A Region in IVS5 of the Human Cardiac L-type Calcium Channel Is Required for the Use-dependent Block by Phenylalkylamines and Benzothiazepines

Mutations in motif IVS5 and IVS6 of the human cardiac calcium channel were made using homologous residues from the rat brain sodium channel 2a. [3H]PN200-110 and allosteric binding assays revealed that the dihydropyridine and benzothiazepine receptor sites maintained normal coupling in the chimeric mutant channels. Whole cell voltage clamp recording from Xenopus oocytes showed a dramatically slowed inactivation and a complete loss of use-dependent block for mutations in the cytoplasmic connecting link to IVS5 (HHT-5371) and in IVS5 transmembrane segment (HHT-5411) with both diltiazem and verapamil. However, the use-dependent block by isradipine was retained by these two mutants. For mutants HHT-5411 and HHT-5371, the residual current appeared associated with a loss of voltage dependence in the rate of inactivation indicating a destabilization of the inactivated state. Furthermore, both HHT-5371 and -5411 recovered from inactivation significantly faster after drug block than that of the wild type channel. Our data demonstrate that accelerated recovery of HHT-5371 and HHT-5411 decreased accumulation of these channels in inactivation during pulse trains and suggest a close link between inactivation gating of the channel and use-dependent block by phenylalkylamines and benzothiazepines and provide evidence of a role for the transmembrane and cytoplasmic regions of IVS5 in the use-dependent block by diltiazem and verapamil.