Localized Calcineurin in Calcium- Dependent Inactivation of L-type Calcium Channels

Abstract

The open probability of CaV1.2 L-type Ca2+ channels is enhanced by cAMP-dependent protein kinase (PKA), which is scaffolded to CaV1.2 channels by A-kinase anchoring proteins (AKAPs). CaV1.2 channels also undergo negative autoregulation via Ca2+-dependent inactivation (CDI). CDI relies upon binding of Ca2+/calmodulin (CaM) to an IQ motif in the carboxy tail of CaV1.2 L-type channels, a mechanism seemingly unrelated to phosphorylation-mediated channel enhancement. In neurons, AKAP79/150 anchors both PKA and the Ca2+-activated phosphatase calcineurin (CaN) to CaV1.2 channels. Using transfected tsA201 cells or neurons, and tools such as the isolated calcineurin autoinhibitory peptide, over-expression of the catalytically-inactive CaNH151A mutant, and RNAi suppression of AKAP79, we have found that channel-linked CaM serves as a Ca2+ sensor for CaN, and that Ca2+/CaM-activated CaN participates in CDI by reversing channel enhancement by kinases such as PKA.We have also observed that I→E substitution in the IQ motif produces a mutant CaV1.2I/EQ channel that - when co-expressed with AKAP79 in tsA201 cells - unexpectedly exhibits ultra-fast inactivation. Ultra-fast inactivation is eliminated in Ca2+-free Na+ external solution, as well as by over-expression of the CaNH151A mutant or stimulation of PKA with forskolin. One interpretation is that the intact IQ motif's affinity for Ca2+/CaM limits the speed of CDI, and that reducing IQ affinity for Ca2+/CaM via I→E substitution allows CDI to proceed at a greatly speeded rate. FRET results with the CaV1.2I/EQ mutant or with AKAP79 lacking the CaN anchoring motif suggest that, during periods of elevated channel activity, the IQ-CaM and AKAP79-CaN interactions are both necessary for CaN-mediated reversal of current enhancement by PKA. In sum, our work supports a synthetic view fusing previous ideas regarding CaM and phosphorylation signaling in CDI.