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A Single Amino Acid Determines the Selectivity and Efficacy of Selective Negative Allosteric Modulators of Ca_V1.3 L-Type Calcium Channels.

ABSTRACT: Ca²⁺ channels with a Ca_V1.3 pore-forming α₁ subunit have been implicated in both neurodegenerative and neuropsychiatric disorders, motivating the development of selective and potent inhibitors of Ca_V1.3 versus Ca_V1.2 channels, the calcium channels implicated in hypertensive disorders. We have previously identified pyrimidine-2,4,6-triones (PYTs) that preferentially inhibit Ca_V1.3 channels, but the structural determinants of their interaction with the channel have not been identified, impeding their development into drugs. By a combination of biochemical, computational, and molecular biological approaches, it was found that PYTs bind to the dihydropyridine (DHP) binding pocket of the Ca_V1.3 subunit, establishing them as negative allosteric modulators of channel gating. Site-directed mutagenesis, based on homology models of Ca_V1.3 and Ca_V1.2 channels, revealed that a single amino acid residue within the DHP binding pocket (M1078) is responsible for the selectivity of PYTs for Ca_V1.3 over Ca_V1.2. In addition to providing direction for chemical optimization, these results suggest that, like dihydropyridines, PYTs have pharmacological features that could make them of broad clinical utility.

SUBMITTER: Cooper G

PROVIDER: S-EPMC7502546 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectiveAn increase of neuronal Cav 1.3 L-type calcium channels (LTCCs) has been observed in various animal models of epilepsy. However, LTCC inhibitors failed in clinical trials of epileptic treatment. There is compelling evidence that paroxysmal depolarization shifts (PDSs) involve Ca2+ influx through LTCCs. PDSs represent a hallmark of epileptiform activity. In recent years, a probable epileptogenic role for PDSs has been proposed. However, the implication of the two neuronal LTCC isoforms, Cav 1.2 and Cav 1.3, in PDSs remained unknown. Moreover, Ca2+ -dependent nonspecific cation (CAN) channels have also been suspected to contribute to PDSs. Nevertheless, direct experimental support of an important role of CAN channel activation in PDS formation is still lacking.MethodsPrimary neuronal networks derived from dissociated hippocampal neurons were generated from mice expressing a dihydropyridine-insensitive Cav 1.2 mutant (Cav 1.2DHP-/- mice) or from Cav 1.3-/- knockout mice. To investigate the role of Cav 1.2 and Cav 1.3, perforated patch-clamp recordings were made of epileptiform activity, which was elicited using either bicuculline or caffeine. LTCC activity was modulated using the dihydropyridines Bay K 8644 (agonist) and isradipine (antagonist).ResultsDistinct PDS could be elicited upon LTCC potentiation in Cav 1.2DHP-/- neurons but not in Cav 1.3-/- neurons. In contrast, when bicuculline led to long-lasting, seizure-like discharge events rather than PDS, these were prolonged in Cav 1.3-/- neurons but not in Cav 1.2DHP-/- neurons. Because only the Cav 1.2 isoform is functionally coupled to CAN channels in primary hippocampal networks, PDS formation does not require CAN channel activity.SignificanceOur data suggest that the LTCC requirement of PDS relates primarily to Cav 1.3 channels rather than to Cav 1.2 channels and CAN channels in hippocampal neurons. Hence, Cav 1.3 may represent a new therapeutic target for suppression of PDS development. The proposed epileptogenic role of PDSs may allow for a prophylactic rather than the unsuccessful seizure suppressing application of LTCC inhibitors.

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A Single Amino Acid Determines the Selectivity and Efficacy of Selective Negative Allosteric Modulators of CaV1.3 L-Type Calcium Channels.

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A Single Amino Acid Determines the Selectivity and Efficacy of Selective Negative Allosteric Modulators of Ca_V1.3 L-Type Calcium Channels.