Abstract

The L-type calcium channel (LTCC) isoforms Ca(v)1.2 and Ca(v)1.3 display similar 1,4-dihydropyridine (DHP) binding properties and are both expressed in mammalian brain. Recent work implicates Ca(v)1.3 channels as interesting drug targets, but no isoform-selective modulators exist. It is also unknown to what extent Ca(v)1.1 and Ca(v)1.4 contribute to L-type-specific DHP binding activity in brain. To address this question and to determine whether DHPs can discriminate between Ca(v)1.2 and Ca(v)1.3 binding pockets, we combined radioreceptor assays and quantitative polymerase chain reaction (qPCR). We bred double mutants (Ca(v)-DM) from mice expressing mutant Ca(v)1.2 channels [Ca(v)1.2DHP(-/-)] lacking high affinity for DHPs and from Ca(v)1.3 knockouts [Ca(v)1.3(-/-)]. (+)-[(3)H]isradipine binding to Ca(v)1.2DHP(-/-) and Ca(v)-DM brains was reduced to 15.1 and 4.4% of wild type, respectively, indicating that Ca(v)1.3 accounts for 10.7% of brain LTCCs. qPCR revealed that Ca(v)1.1 and Ca(v)1.4 alpha(1) subunits comprised 0.08% of the LTCC transcripts in mouse whole brain, suggesting that they cannot account for the residual binding. Instead, this could be explained by low-affinity binding (127-fold K(d) increase) to the mutated Ca(v)1.2 channels. Inhibition of (+)-[(3)H]isradipine binding to Ca(v)1.2DHP(-/-) (predominantly Ca(v)1.3) and wild-type (predominantly Ca(v)1.2) brain membranes by unlabeled DHPs revealed a 3- to 4-fold selectivity of nitrendipine and nifedipine for the Ca(v)1.2 binding pocket, a finding further confirmed with heterologously expressed channels. This suggests that small differences in their binding pockets may allow development of isoform-selective modulators for LTCCs and that, because of their very low expression, Ca(v)1.1 and Ca(v)1.4 are unlikely to serve as drug targets to treat CNS diseases.