L-type calcium channels: Asymmetrical intramembrane binding domain revealed by variable length, permanently charged 1,4-dihydropyridines

We have used an homologous series of dihydropyridine (DHP) derivatives to determine the location of the binding domain for DHPs on cardiac L-type calcium channels, relative to the extracellular and intracellular membrane surfaces. The series of test molecules consisted of DHP analogs in which the DHP moiety was linked to either a neutral (-CH₂CH₃) or permanently charged [-⁺N(CH₃)₃] headgroup and the distance between the headgroup and the active moiety was systematically varied with alkyl spacer chains containing 2, 6, 8, 10, 12, or 16 methylene (-CH₂) groups. These compounds were previously shown, by radioligand binding experiments, to interact with the high affinity DHP binding site in intact neonatal rat heart cells. In the present experiments, access to the DHP binding site was assayed by inhibition of L-type calcium channel currents using whole-cell patch-clamp procedures in guinea pig ventricular myocytes. Intracellular application was achieved by dialysis via charged DHP-containing whole-cell patch pipettes, and cell dialysis was monitored by using a charged DHP labeled with a rhodamine fluorophore. Our results show that access of extracellularly applied charged, but not neutral, DHPs to the DHP binding domain depends markedly on the alkyl spacer chain, with the optimal length being near 10 methylene groups. Intracellular application failed to inhibit channel activity for spacer chain lengths up to 16 methylene groups. From our results, we conclude that the DHP binding domain of cardiac L-type calcium channels is not on the extracellular membrane surface but is probably within the lipid bilayer, approximately 11- 14 Å from the extracellular surface.