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 intracellula r membrane surfaces. The series of test molecules consisted of DHP ana logs in which the DHP moiety was linked to either a neutral (-CH2CH3) or permanently charged [-N-+(CH3)(3)] headgroup and the distance betwe en 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 i ntact neonatal rat heart cells. In the present experiments, access to the DHP binding site was assayed by inhibition of L-type calcium chann el currents using whole-cell patch-clamp procedures in guinea pig vent ricular myocytes. Intracellular application was achieved by dialysis v ia charged DHP-containing whole-cell patch pipettes, and cell dialysis was monitored by using a charged DHP labeled with a rhodamine fluorop hore. Our results show that access of extracellularly applied charged, but not neutral, DHPs to the DHP binding domain depends markedly on t he 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 chan nels is not on the extracellular membrane surface but is probably with in the lipid bilayer, approximately 11-14 Angstrom from the extracellu lar surface.