The L-type voltage-dependent calcium channel is an important link in excitation-contraction coupling of muscle cells (reviewed in refs 2 and 3). The channel has two functional characteristics: calcium permeation and receptor sites for calcium antagonists. In skeletal muscle the channel is a complex of five subunits, alpha 1, alpha 2, beta, gamma and delta. Complementary DNAs to these subunits have been cloned and their amino-acid sequences deduced. The skeletal muscle alpha 1 subunit cDNA expressed in L cells manifests as specific calcium-ion permeation, as well as sensitivity to the three classes of organic calcium-channel blockers. We report here that coexpression of the alpha 1 subunit with other subunits results in significant changes in dihydropyridine binding and gating properties. The available number of drug receptor sites increases 10-fold with an alpha 1 beta combination, whereas the affinity of the dihydropyridine binding site remains unchanged. Also, the presence of the beta subunit accelerates activation and inactivation kinetics of the calcium-channel current.

A. Targeted disruption of the voltage-dependent calcium channel ␣2/␦-1-subunit. Cardiac L-type voltage-dependent Ca 2ϩ channels are heteromultimeric polypeptide complexes of ␣1-, ␣2/␦-, and ␤-subunits. The ␣2/␦-1-subunit possesses a stereoselective, high-affinity binding site for gabapentin, widely used to treat epilepsy and postherpetic neuralgic pain as well as sleep disorders. Mutations in ␣2/␦-subunits of voltage-dependent Ca 2ϩ channels have been associated with different diseases, including epilepsy. Multiple heterologous coexpression systems have been used to study the effects of the deletion of the ␣2/␦-1-subunit, but attempts at a conventional knockout animal model have been ineffective. We report the development of a viable conventional knockout mouse using a construct targeting exon 2 of ␣2/␦-1. While the deletion of the subunit is not lethal, these animals lack high-affinity gabapentin binding sites and demonstrate a significantly decreased basal myocardial contractility and relaxation and a decreased L-type Ca 2ϩ current peak current amplitude. This is a novel model for studying the function of the ␣2/␦-1-subunit and will be of importance in the development of new pharmacological therapies. cardiac calcium channel; murine knockout model; gabapentin binding; myocardial contractility CARDIAC L-type voltage-dependent Ca 2ϩ channels (L-VDCCs) are heteromultimeric polypeptide complexes of ␣ 1 -, ␣ 2 /␦-, and ␤-subunits. The ␣ 1 -subunit is autoregulatory and harbors the channel pore, gating machinery, and modulatory drug binding sites (30). The accessory subunits (␣ 2 /␦ and ␤) affect channel kinetics and are involved in the trafficking and insertion of the ␣ 1 -subunit into the membrane. The ␣ 2 -subunit is closely associated with an extracellular loop of the ␣ 1 -subunit (15) and linked to a small protein called ␦ (2, 9). Both the ␣ 2 and ␦ are encoded by the same gene, separated by proteolytic cleavage, and extracellularly linked through a disulfide bridge (9). Currently, four ␣ 2 /␦-subunits, each encoded by separate genes, have been identified (4). The ␣ 2 /␦-1, originally cloned from skeletal muscle (10), is ubiquitously distributed (18), with high levels of protein expression in brain, heart, skeletal, and

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Conditions of stress, such as myocardial infarction, stimulate up-regulation of heme oxygenase (HO-1) to provide cardioprotection. Here, we show that CO, a product of heme catabolism by HO-1, directly inhibits native rat cardiomyocyte L-type Ca 2؉ currents and the recombinant ␣ 1C subunit of the human cardiac L-type Ca 2؉ channel. CO (applied via a recognized CO donor molecule or as the dissolved gas) caused reversible, voltage-independent channel inhibition, which was dependent on the presence of a spliced insert in the cytoplasmic C-terminal region of the channel. Sequential molecular dissection and point mutagenesis identified three key cysteine residues within the proximal 31 amino acids of the splice insert required for CO sensitivity. CO-mediated inhibition was independent of nitric oxide and protein kinase G but was prevented by antioxidants and the reducing agent, dithiothreitol. Inhibition of NADPH oxidase and xanthine oxidase did not affect the inhibitory actions of CO. Instead, inhibitors of complex III (but not complex I) of the mitochondrial electron transport chain and a mitochondrially targeted antioxidant (Mito Q) fully prevented the effects of CO. Our data indicate that the cardioprotective effects of HO-1 activity may be attributable to an inhibitory action of CO on cardiac L-type Ca 2؉ channels. Inhibition arises from the ability of CO to promote generation of reactive oxygen species from complex III of mitochondria. This in turn leads to redox modulation of any or all of three critical cysteine residues in the channel's cytoplasmic C-terminal tail, resulting in channel inhibition.CO is an established and important signaling molecule in both the heart and vasculature as well as other tissues (1, 2). Cardiac atrial and ventricular myocytes express heme oxygenases HO-1 4 and HO-2, which generate CO along with biliverdin and free Fe 2ϩ by heme catabolism, and HO-1 levels can be increased by various stress factors (3), including myocardial infarction (4). CO limits the cellular damage of ischemia/reperfusion injury in the heart (5). Indeed, greater cardiac damage is seen following ischemia/reperfusion injury in HO-1 knock-out mice (6). Conversely, HO-1 overexpression in the heart reduces infarct size and other markers of damage following ischemia/ reperfusion injury (7). CO also improves cardiac blood supply through coronary vessel dilation (8, 9) and reduces cardiac contractility (9). However, the mechanisms underlying this cardioprotective effect of CO are not understood.In the vasculature, CO also exerts numerous beneficial effects. Its ability to dilate blood vessels is long established (9 -11) and endothelium-independent (12) and not due to development of hypoxia through displacement of O 2 (see Ref. 13). CO has clear, protective effects in various vascular diseases, such as systemic and pulmonary hypertension, development of atherosclerosis, and neointimal hyperplasia due to proliferation of vascular smooth muscle cells following vascular injury (all reviewed in Refs. 2, 13, and 14). Import...

L-Type calcium channels are multiprotein complexes composed of pore-forming (Ca V 1.2) and modulatory auxiliary ␣ 2 ␦-and ␤-subunits. We demonstrate expression of two different isoforms for the ␤ 2 -subunit (␤ 2a , ␤ 2b ) and the ␤ 3 -subunit (␤ 3a , ␤ 3trunc ) in human non-failing and failing ischemic myocardium. Quantitatively, in the left ventricle expression of ␤ 2b transcripts prevails in the order of > ␤ 3 > > ␤ 2a . The expressed cardiac full-length ␤ 3 -subunit is identical to the ␤ 3a -isoform, and ␤ 3trunc results from deletion of exon 6 (20 nn) entailing a reading frameshift and translation stop at nucleotide position 495. In failing ischemic myocardium ␤ 3trunc expression increases whereas overall ␤ 3 expression remains unchanged. Heterologous coexpression studies demonstrated that ␤ 2 induced larger currents through rabbit and human cardiac Ca V 1.2 pore subunits than ␤ 3 isoforms. All ␤-subunits increased channel availability at single channel level, but ␤ 2 exerted an additional, marked stimulation of rapid gating (open and closed times, first latency), leading to higher peak current values. We conclude that cardiac ␤-subunit isoforms differentially modulate calcium inward currents because of regulatory effects within the channel protein complex. Moreover, differences in the various ␤-subunit gene products present in human heart might account for altered single channel behavior found in human heart failure.