1,5-BENZOTHIAZEPINE BINDING DOMAIN IS LOCATED ON THE EXTRACELLULAR SIDE OF THE CARDIAC L-TYPE CA2+ CHANNEL

To determine whether 1,5-benzothiazepine Ca2+ channel blocker approach es its binding domain within the cardiac L-type Ca2+ channel from insi de or outside of the membrane, we tested the effects of a novel potent 1,5-benzothiazepine derivative (DTZ323) and its quaternary ammonium d erivative (DTZ417) on guinea pig ventricular myocytes by using the who le-cell patch-clamp technique. The extracellular application of DTZ417 suppressed the L-type Ca2+ channel currents (I-Ca(L)) with an IC50 va lue of 1.2 +/- 0.02 mu M, which was close to the IC50 value of diltiaz em (0.63 +/- 0.01 mu M). The suppression of I-Ca(L) by DTZ417 was volt age and use dependent but lacked tonic block, which allowed us to inve stigate the onset of the effect on I-Ca(L) by changing the holding pot ential (HP) from -90 to -50 mV in the presence of DTZ417. DTZ417 did n ot have significant effects on I-Ca(L) at an HP of -90 mV. At -50 mV, DTZ417 (50 mu M) applied from the extracellular side completely suppre ssed I-Ca(L), whereas it had no effect from the intracellular side. DT Z323 (1 mu M) also inhibited I-Ca(L) only from the extracellular side, without any effects by the intracellular application of less than or equal to 10 mu M. However, a quaternary phenylalkylamine derivative, D 890 (0.1 mM), acted only from the intracellular side. These results su ggest that in contrast to the phenylalkylamine binding site, in cardia c myocytes the 1,5-benzothiazepine binding site is accessible from the extracellular side of the L-type Ca2+ channel.