Dihydropyridine binding and Ca(2+)-channel characterization in clonal calcitonin-secreting cells.
1,4-Dihydropyridine-sensitive voltage-dependent Ca2+ channels play a crucial role in the extracellular Ca(2+)-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 Ca(2+)-sensitive C-cells (rat C-cell line rMTC 44-2) in comparison with 'defective' Ca(2+)-insensitive C-cells (human C-cell line TT). In membranes of rMTC cells, we detected a high-affinity, stereoselective and Ca(2+)-dependent binding site for the Ca(2+)-channel-blocking 1,4-dihydropyridine, (+)-[3H]PN 200-110. Radioligand binding was saturable (Bmax. = 18 +/- 2 fmol/mg of protein), reversible [Ki 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 alpha 1-subunit of L-type Ca2+ channels. In contrast, in membranes of TT cells, which are known to lack Ca(2+)-sensitivity, no Ca(2+)-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 Ca(2+)-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 Ca(2+)-sensitive rMTC cells, but not in Ca(2+)-insensitive TT cells, reflects the electrophysiological detection of functional Ca2+ channel in rMTC cells, but not in TT cells.
Pharmakologisches Institut, Freien Universität Berlin, Federal Republic of Germany.
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D
Krautwurst
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H
Scherübl
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Kleppisch
J
J
Hescheler
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G
Schultz
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001464:Barium / Q000378:metabolism; D044182:Benzylisoquinolines; D001667:Binding, Competitive; D002116:Calcitonin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D002460:Cell Line / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D004095:Dihydropyridines / Q000378:metabolism*; D004553:Electric Conductivity; D007700:Kinetics; D008839:Microelectrodes; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism
D000470:Alkaloids; D044182:Benzylisoquinolines; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004095:Dihydropyridines; D001464:Barium; C009438:tetrandrine; C038806:1,4-dihydropyridine; D002116:Calcitonin; D002118:Calcium