Inactivation determinant in the I‐II loop of the Ca2+ channel α1‐subunit and β‐subunit interaction affect sensitivity for the phenylalkylamine (−)gallopamil

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 (α1A-PAA). Use-dependent barium current (IBa) inhibition of α1A-PAA by (−)gallopamil and Ca2+ channel recovery from inactivation and block were studied with two-microlectrode voltage clamp after expression of α1A-PAA and auxiliary α2-δ- and β1a- or β2a-subunits in Xenopus oocytes. Mutation Arg387Glu (α1A numbering) in the intracellular loop connecting domains I and II of α1A-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 α1A-PAA/R-E/β1a channels by 100 μm (−)gallopamil (53 ± 2 %, α1A-PAA/β1avs. 31 ± 2 %, α1A-PAA/R-E/β1a, n ≥ 4). This amino acid substitution simultaneously accelerated the recovery of channels from inactivation and from block by (−)gallopamil. Coexpression of α1A-PAA with the β2a-subunit reduced fast IBa inactivation and induced a substantial reduction in use-dependent IBa inhibition by (−)gallopamil (25 ± 4 %, α1A-PAA/β2a; 13 ± 1 %, α1A-PAA/R-E/β2a). The time constant of recovery from block at rest was not significantly affected. These results demonstrate that changes in channel inactivation induced by Arg387Glu or β2a-α1-subunit interaction affect the drug-channel interaction. Calcium (Ca2+) channel inhibition by drugs such as phenylalkylamines (PAAs), benzothiazepines (BTZs) and mibefradil increases during repetitive depolarisation of the membrane (Lee & Tsien 1983; McDonald et al. 1984; Bezprozvanny & Tsien 1995; Aczel et al. 1998). Such a ‘use-dependent’ channel inhibition reflects distinct drug interactions with the resting, open and inactivated channel states. It is believed that state-dependent Ca2+ channel block plays an important role in the therapeutic action of PAAs and BTZs as antiarrhythmics (Hondeghem & Katzung, 1984). Functional studies on mutant Ca2+ channels enabled the first insights into the molecular architecture of the Ca2+ channel drug-binding domains (see Hockerman et al. 1997b and Striessnig et al. 1998 for review). The available data suggest that three different classes of Ca2+ channel antagonists (PAAs, BTZs and 1,4-dihydropyridines) bind in close proximity within the pore region of L-type Ca2+ channel α1-subunits (Striessnig et al. 1998). Three amino acids in segment IVS6 (Tyr1463, Ala1467, Ile1470) and four residues in transmembrane segment IIIS6 (Tyr1152, Ile1153, Phe1164 and Val1165) have been identified as crucial L-type determinants of the PAA sensitivity (Hockerman et al. 1995, 1997a). Insertion of three ‘L-type-specific’ residues (Tyr1463, Ala1467 and Ile1470) into segment IVS6 of the only weakly PAA sensitive class A (α1A) Ca2+ channel transferred PAA sensitivity to the corresponding α1A mutant (here called α1A-PAA; Hering et al. 1996). An unequivocal identification of the PAA binding determinants by mutational analysis of α1 Ca2+ channel subunits is, however, complicated by an apparent interdependence between Ca2+ channel block and inactivation gating (see Hering et al. 1998 for review). In particular, transfer of the IVS6 L-type determinants of PAA sensitivity (Tyr1463, Ala1467 and Ile1470) to class A Ca2+ channels accelerated inactivation (Hering et al. 1996; Degtiar et al. 1997). Moreover, introduction of an additional L-type amino acid (Met1464 into IVS6 of α1A-PAA) facilitated channel inactivation and enhanced use-dependent channel block by (−)gallopamil (Hering et al. 1996). Accordingly, substitution of a PAA determinant in α1A-PAA (Ile1470 by the corresponding class A channel Met, α1C-a numbering) which substantially reduced channel inactivation induced an about 30-fold decrease of the apparent association rate for (−)devapamil (Degtiar et al. 1997) and alanine substitutions of three L-type amino acids localised close to the inner channel mouth on segment IIIS6 and IVS6 reduced Ca2+ channel inactivation and simultaneously BTZ and PAA sensitivity (Hering et al. 1997; Berjukow et al. 1999). In our previous studies on the role of Ca2+ channel inactivation in channel block by PAAs and BTZs we have focused on residues that are located on segments IIIS6 and IVS6 (Degtiar et al. 1997; Hering et al. 1997; Berjukow et al. 1999). Here we analyse in a PAA-sensitive class A Ca2+ channel mutant (α1A-PAA) expressed in Xenopus oocytes if inactivation determinants localised outside the channel pore of an α1-subunit influence Ca2+ channel block by (−)gallopamil. We demonstrate that a single amino acid substitution (Arg387Glu, α1A numbering) in the intracellular loop between domains I and II slows channel inactivation and reduces sensitivity for (−)gallopamil. Furthermore, a reduced inactivation caused by coexpression of α1A-PAA with β2a- instead of the β1a-subunit reduced Ca2+ channel block by (−)gallopamil even more dramatically. Our study clearly demonstrates that inactivation determinants that are localised outside the putative drug binding regions in the channel pore affect the molecular mechanism of use-dependent Ca2+ channel block by (−)gallopamil.