Temperature-dependent regulation of d-cis-[H]diltiazem binding to Ca channels by 1,4-dihydropyridine channel agonists and antagonists

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. EC₅₀-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.