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Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents
Published on: July 3, 2013
High-affinity binding of [3H]DTZ323 to the diltiazem-binding site of L-type Ca2+ channels
Masafumi Hagiwara1, Satomi Adachi-Akahane, Taku Nagao
1Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Bunkyo, Japan.
Abstract:
D-cis-[N-Methyl-3H]-3-(acetyloxy)-5-[2-[[2-(3,4-dimethoxyphenyl)ethyl]-methylamino]ethyl]-2,3-dihydro-2-(4-methoxyphenyl)-1,5-benzothiazepine-4(5H)-one ([3H]DTZ323), a novel 1,5-benzothiazepine radioligand, was characterized in a ligand-receptor binding study. Specific binding of [3H]DTZ323 to rabbit skeletal muscle T-tubule membranes was saturable and reversible. Scatchard analysis indicated a single binding site with a K(d) value of 1.4 and 1.8 nM at 25 and 37 degrees C, respectively. DTZ323 and diltiazem derivatives inhibited specific [3H]DTZ323 binding with a rank order of DTZ323>DTZ417 (quaternary ammonium derivative of DTZ323)>diltiazem>L-cis-DTZ323. The affinity of DTZ323 was 51 times higher than that of diltiazem. [3H]DTZ323 binding was also completely inhibited by verapamil and tetrandrine, thus revealing the unique nature of the diltiazem-binding site. Specific [3H]DTZ323 binding to crude guinea pig ventricular membranes was inhibited by diltiazem, DTZ323 and its derivatives with IC(50) values close to those previously reported for the blockade of L-type Ca(2+) channel currents. These results indicate that [3H]DTZ323 is a potent and selective radioligand for the diltiazem-binding site of L-type Ca(2+) channels.
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