Differential Regulated Interactions of Calcium/Calmodulin-Dependent Protein Kinase II with Isoforms of Voltage-Gated Calcium Channel β Subunits†

Voltage-gated Ca2+ channel (VGCC) ion selectivity and responsiveness to pharmacological antagonist ligands are defined by the identity of the pore forming α1 subunit. The biophysical properties are generally modified by differential association of auxiliary β, α2δ and γ subunits (1–3). Four genes encoding β isoforms have been identified (β1–4) each having multiple mRNA splice variants, which differentially modulate the properties and cell surface expression of VGCC complexes (4–6). In addition, VGCC complexes are further modulated by a variety of posttranslational modifications. The regulatory properties of Ca2+/calmodulin-dependent protein kinase II (CaMKII) allow integration of signals conveyed by changes in the frequency, duration and amplitude of intracellular Ca2+ transients (7). This feature critically depends on Ca2+/calmodulin-dependent autophosphorylation of Thr286 in CaMKIIα (or Thr287 in other CaMKII isoforms) in the autoregulatory domain, which confers subsequent autonomous kinase activity (reviewed in (8, 9, 10 )). Thus, Ca2+ transients induce more prolonged kinase activation depending on specific parameters of Ca2+ signals in the local environment. Recent studies demonstrated that localization of CaMKII to specific subcellular microdomains confers distinct downstream phosphorylation events (11, 12). These studies are consistent with the emerging concept that direct interactions of signaling molecules ensures accurate and timely responses to cell stimulation (13, 14). Determining the mechanisms for CaMKII binding to target proteins, such as VGCCs, is thus an important goal for understanding the role of CaMKII in excitable cells. CaMKII phosphorylates the α and/or β subunits of a variety of VGCCs to modulate Ca2+ entry. For example, CaMKII regulates T-type Ca2+ channels by binding to and phosphorylating the II-III intracellular loop in the CaV3.2 α subunit (15, 16). The EF hand motif in the C-terminal domain of the L-type Ca2+ channel (LTCC) CaV1.3 α subunit is also phosphorylated by CaMKII, causing a negative voltage shift in LTCC current activation (17). CaMKII also phosphorylates multiple sites in the LTCC CaV1.2 α1c subunit to promote both Ca2+- and voltage-dependent facilitation in heterologous cells (18–20). In addition to these roles in feedback regulation, CaMKII is involved in crosstalk between Ca2+ channels: for example, LTCC activation leads to depression of R-type Ca2+ channels in dendritic spines via a poorly defined CaMKII-dependent mechanism (21). We recently showed that CaMKII-dependent facilitation of cardiac CaV1.2 LTCCs is mediated by phosphorylation of the β2a subunit at Thr498 in cardiomyocytes (22). Moreover, β2a acts as a CaMKII associated protein (CaMKAP) that directly interacts with CaMKII in vitro and in intact cells. Our findings showed that VGCC regulation may be strongly enhanced or modified by association of CaMKII with the β2a subunit. In the current study, we demonstrate that CaMKII phosphorylates all of the β subunit isoforms, but interacts with β subunits in an isoform specific manner; this CaMKII-β subunit interaction is negatively modulated by phosphorylation of the β subunit.