Molecular mechanism of calcium channel block by isradipine. Role of a drug-induced inactivated channel conformation.

The role of the inactivated channel conformation in the molecular mechanism of Ca(2+) channel block by the 1,4-dihydropyridine (DHP) (+)-isradipine was analyzed in L-type channel constructs (alpha(1Lc); Berjukow, S., Gapp, F., Aczel, S., Sinnegger, M. J., Mitterdorfer, J., Glossmann, H., and Hering, S. (1999) J. Biol. Chem. 274, 6154-6160) and a DHP-sensitive class A Ca(2+) channel mutant (alpha(1A-DHP); Sinnegger, M. J., Wang, Z., Grabner, M., Hering, S., Striessnig, J., Glossmann, H., and Mitterdorfer, J. (1997) J. Biol. Chem. 272, 27686-27693) carrying the high affinity determinants of the DHP receptor site but inactivating at different rates. Ca(2+) channel inactivation was modulated by coexpressing the alpha(1A-DHP)- or alpha(1Lc)-subunits in Xenopus oocytes with either the beta(2a)- or the beta(1a)-subunit and amino acid substitutions in L-type segment IVS6 (I1497A, I1498A, and V1504A). Contrary to a modulated receptor mechanism assuming high affinity DHP binding to the inactivated state we observed no clear correlation between steady state inactivation and Ca(2+) channel block by (+)-isradipine: (i) a 3-fold larger fraction of alpha(1A-DHP)/beta(1a) channels in steady state inactivation at -80 mV (compared with alpha(1A-DHP)/beta(2a)) did not enhance the block by (+)-isradipine; (ii) different steady state inactivation of alpha(1Lc) mutants at -30 mV did not correlate with voltage-dependent channel block; and (iii) the midpoint-voltages of the inactivation curves of slowly inactivating L-type constructs and more rapidly inactivating alpha(1Lc)/beta(1a) channels were shifted to a comparable extent to more hyperpolarized voltages. A kinetic analysis of (+)-isradipine interaction with different L-type channel constructs revealed a drug-induced inactivated state. Entry and recovery from drug-induced inactivation are modulated by intrinsic inactivation determinants, suggesting a synergism between intrinsic inactivation and DHP block.

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.

Sequence differences between alpha1C and alpha1S Ca2+ channel subunits reveal structural determinants of a guarded and modulated benzothiazepine receptor.

The molecular basis of the Ca2+ channel block by (+)-cis-diltiazem was studied in class A/L-type chimeras and mutant alpha1C-a Ca2+ channels. Chimeras consisted of either rabbit heart (alpha1C-a) or carp skeletal muscle (alpha1S) sequence in transmembrane segments IIIS6, IVS6, and adjacent S5-S6 linkers. Only chimeras containing sequences from alpha1C-a were efficiently blocked by (+)-cis-diltiazem, whereas the phenylalkylamine (-)-gallopamil efficiently blocked both constructs. Carp skeletal muscle and rabbit heart Ca2+ channel alpha1 subunits differ with respect to two nonconserved amino acids in segments IVS6. Transfer of a single leucine (Leu1383, located at the extracellular mouth of the pore) from IVS6 alpha1C-a to IVS6 of alpha1S significantly increased the (+)-cis-diltiazem sensitivity of the corresponding mutant L1383I. An analysis of the role of the two heterologous amino acids in a L-type alpha1 subunit revealed that corresponding amino acids in position 1487 (outer channel mouth) determine recovery of resting Ca2+ channels from block by (+)-cis-diltiazem. The second heterologous amino acid in position 1504 of segment IVS6 (inner channel mouth) was identified as crucial inactivation determinant of L-type Ca2+ channels. This residue simultaneously modulates drug binding during membrane depolarization. Our study provides the first evidence for a guarded and modulated benzothiazepine receptor on L-type channels.