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...

Abstract-Hypoxic pulmonary vasoconstriction is initiated by inhibiting one or more voltage-gated potassium (Kv) channel in the vascular smooth muscle cells (VSMCs) of the small pulmonary resistance vessels. Although progress has been made in identifying which Kv channel proteins are expressed in pulmonary arterial (PA) VSMCs, there are conflicting reports regarding which channels contribute to the native O 2 -sensitive K ϩ current. In this study, we examined the effects of hypoxia on the Kv1.2, Kv1.5, Kv2.1, and Kv9.3 ␣ subunits expressed in mouse L cells using the whole-cell patch-clamp technique. Hypoxia (PO 2 ϭϷ30 mm Hg) reversibly inhibited Kv1.2 and Kv2.1 currents only at potentials more positive than 30 mV. In contrast, hypoxia did not alter Kv1.5 current. Currents generated by coexpression of Kv2.1 with Kv9.3 ␣ subunits were reversibly inhibited by hypoxia in the voltage range of the resting membrane potential (E M ) of PA VSMCs (Ϸ28% at Ϫ40 mV). Coexpression of Kv1.2 and Kv1.5 ␣ subunits produced currents that displayed kinetic and pharmacological properties distinct from Kv1.2 and Kv1.5 channels expressed alone. Moreover, hypoxia reversibly inhibited Kv1.2/Kv1.5 current activated at physiologically relevant membrane potentials (Ϸ65% at Ϫ40 mV). These results indicate that (1) hypoxia reversibly inhibits Kv1.2 and Kv2.1 but not Kv1.5 homomeric channels, (2) Kv1.2 and 1.5 ␣ subunits can assemble to form an O 2 -sensitive heteromeric channel, and (3) Key Words: Kv channel Ⅲ hypoxia Ⅲ pulmonary artery Ⅲ heteromeric H ypoxia induces constriction of the small pulmonary resistance arteries, a process known as hypoxic pulmonary vasoconstriction (HPV). 1 This constrictor response is the opposite of that which occurs in resistance vessels of the systemic circulation. In these vessels, hypoxia results in vasodilation. 2 In the fetus, HPV contributes to high pulmonary arterial resistance by diverting blood flow through the ductus arteriosus. 3 In the adult, HPV reduces blood flow through atelectatic or underventilated areas of the lung where ventilation is not adequate for oxygenation. 3 In this way, short-term HPV is an essential mechanism that helps match ventilation to perfusion, diverting blood flow away from poorly ventilated regions of the lung to maximize arterial saturation. 4 However, when hypoxia becomes more generalized, as seen in patients suffering from either long-term obstructive lung diseases or high altitude exposure, 4,5 the subsequent pulmonary vasoconstriction causes an increase in pulmonary arterial pressure that can lead to the development of pulmonary hypertension.In pulmonary arterial (PA) vascular smooth muscle cells (VSMCs), voltage-gated potassium (Kv) channels play an important role in setting the resting membrane potential (E M ϭϷϪ55 mV) and, consequently, vascular tone. 6 -8 It is thought that hypoxia reversibly inhibits Kv channels and, thereby, regulates vasoconstriction. 7,9 -12 This hypothesis is supported by the observation that hypoxia inhibits whole-cell K ϩ currents and ca...

During the fight-or-flight response, epinephrine and norepinephrine released by the sympathetic nervous system increase L-type calcium currents conducted by Ca V1.2a channels in the heart, which contributes to enhanced cardiac performance. Activation of ␤-adrenergic receptors increases channel activity via phosphorylation by cAMP-dependent protein kinase (PKA) tethered to the distal C-terminal domain of the ␣1 subunit via an A-kinase anchoring protein (AKAP15). Here we measure phosphorylation of S1928 in dissociated rat ventricular myocytes in response to ␤-adrenergic receptor stimulation by using a phosphospecific antibody. Isoproterenol treatment increased phosphorylation of S1928 in the distal C-terminal domain, and a similar increase was observed with a direct activator of adenylyl cyclase, forskolin, confirming that the cAMP and PKA are responsible. Pretreatment with selective ␤1-and ␤2-adrenergic antagonists reduced the increase in phosphorylation by 79% and 42%, respectively, and pretreatment with both agents completely blocked it. In contrast, treatment with these agents in the presence of 1,2-bis(2-aminophenoxy)ethane-N,Ntetraacetic acid (BAPTA)-acetoxymethyl ester to buffer intracellular calcium results in only ␤1-stimulated phosphorylation of S1928. Whole-cell patch clamp studies with intracellular BAPTA demonstrated that 98% of the increase in calcium current was attributable to ␤1-adrenergic receptors. Thus, ␤-adrenergic stimulation results in phosphorylation of S1928 on the Ca V1.2 ␣1 subunit in intact ventricular myocytes via both ␤1-and ␤2-adrenergic receptor pathways, but the ␤2-dependent increase in phosphorylation depends on elevated intracellular calcium and does not contribute to regulation of whole-cell calcium currents at basal calcium levels. Our results correlate phosphorylation of S1928 with ␤1-adrenergic functional up-regulation of cardiac calcium channels in the presence of BAPTA in intact ventricular myocytes.calcium channel ͉ cAMP ͉ heart ͉ protein kinase ͉ sympathetic regulation T he primary calcium current in cardiac myocytes is conducted by Ca v 1.2a channels (1, 2). Calcium entering through these channels triggers release of calcium from the sarcoplasmic reticulum, which in turn activates contraction. Modulation of calcium entry through the Ca V 1.2 channel is a key point of regulation of cardiac contraction by the sympathetic nervous system, primarily through epinephrine and norepinephrine acting at ␤-adrenergic receptors (1, 3). Activation of ␤-adrenergic signaling leads to 3-to 4-fold increases in calcium current (2), mediated by protein kinase A (PKA) phosphorylation (1-3). Increases in calcium current are produced by activation of adenylyl cyclase with forskolin, inhibition of phosphodiesterases, and activation of PKA with cyclic nucleotide analogs (4-6).Cardiac calcium channels are heteromeric protein complexes (7). The pore-forming ␣1 subunit consists of four homologous domains surrounding an ion conduction pore and contains the gating machinery and the receptor sites for...