THE BENZAZEPINE BENZOTHIAZEPINE BINDING DOMAIN OF THE CARDIAC L-TYPE CA2+ CHANNEL IS ACCESSIBLE ONLY FROM THE EXTRACELLULAR SIDE

The whole-cell tight seal recording technique was used to investigate location of benzothiazepine binding site of the cardiac L-type Ca2+ channel. For this we utilized a permanently charged compound, SQ 32.428, out of a series of benzazepine drugs which have been characterized as competitive inhibitors of diltiazem binding. The non-permanently charged derivative SQ 32.910 was initially tested to electrophysiologically establish Ca2+ antagonistic properties of benzazepines. Upon extracellular application, either compound was able to completely block Ca2+ currents. At a stimulating frequency of 0.2 Hz IC50 concentrations of SQ 32.910 and SQ 32.428 were determined as 35 nM and 15 muM, respectively. Intracellular application of SQ 32.428 was then compared to control experiments in the absence of drug. Initially, adequate drug dialysis was confirmed with 100 muM D890, which produced a progressive inhibition of Ca2+ currents within 10 min after whole-cell access. In contrast, internal application of 100 muM SQ 32.428 did not change time-course of Ca2+ currents compared to control run-down. These results show that the benzazepine/benzothiazepine binding domain of the cardiac L-type Ca2+ channel is accessible only from the extracellular side and therefore suggest an extracellular location on the alpha1-subunit of the Ca2+ channel protein.