Abstract

In neurons, L-type voltage-gated Ca2+ channels (L-channels) provide Ca2+ signals that help control gene expression. Consequently, regulation of L-channel activity via processes such as Ca2+-dependent inactivation (CDI) is thought to be important in neuronal function. CDI of CaV1.2 L-channels relies upon proteins associated with the channel's C-terminus, such as the Ca2+-sensor calmodulin. The C-terminus of the CaV1.2 α1 subunit also interacts with the A-kinase anchoring protein AKAP79/150 (79 = human, 150 = rodent), which binds both protein kinase A (PKA) and the Ca2+/calmodulin-activated phosphatase calcineurin (CaN). To study the roles of AKAP79/150-anchored CaN and PKA in CDI, we recorded pharmacologically-isolated L-type Ca2+ currents from rodent hippocampal neurons maintained in culture for up to 6 days. Rates of Ca2+-dependent inactivation (1/τ) were estimated by curve-fitting the fast component of the decay of Ca2+ current (10 mM) that occurred during 500-msec step depolarizations (0.067 Hz) from the holding potential (−60 mV) to 0 mV. In control neurons L-current underwent CDI at a rate of ∼40 sec−1. CDI was slowed to < 10 sec−1 by disruption of CaN anchoring or activity via (1) RNAi knock-down of AKAP150 coupled with over-expression of a mutant human AKAP lacking the CaN binding motif (AKAP79ΔPIX); (2) block of CaN-AKAP150 interaction by an anchoring competitor (VIVIT peptide); (3) the CaN inhibitor cyclosporine A; or (4) expression of AKAP150ΔPIX in transgenic mice. CDI was slowed to less than 20 sec−1 by perturbation of the PKA-AKAP interaction via (1) AKAP150 knock-down coupled with over-expression of anchoring protein lacking the PKA binding site (AKAP79ΔPKA); (2) block of PKA-AKAP150 interaction with an anchoring competitor (Ht31 peptide); or (3) expression of AKAP150ΔPKA in transgenic mice. The PKA inhibitor PKI slowed CDI to < 7 sec−1.

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