Voltage-gated Ca2+ channels of the Ca V 1 family initiate excitation-contraction coupling in cardiac, smooth, and skeletal muscle and are primary targets for regulation by the sympathetic nervous system in the 'fight-or-flight' response. In the heart, activation of β-adrenergic receptors greatly increases the L-type Ca 2+ current through Ca V 1.2 channels, which requires phosphorylation by cyclic AMP-dependent protein kinase (PKA) anchored via an A-kinase anchoring protein (AKAP15). Surprisingly, the site of interaction of PKA and AKAP15 lies in the distal C-terminus, which is cleaved from the remainder of the channel by in vivo proteolytic processing. Here we report that the proteolytically cleaved distal C-terminal domain forms a specific molecular complex with the truncated α 1 subunit and serves as a potent autoinhibitory domain. Formation of the autoinhibitory complex greatly reduces the coupling efficiency of voltage sensing to channel opening and shifts the voltage dependence of activation to more positive membrane potentials. Ab initio structural modelling and site-directed mutagenesis revealed a binding interaction between a pair of arginine residues in a predicted α-helix in the proximal C-terminal domain and a set of three negatively charged amino acid residues in a predicted helix-loop-helix bundle in the distal C-terminal domain. Disruption of this interaction by mutation abolished the inhibitory effects of the distal C-terminus on Ca V 1.2 channel function. These results provide the first functional characterization of this autoinhibitory complex, which may be a major form of the Ca V 1 family Ca 2+ channels in cardiac and skeletal muscle cells, and reveal a unique ion channel regulatory mechanism in which proteolytic processing produces a more effective autoinhibitor of Ca V 1.2 channel function.

.2 channels and PKA in the transverse tubules of isolated ventricular myocytes. Site-directed mutagenesis studies reveal that AKAP15 directly interacts with the distal C terminus of the cardiac Ca V1.2 channel via a leucine zipper-like motif. Disruption of PKA anchoring to Ca V1.2 channels via AKAP15 using competing peptides markedly inhibits the ␤-adrenergic regulation of CaV1.2 channels via the PKA pathway in ventricular myocytes. These results identify a conserved leucine zipper motif in the C terminus of the Ca V1 family of Ca 2؉ channels that directly anchors an AKAP15-PKA signaling complex to ensure rapid and efficient regulation of L-type Ca 2؉ currents in response to ␤-adrenergic stimulation and local increases in cAMP.V oltage-gated L-type calcium (Ca 2ϩ ) channels play a pivotal role in the regulation of a wide range of cellular processes, including membrane excitability, Ca 2ϩ homeostasis, protein phosphorylation, and gene regulation. In cardiac myocytes, Ca 2ϩ influx through Ca V 1.2 channels contributes to the plateau phase of the cardiac action potential and is responsible for initiating excitation-contraction coupling (1-3). Voltage-gated L-type Ca 2ϩ channels are multisubunit complexes composed of a poreforming ␣ 1 subunit and auxiliary ␤ and ␣ 2 ␦ subunits (4). In cardiac muscle, a distinct ␣ 1 subunit (␣ 1 1.2a) (5, 6), an ␣ 2 ␦ subunit (7), and several isoforms of ␤ subunits [␤ 1b and ␤ 2a-d (8-11)] have been identified and implicated to form the Ca V 1.2 channel. As for the skeletal muscle Ca V 1.1 channel (12, 13), two size forms of the ␣ 1 1.2a subunit of Ca V 1.2 channels, Ϸ240 and 210 kDa, are present in cardiac muscle and differ by truncation at the C terminus (14). Whereas the majority of Ca 2ϩ channel ␣ 1 subunits isolated from cardiac muscle are truncated (11,14,15), the cleaved distal C terminus remains associated with the truncated ␣ 1 subunit of Ca V 1.2 after proteolytic processing, and peptides derived from it can regulate channel activity (16,17). Ca V 1.2 channels can be modulated by a variety of receptormediated processes, including stimulation through activation of the ␤-adrenergic receptor͞cAMP signaling pathway (4, 18-21). Activation of ␤-adrenergic receptors increases cardiac L-type Ca 2ϩ currents through cAMP-dependent protein kinase A (PKA)-mediated phosphorylation of the Ca V 1.2 channel protein and͞or associated proteins (22, 23). As for skeletal muscle Ca v 1.1 channels (24, 25), both the ␣ 1 and ␤ subunits of Ca V 1.2 channels are substrates for phosphorylation by PKA (14,(26)(27)(28). PKA phosphorylates only the full-length form of the ␣ 1 subunit, on a single site containing serine 1928 in the C-terminal domain (14,26). In contrast, the C-terminal truncated ␣ 1 subunit is not a substrate for phosphorylation by PKA in vitro (14,26). Ca V 1.2 channels consisting of only ␣ 1 subunits can be regulated by PKA, implicating phosphorylation of serine 1928 in channel regulation (29, 30). Mutation of serine 1928 to alanine prevents PKAdependent phosphorylation and the low l...

In skeletal muscle cells, voltage-dependent potentiation of Ca 2؉ channel activity requires phosphorylation by cAMP-dependent protein kinase (PKA) anchored via an A-kinase anchoring protein (AKAP15), and the most rapid sites of phosphorylation are located in the C-terminal domain. Surprisingly, the site of interaction of the complex of PKA and AKAP15 with the ␣1-subunit of CaV1.1 channels lies in the distal C terminus, which is cleaved from the remainder of the channel by in vivo proteolytic processing. Here we report that the distal C terminus is noncovalently associated with the remainder of the channel via an interaction with a site in the proximal C-terminal domain when expressed as a separate protein in mammalian nonmuscle cells. Deletion mapping of the C terminus of the ␣1-subunit using the yeast two-hybrid assay revealed that a distal C-terminal peptide containing amino acids 1802-1841 specifically interacts with a region in the proximal C terminus containing amino acid residues 1556 -1612. Analysis of the purified ␣1-subunit of CaV1.1 channels from skeletal muscle by saturation sequencing of the intracellular peptides by tandem mass spectrometry identified the site of proteolytic processing as alanine 1664. Our results support the conclusion that a noncovalently associated complex of the ␣1-subunit truncated at A1664 with the proteolytically cleaved distal C-terminal domain, AKAP15, and PKA is the primary physiological form of CaV1.1 channels in skeletal muscle cells.calcium channels ͉ contraction coupling ͉ excitation ͉ protein kinase ͉ proteolysis