In the heart, activation of β-adrenergic receptors increases the activity of L-type CaV1.2 channels through cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We have recently demonstrated that an A-kinase anchoring protein (AKAP15) targets PKA to the carboxy (C-) terminus of skeletal muscle L-type CaV1.1 channels through modified leucine zipper (LZ) interactions (Hulme et al. 2002). Sequence alignment of the LZ-like region of CaV1.1 with other members of the CaV1 family revealed a striking conservation of LZ-like motifs, suggesting that AKAP15 may also target PKA to CaV1.2 channels in the heart.
Animals were humanely killed and procedures were approved by the Institutional Animal Care and Use Committee at the University of Washington. Immunoprecipitation experiments using rat heart extracts revealed that AKAP15 co-immunoprecipitates with CaV1.2 channels. Immunohistochemical staining of isolated rat ventricular myocytes showed CaV1.2 channels and AKAP15 to be localised in the transverse tubules. Site-directed mutagenesis studies in transfected HEK293 cells confirmed that AKAP15 directly interacts with the distal C-terminus of the cardiac CaV1.2 channel (residues 2057-2115) via a LZ interaction similar to that identified in the skeletal muscle CaV1.1 channel (Hulme et al. 2002). The functional importance of AKAP15 binding to the C-terminus of CaV1.2 channels with regards to β-adrenergic regulation of L-type Ca2+ currents in rat ventricular myocytes was examined using the whole cell patch clamp technique. In control cells, isoprenaline (1 µM) caused a marked increase in the amplitude of the Ca2+ current and a hyperpolarising shift in the current-voltage (I-V) relationship. In contrast, in cells dialysed with a synthetic peptide corresponding to the LZ motif of AKAP15 (AKAP15LZ(38-54)), the isoprenaline-induced increase in Ca2+ current was greatly attenuated, and the hyperpolarising shift in the voltage dependence of activation was significantly blocked. Moreover, dialysis of a mutant form of the same peptide had no significant effect on the isoprenaline-induced increase in the amplitude of the Ca2+ current.
These data indicate that targeting of PKA to Ca2+ channels via a LZ-like interaction between AKAP15 and the CaV1.2 channel plays an essential role in the β-adrenergic-dependent regulation of Ca2+ channels in rat ventricular myocytes.