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 CaV1.2 L-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). CaV1.2 channels 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 IQ 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 over-expression of an AKAP79/150 mutant incapable of binding CaN (ΔPIX) 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.

AKAP-Anchored PKA Maintains Neuronal L-type Calcium Channel Activity and NFAT Transcriptional Signaling

“…Past and present work clearly shows that the phosphatase CaN must be targeted near the LTCC to provide efficient and specific activation of NFAT signaling via channel Ca 2+ influx (Li et al, 2012; Oliveria et al, 2007; Zhang and Shapiro, 2012). Yet sequestering CaN within the LTCC nano-domain also promotes a robust phosphatase mediated negative-feedback on PKA-enhanced channel activity, both through suppression of peak current amplitude and promotion of Ca 2+ -dependent inactivation (CDI) of neuronal LTCCs (Dittmer et al, 2014; Oliveria et al, 2007; Oliveria et al, 2012). Indeed, while previous studies in hippocampal neurons demonstrated that LTCC current and Ca 2+ influx can be enhanced by activation of cAMP-PKA signaling (Hoogland and Saggau, 2004; Kavalali et al, 1997), the degree of PKA enhancement of LTCC current is constrained by AKAP79/150-anchored CaN activity (Oliveria et al, 2007).…”

“…Neuronal membrane depolarization initiates NFAT signaling by triggering Ca 2+ influx through LTCCs to activate calmodulin (CaM) molecules tethered to the intracellular C-terminal domain of the channel (Peterson et al, 1999; Zuhlke et al, 1999). Ca 2+ -CaM promotes rapid activation of CaN, which is recruited to the LTCC through AKAP79/150 anchoring (Oliveria et al, 2007; Oliveria et al, 2012; Zhang and Shapiro, 2012). Organization of the LTCC-AKAP-CaN macromolecular complex at the plasma membrane arises in part through additional interaction of modified leucine zipper (LZ) motifs on AKAP79/150 and the C-terminal tail of Ca V 1.2 (Hulme et al, 2003; Oliveria et al, 2007).…”

“…In addition to CaN, AKAP79/150 anchors PKA near the LTCC to promote phosphorylation-mediated enhancement of channel activity that is opposed by CaN dephosphorylation, likely through modification of serine residues in the Ca V 1.2 C-terminus (De Jongh et al, 1996; Fuller et al, 2010; Gao et al, 1997; Hall et al, 2007; Oliveria et al, 2007; Oliveria et al, 2012). Thus, AKAP-anchored CaN paradoxically serves as both a negative feedback regulator of LTCC activity and a positive downstream transducer of LTCC Ca 2+ signaling to NFAT.…”

A Calcineurin Docking Motif (LXVP) in Dynamin-related Protein 1 Contributes to Mitochondrial Fragmentation and Ischemic Neuronal Injury

“…Mechanisms targeting CaN to different subcellular locales exist and have been shown to be an important part of CaN signaling. For example, AKAP79/150 targets CaN to the post-synaptic density where it regulates ionotropic glutamate receptors and L-type Ca 2ϩ channels (52)(53)(54)(55). It is therefore possible that similar scaffolding interactions target CaN to the OMM to amplify Drp1 Ser-656 dephosphorylation.…”

Methods of activation and the role of calcium/calmodulin-dependent protein kinase II in the regulation of acetylcholine secretion in the motor synapses of mice

“…At present, it is universally recognized that regard less of the neuron or terminal type, L type calcium channels are the target of various phosphatase-kinase and voltage dependent regulations [27,[32][33][34], although, in motor terminals, inhibitory phosphatase actions dominate resulting in the inhibition of L type calcium channels [10,20]. However, in cases where L type calcium channels are disinhibited, CaMKII activity is generally present in the cell; quite often, it is a form of positive feedback for the maintenance of the functioning of a channel itself [21,30,35].…”