EXTRACELLULAR LOCALIZATION OF THE BENZOTHIAZEPINE BINDING DOMAIN OF L-TYPE CA(2+) CHANNELS

To determine which side of L-type Ca2+ channels forms the benzothiazep ine binding domain, we tested the effects of a membrane-impermeable, d iltiazem-like, Ca2+ antagonist, SQ32,428 [2-trimethylammonio)ethyl]-2H -1-benzazepin-2-one], on Ca2+ channels in smooth muscle-like cells (A7 r5 cells) and skeletal muscle-like cells (differentiated BC3H1 cells). This permanently charged, quaternary benzazepine bound to the benzoth iazepine-selective domain of skeletal muscle Ca2+ channels with a K(i) of 1.2 +/- 0.1 mum. Extracellular application of SQ32,428 reversibly blocked whole-cell barium currents through L-type Ca2+ channels in A7r 5 and BC3H1 cells with similar potencies (A7r5, IC50 = 86 mum; BC3H1, IC50 = 50 mum). Block was fully reversible, was independent of stimula tion frequency, and did not affect steady state inactivation of the ch annel in A7r5 cells. Intracellular dialysis of the cells with 100 mum SQ32,428 was without effect, but the same concentration of the quatern ary phenylalkylamine D890 blocked channel activity from the cytoplasmi c side. Our data demonstrate that the benzothiazepine binding domain o f L-type Ca2+ channels binds diltiazem-like benzazepine Ca2+ antagonis ts and is formed by amino acid residues exposed to the extracellular c hannel surface.