Functional Roles of a C-terminal Signaling Complex of CaV1 Channels and A-kinase Anchoring Protein 15 in Brain Neurons

Abstract

Regulation of Ca V 1.2 channels in cardiac myocytes by the ␤-adrenergic pathway requires a signaling complex in which the proteolytically processed distal C-terminal domain acts as an autoinhibitor of channel activity and mediates up-regulation by the ␤-adrenergic receptor and PKA bound to A-kinase anchoring protein 15 (AKAP15). We examined the significance of this distal C-terminal signaling complex for Ca V 1.2 and Ca V 1.3 channels in neurons. AKAP15 co-immunoprecipitates with Ca V 1.2 and Ca V 1.3 channels. AKAP15 has overlapping localization with Ca V 1.2 and Ca V 1.3 channels in cell bodies and proximal dendrites and is closely co-localized with Ca V 1.2 channels in punctate clusters. The neuronal AKAP MAP2B, which also interacts with Ca V 1.2 and Ca V 1.3 channels, has complementary localization to AKAP15, suggesting different functional roles in calcium channel regulation. Studies with mice that lack the distal C-terminal domain of Ca V 1.2 channels (Ca V 1.2⌬DCT) reveal that AKAP15 interacts with neuronal Ca V 1.2 channels via their C terminus in vivo and is co-localized in punctate clusters of Ca V 1.2 channels via that interaction. Ca V 1.2⌬DCT neurons have reduced L-type calcium current, indicating that the distal C-terminal domain is required for normal functional expression in vivo. Deletion of the distal C-terminal domain impairs calcium-dependent signaling from Ca V 1.2 channels to the nucleus, as shown by reduction in phosphorylation of the cAMP response element-binding protein. Our results define AKAP signaling complexes of Ca V 1.2 and Ca V 1.3 channels in brain and reveal three previously unrecognized functional roles for the distal C terminus of neuronal Ca V 1.2 channels in vivo: increased functional expression, anchoring of AKAP15 and PKA, and initiation of excitation-transcription coupling.Voltage-gated calcium channels of the Ca V 1 subfamily conduct L-type calcium currents that transduce cell-surface depolarization into calcium transients and initiate excitation-contraction coupling, excitation-secretion coupling, protein phosphorylation, and gene regulation (1-5). Calcium influx via postsynaptic Ca V 1 channels supports sustained phosphorylation of cAMP response element-binding protein (CREB) 3 and CREB-dependent gene expression in hippocampal neurons (6 -13).Functional Ca V 1 channels are multimeric complexes composed of pore-forming ␣ 1 and associated ␣ 2 ␦, ␤, and in some cases, ␥ subunits (14 -19). These channels have an extended C terminus containing many protein interaction sites for regulation (5). In brain, Ca V 1 channels are composed of 70% Ca V 1.2 and 22% Ca V 1.3 with minor contributions from other Ca V 1 channels, as indicated by immunoprecipitation with specific antibodies (20). Ca V 1.2 and Ca V 1.3 channels are primarily localized in the soma and proximal dendrites (20, 21).The ␤-adrenergic pathway activates cAMP-dependent protein kinase (PKA) and increases the activity of Ca V 1 channels in skeletal and cardiac myocytes and neurons (1-5, 22, 23). PKAmediated regulatio...

Cardiomyocytes from AKAP7 knockout mice respond normally to adrenergic stimulation

“…channels (42). AKAP7α has not been reported to interact with any proteins other than ion channels and PKA.…”

The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential

“…A kinase-anchoring proteins are also found in a complex with native Ca V 1.3 channels (Marshall et al, 2011) Pharmacology of Voltage-Gated Calcium Channels 837 and are stimulated by PKA. This has been shown in adrenal chromaffin cells (Mahapatra et al, 2012) and in the SAN (Mangoni et al, 2003).…”

Regulation of Cardiac L-Type Ca 2+ Channel Ca V 1.2 Via the β-Adrenergic-cAMP-Protein Kinase A Pathway

“…Direct intracellular perfusion (dialysis) of the myocytes through the patch pipette often yielded incomplete inhibition of the β-AR effect: 50% to 70% with 1 µmol/L PKI, 0.5 mmol/L Rp-cAMPS, or a cocktail of PKA and phosphorylation inhibitors. [87][88][89][90] Other works reported full inhibition of effects of β-AR agonists in frog and rat ventricular cardiomyocytes by dialyzing cells with 2 to 5 mmol/L Rp-cAMPS, 16 µmol/L PKI (which is >3 orders of magnitude higher than its K i ), 20 µmol/L PKI peptide (PKI [15][16][17][18][19][20][21][22] ; K i =36 nmol/L), [91][92][93] or with 1 µmol/L H-89 applied externally. 94 Although the latter studies used rather high doses of inhibitors, the accumulated data strongly support the view that a major part and possibly all of the β-AR enhancement of cardiac Ca V 1.2 is mediated by activation of PKA.…”

“…One is the role of PKA in basal I Ca . In the best reconstitution setting (α 1C Δ1800 coexpressed with dCT and AKAP7), the reduction of basal I Ca was ≈30% with 1 µmol/L PKI [14][15][16][17][18][19][20][21][22] peptide and ≈50% with a mixture of higher concentrations of 2 PKI peptides. Such sensitivity to blockers resembles embryonic cardiomyocytes or other heterologous expression studies but is somewhat higher than in adult cardiomyocytes.…”

“…[179][180][181] The AKAP-PKA complex is attached to α 1C , which is considered to be the core of the signaling complex, via the dCT. 16,178,182 AKAP5 directly interacts with the CT and additional cytosolic segments of the α 1C subunit of Ca V 1.2 in neurons. 183 AKAP7 interacts with Ca V 1.2 via leucine zipper-like motif within the dCT (a.a. 2057-2115).…”