Abstract
The L-type calcium channel (LTCC) isoforms Cav1.2 and Cav1.3 display similar 1,4-dihydropyridine (DHP) binding properties and are both expressed in mammalian brain. Recent work implicates Cav1.3 channels as interesting drug targets, but no isoform-selective modulators exist. It is also unknown to what extent Cav1.1 and Cav1.4 contribute to L-type-specific DHP binding activity in brain. To address this question and to determine whether DHPs can discriminate between Cav1.2 and Cav1.3 binding pockets, we combined radioreceptor assays and quantitative polymerase chain reaction (qPCR). We bred double mutants (Cav-DM) from mice expressing mutant Cav1.2 channels [Cav1.2DHP(-/-)] lacking high affinity for DHPs and from Cav1.3 knockouts [Cav1.3(-/-)]. (+)-[3H]isradipine binding to Cav1.2DHP(-/-) and Cav-DM brains was reduced to 15.1 and 4.4% of wild type, respectively, indicating that Cav1.3 accounts for 10.7% of brain LTCCs. qPCR revealed that Cav1.1 and Cav1.4 α1 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 Kd increase) to the mutated Cav1.2 channels. Inhibition of (+)-[3H]isradipine binding to Cav1.2DHP(-/-) (predominantly Cav1.3) and wild-type (predominantly Cav1.2) brain membranes by unlabeled DHPs revealed a 3- to 4-fold selectivity of nitrendipine and nifedipine for the Cav1.2 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, Cav1.1 and Cav1.4 are unlikely to serve as drug targets to treat CNS diseases.
Footnotes
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This work was supported by the Austrian Research Fund [Grants P17159, P17807], the Tyrolean Research Fund, and the University of Innsbruck.
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M.J.S.-B. and I.G.H. contributed equally to this work.
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ABBREVIATIONS: LTCC, L-type Ca2+ channels; DHP, 1,4-dihydropyridine; qPCR, quantitative real-time PCR; WT, wild-type; ANOVA, analysis of variance.
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↵1 Current affiliation: Department of Neuroscience, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
- Received June 30, 2008.
- Accepted November 24, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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