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

Abstract

Mutations in motif IVS5 and IVS6 of the human cardiac calcium channel were made using homologous residues from the rat brain sodium channel 2a.[ 3 H]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 usedependent 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.Voltage-dependent calcium channels mediate calcium influx in response to membrane depolarization and play a critical role in cellular activities such as excitation-contraction coupling, neurotransmitter release, and hormone secretion (1, 2). L-type voltage-dependent calcium channels are characterized pharmacologically by high sensitivity to the calcium blocking drugs that include dihydropyridines (DHP), 1 phenylalkylamines (PAA), and benzothiazepines (BZT) (3, 4). Photolabeling studies of the purified ␣ 1 subunit and identification of the proteolyzed fragments by site-directed antibodies demonstrate that the ␣ 1 subunit harbors all three classes of binding sites (3). The results from these studies provide substantial evidence that motifs IIIS6 and IVS6 contain the major binding regions for these drugs.In more recent studies, the molecular identification of the binding sites on the ␣ 1 subunit for PAA (5-8), BZT (9, 10), and DHPs (7,(11)(12)(13)(14) were revealed. The region YMAI in IVS6 is a common binding site for both BZT (9) and PAA (7, 15). A part of this region in addition to amino acid residues within IIIS5 (16, 17) and IIIS6 (8) contribute to the DHP pocket (18,19). When the YMAI region was mutated to the corresponding amino acids in the DHP-insensitive channel ␣ 1A, the resultant channel was still sensitive to PAAs and BZTs (20). Thus, the high affinity binding site YMAI, however, is not the minimal requirement for drug binding.Less detailed structural information is known regarding the mechanism of use-dependent block, a feature that is critical to the therapeutically useful calcium antago...

On the Role of Ca 2+ - and Voltage-Dependent Inactivation in Ca v 1.2 Sensitivity for the Phenylalkylamine (-)Gallopamil

“…A comparison of the PAA sensitivities of Ca v 2.1 mutants and chimeric channel constructs inactivating at different rates revealed a strong correlation between the rates of fast voltage-dependent inactivation and apparent PAA sensitivities. [11][12][13] Similar findings were reported for Ca 2ϩ channel block by diltiazem. 11,14 Subsequently, the studies concentrated on the localization of inactivation determinants that are relevant for channel inhibition.…”

“…[2][3][4]6 Since then, numerous studies in myocardial and smooth muscle cells have shown that Ca 2ϩ channel inhibition by these compounds, as well as by the new compound mibefradil, reflect a balance between channel shut-off during membrane depolarization and subsequent recovery at rest. [7][8][9][10][11] Over the last three years there has been substantial progress toward understanding the role of inactivation in Ca 2ϩ channel inhibition by Ca 2ϩ antagonists. Site directed mutagenesis enabled a detailed analysis of the impact of this process in PAA sensitivity.…”

The L-type calcium channel in the heart: the beat goes on

“…Hering et al (95) were among the first to establish a relationship between Ca 2+ channel inactivation and use-dependent Ca 2+ channel block by PAA. Since that time, single amino acids have been identified as inactivation determinants in motifs IIIS6, IVS6, and IVS5, with some of them also serving as high-affinity determinants for the DHP receptor site (94)(95)(96).…”

The Role of Region IVS5 of the Human Cardiac Calcium Channel in Establishing Inactivated Channel Conformation

“…Identical pulse protocols were used in the presence of drugs. Diltiazem (racemic) was perfused in the bath (for a 2.5-min period) at concentrations of 200 M. This concentration was selected because it provided sufficient block for the use-dependent measurements in Xenopus oocytes (13).…”

“…Our previous finding showed that a segment in IVS5 of the human ␣ 1C (Ca v 1.2) subunit is critically involved in inactivation of the channel (13). Consequently, mutants constructed in this region lost the characteristic use-dependent block by PAA and BTZ and recovered from inactivation significantly faster after drug block compared with the wild type channel.…”