Extra- and intracellular action of quaternary devapamil on muscle L-type Ca2+-channels

1 The quaternary derivative of the potent verapamiL-analogue, (-)-D888, (qD888, 4-cyano-4-(3,4-dimethoxyphenyl)-N-[2-(3-methoxy phenyl)ethyl]-N,N,5-trimethyl-1-hexanaminium) was synthesized as a novel membrane-impermeable probe to study the localization of phenylalkylamine (PAA) effector domains on L-type Ca2+ channels. Channel block by qD888 was investigated in smooth muscle-like (A7r5) cells after extra- and intracellular application by use of the whole-cell configuration of the patch clamp technique. 2 Extracellularly applied qD888 inhibited Sr2+ (I-Sr) (IC50=90 mu M) and Na+ (IC50=27 mu m) inward currents through L-type Ca2+-channels mainly in a resting-state-dependent manner. Structurally closely related quaternary PAAs (e.g. D890) were ineffective after extracellular application. 3 QD888 also blocked I-Sr from the cytoplasmic side, as did other quaternary PAAs (D890, D575). Intracellular block was clearly dependent on channel opening, which resulted in pronounced use-dependence. 4 We conclude that qD888 blocks L-type Ca2+ channels not only from the intracellular side, via interaction with the classical PAA binding domain, but also from the extracellular channel surface. The properties of Ca2+ channel block together with previous biochemical and structural data suggest that extracellular block may be mediated by a site that also confers tonic block by quaternary benzothiazepines.