Localized calcineurin confers Ca2+-dependent inactivation on neuronal L-type Ca2+ channels

Abstract

Excitation-driven entry of Ca2+ through L-type voltage-gated Ca2+ channels controls gene expression in neurons and a variety of fundamental activities in other kinds of excitable cells. The probability of opening of CaV1.2L-type channels is subject to pronounced enhancement by cAMP-dependent protein kinase(PKA), which is scaffolded to CaV1.2 channels by A-kinase anchoring proteins (AKAPs). CaV1.2channels also undergo negative autoregulation via Ca2+ -dependent inactivation (CDI), which strongly limits Ca2+ entry. An abundance of evidence indicates that CDI relies upon binding of Ca2/calmodulin (CaM) to an isoleucine-glutamine motif in the carboxy tail of CaV1.2 L-type channels, a molecular mechanism seemingly unrelated to phosphorylation-mediated channel enhancement. But our work reveals, in cultured hippocampal neurons and a heterologous expression system, that the Ca2+ /CaM-activated phosphatase calcineurin (CaN) is scaffolded to CaV1.2 channels by the neuronal anchoring protein AKAP79/150, and that overexpression of an AKAP79/150 mutant incapable of binding CaN (ΔPIX; CaN-binding PXIXIT motif deleted) impedes CDI. Interventions that suppress CaN activity-mutation in its catalytic site, antagonism with cyclosporine A or FK506, or intracellular perfusion with a peptide mimicking the sequence of the phosphatase's autoinhibitory domain-interfere with normal CDI. In cultured hippocampal neurons from a ΔPIX knock-in mouse, CDI is absent. Results of experiments with the adenylyl cyclase stimulator forskolin and with the PKA inhibitor PKI suggest that Ca2+/CaM-activated CaN promotes CDI by reversing channel enhancement effectuated by kinases such as PKA. Hence, our investigation of AKAP79/150-anchored CaN reconciles the CaM-based model of CDI with an earlier, seemingly contradictory model based on dephosphorylation signaling. © 2012 the authors.