Temperature‐dependent regulation of d‐cis‐[3H]diltiazem binding to Ca2+ channels by 1,4‐dihydropyridine channel agonists and antagonists

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Abstract
The binding of the Ca2+‐channel blocker d‐cis‐[3H]diltiazem to guinea pig skeletal muscle microsomes is temperature‐dependent. At 2°C the K D is 39 nM and B max is 11 pmol/mg protein. The binding is fully reversible (K −1 = 0.02 min−1). The binding sites discriminate between the diastereoisomers 1‐ and d‐cis‐diltiazem, recognize verapamil, gallopamil and tiapamil, and are sensitive to La3+‐inhibition. At 30°C the K D is 37 nM and the B max is 2.9 pmol/mg protein. D‐cis‐diltiazem‐labelling is regulated by the 1,4‐dihydropyridine Ca2+‐channel blockers and a novel Ca2+‐channel activator in a temperature‐dependent manner. At 30°C an enhancement of d‐cis‐diltiazem binding by the channel blockers is observed. This is attributed to a B max increase. EC 50‐values for enhancement and the maximal enhancement differ for the individual 1,4‐dihydropyridines. At 2°C 1,4‐dihydropyridines inhibit d‐cis‐[3H]diltiazem binding. This is attributed to a B max decrease. We have directly labelled one of the drug receptor sites within the Ca2+‐channel which can allosterically interact with the 1,4‐dihydropyridine binding sites.