IDENTIFICATION OF BENZ(OTHI)AZEPINE-BINDING REGIONS WITHIN L-TYPE CALCIUM-CHANNEL ALPHA-1 SUBUNITS

To identify the binding domain for diltiazem-like Ca2+ antagonists on L-type Ca2+ channel alpha 1 subunits we synthesized the benzazepine [H -3]benziazem as a novel photoaffinity probe. [H-3]Benziazem reversibly labeled the benzothiazepine (BTZ)-binding domain of partially purifie d skeletal muscle Ca2+ channels with high affinity (K-d = 12 nM) and p hotoincorporated into its binding domain with high yield (>66%). Antib ody mapping of proteolytic labeled fragments revealed specific labelin g of regions associated with transmembrane segments S6 in repeats III and IV. More than 50% of the labeling was found in the tryptic fragmen t alanine 1023-lysine 1077 containing IIIS6 together with extracellula r and intracellular amino acid residues. The remaining labeling was id entified in a second site comprising segment S6 in repeat IV and adjac ent residues. Unlike for dihydropyridines, no labeling was observed in the connecting IIIS5-IIIS6 linker. The [H-3]benziazem photolabeled re gions must be in close contact to the drug molecule when bound to the channel. We propose that the determinants for high affinity BTZ bindin g are located within or in close proximity to segments IIIS6 and/or IV S6. Therefore the binding domain for BTZs, like for the other main cla sses of Ca2+ antagonists, must be located in close proximity to pore-f orming regions of the channel.