Dihydropyridine [methyl‐³H]PN 200–110 Binding and Myogenesis in Intact Muscle Cells In Vitro

Abstract: The radioligand dihydropyridine [methyl‐³H]PN 200–110 binds to contracting myotubes in culture derived from chick embryo pectoralis muscle. [methyl‐³H]PN 200–110 binds specifically to high‐affinity sites, with nonspecific binding only between 15 and 30% of the total binding. A Scatchard plot of the specific binding revealed a single high‐affinity binding site with a K_D (dissociation constant) of 0.5 nM± 0.2 nM and B_max (number of binding sites) of 100 fmol/10⁶ nuclei. We employed this sensitive assay to probe the appearance of high‐affinity [methyl‐³H]PN 200–110 binding sites during myogenesis. The time course of appearance of high‐affinity binding sites lags behind that of fusion. Low‐calcium media prevented the differentiation of myoblasts and blocked the appearance of high‐affinity sites. Chelation of intracellular calcium before or after fusion of myoblasts with the calcium indicator Quin 2 prevented the appearance of dihydropyridine binding sites. These findings are consistent with the view that the expression of dihydropyridine receptors is modulated by the intracellular calcium.