Targeting of Protein Phosphatases PP2A and PP2B to the C Terminus of L-Type Calcium Channel CaV1.2

The L-type Ca2+ channel CaV1.2 lies within a macromolecular complex including β2- adrenergic receptor, trimeric GS protein, adenylyl cyclase, and cAMP-dependent protein kinase (PKA). This complex mediates highly spatially and temporally regulated signaling, both acute (cardiac excitation contraction coupling, synaptic transmission, neurosecretion) and long-term (gene expression). Protein phosphatases PP2A (PPP2CA) and PP2B (PPP3CA; calcineurin) are also constitutive complex members. Acutely, either phosphatase counteracts PKA-dependent increase in Ca current by PKA. We here report that PP2A binds independently to two separate specific regions in the C-terminus of the central pore-forming α1 subunit of CaV1.2: one region spans residues 1795-1818 and the other residues 1965-1971. PP2B binds independently to a distinct region immediately downstream of residue 1971. To determine the binding regions for these phosphatases we assayed their ability to bind α1 C-terminal fragments with appropriate subregions cleaved. Further, we assayed a pallette of small peptides and identified those able to compete with PP2A and PP2B binding in pull-down assays. With these interfering peptides, we investigated PP2A and PP2B function, assayed by recording ICa(L) in acutely isolated rabbit cardiomyocytes. Using whole-cell β-escin perforated patch, which allowed pipette introduction of peptides while limiting rundown, we found that the PP2B competing peptide increased both basal and isoproterenol-stimulated ICa(L), while a PP2A competing peptide had no effect vs a control peptide or no peptide. Together, the biochemical and electrophysiological results suggest that while PP2A and PP2B are anchored at their respective binding sites on cardiac CaV1.2 α1 and negatively regulate ICa(L) (in this case by counterbalancing PKA-mediated phosphorylation), a fine dynamic balance between phosphorylation and dephosphorylation responses exists. This would be important to the ability of ICa(L) flux to efficiently regulate end effects.