The benzazepine/benzothiazepine binding domain of the cardiac L-type Ca²⁺ channel is accessible only from the extracellular side

Abstract

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 μM, 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 μM D890, which produced a progressive inhibition of Ca²⁺ currents within 10 min after whole-cell access. In contrast, internal application of 100 μM 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 α ₁-subunit of the Ca²⁺ channel protein.