. Digitonin-solubilized binding activity has a sedimentation coefficient of 12 S on a sucrose gradient.