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.
Motoike
H K
HK
Institute of Molecular Pharmacology and Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0828, USA.
Bodi
I
I
Nakayama
H
H
Schwartz
A
A
Varadi
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001464:Barium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020746:Calcium Channels, L-Type; D006801:Humans; D017275:Isradipine / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D051381:Rats; D015222:Sodium Channels / Q000378:metabolism; D013329:Structure-Activity Relationship; D014700:Verapamil / Q000378:metabolism; D014981:Xenopus
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D015222:Sodium Channels; C062217:benzothiazepine receptor; D001464:Barium; D014700:Verapamil; D017275:Isradipine