Dihydropyridine binding and Ca²⁺-channel characterization in clonal calcitonin-secreting cells

1,4-Dihydropyridine-sensitive voltage-dependent Ca²⁺ channels play a crucial role in the extracellular Ca²⁺-sensing of calcitonin-secreting parafollicular cells of the thyroid (C-cells). To characterize the Ca²⁺ channels in C-cells, we studied 1,4-dihydropyridine binding and performed electrophysiological experiments with Ca²⁺-sensitive C-cells (rat C-cell line rMTC 44-2) in comparison with 'defective' Ca²⁺-insensitive C-cells (human C-cell line TT). In membranes of rMTC cells, we detected a high-affinity, stereoselective and Ca²⁺-dependent binding site for the Ca²⁺-channel-blocking 1,4-dihydropyridine, (+)-[³H]PN 200-110. Radioligand binding was saturable (B(max) = 18 ± 2 fmol/mg of protein), reversible [K for (+)-PN 200-110 = 37 ± 1 pM) and allosterically modulated by the phenylalkylamine (-)-desmethoxyverapamil [(-)-D888] as well as the bis-benzylisoquinoline alkaloid (+)-tetrandrine. Thus the 1,4-dihydropyridine binding in rMTC cells featured all characteristics of binding to the α₁-subunit of L-type Ca²⁺ channels. In contrast, in membranes of TT cells, which are known to lack Ca²⁺-sensitivity, no Ca²⁺-channel-specific (+)[³H]PN 200-110 binding was detected. In voltage-clamp experiments, rMTC cells exhibited slowly inactivating Ca²⁺ currents which proved sensitive to (+)-PN 200-110, (-)-D888 and (+)-tetrandrine. These L-type Ca²⁺-channel blockers did not affect the Ca²⁺ currents in TT cells. The numbers of 1,4-dihydropyridine-sensitive Ca²⁺ channels in rMTC cells as calculated from both the binding studies and the whole-cell/single-channel recordings were 2000 and 7000/cell respectively. Thus qualitative and quantitative detection of 1,4-dihydropyridine-sensitive Ca²⁺ channels by radioligand-binding in Ca²⁺-sensitive rMTC cells, but not in Ca²⁺-insensitive TT cells, reflects the electrophysiological detection of functional Ca²⁺ channels in rMTC cells, but not in TT cells.