Inactivation determinant in the I-II loop of the Ca2+ channel alpha1-subunit and beta-subunit interaction affect sensitivity for the phenylalkylamine (-)gallopamil.
1. The role of calcium (Ca2+) channel inactivation in the molecular mechanism of channel block by phenylalkylamines (PAAs) was analysed in a PAA-sensitive rabbit brain class A Ca2+ channel mutant (alpha1A-PAA). Use-dependent barium current (IBa) inhibition of alpha1A-PAA by (-)gallopamil and Ca2+ channel recovery from inactivation and block were studied with two-microlectrode voltage clamp after expression of alpha1A-PAA and auxiliary alpha2-delta- and beta1a- or beta2a-subunits in Xenopus oocytes. 2. Mutation Arg387Glu (alpha1A numbering) in the intracellular loop connecting domains I and II of alpha1A-PAA slowed the inactivation kinetics and reduced use-dependent inhibition (100 ms test pulses at 0.2 Hz from -80 to 20 mV) of the resulting mutant alpha1A-PAA/R-E/beta1a channels by 100 microM (-)gallopamil (53 +/- 2 %, alpha1A-PAA/beta1a vs. 31 +/- 2 %, alpha1A-PAA/R-E/beta1a, n >= 4). This amino acid substitution simultaneously accelerated the recovery of channels from inactivation and from block by (-)gallopamil. 3. Coexpression of alpha1A-PAA with the beta2a-subunit reduced fast IBa inactivation and induced a substantial reduction in use-dependent IBa inhibition by (-)gallopamil (25 +/- 4 %, alpha1A-PAA/beta2a; 13 +/- 1 %, alpha1A-PAA/R-E/beta2a). The time constant of recovery from block at rest was not significantly affected. 4. These results demonstrate that changes in channel inactivation induced by Arg387Glu or beta2a-alpha1-subunit interaction affect the drug-channel interaction.
Sokolov
S
S
Institut für Biochemische Pharmakologie, Peter-Mayr-Strasse 1, A-6020 Innsbruck, Austria.
Weiss
R G
RG
Kurka
B
B
Gapp
F
F
Hering
S
S
D000465:Algorithms; D019943:Amino Acid Substitution; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects* / Q000235:genetics; D020746:Calcium Channels, L-Type / Q000187:drug effects* / Q000235:genetics; D004594:Electrophysiology; D005260:Female; D005711:Gallopamil / Q000494:pharmacology*; D006801:Humans; D009154:Mutation; D011817:Rabbits; D014982:Xenopus laevis
C107597:CACNB2 protein, human; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D005711:Gallopamil