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

Transgenic mice developed hypertrophy and severe cardiomyopathy as a function of age, thus confirming that changes in channel density are sufficient to induce disease. The small, sustained increase in the ingress of Ca(2+) through the calcium channel elevated protein kinase Calpha before the development of hypertrophy, suggesting that protein kinase Calpha plays an important role in triggering hypertrophy.

The L-type voltage-dependent calcium channel (L-VDCC) regulates calcium influx in cardiac myocytes. Activation of the ␤-adrenergic receptor (␤AR) pathway causes phosphorylation of the L-VDCC and that in turn increases Ca 2؉ influx. Targeted expression of the L-VDCC ␣ 1 subunit in transgenic (Tg) mouse ventricles resulted in marked blunting of the ␤AR pathway. Inotropic and lusitropic responses to isoproterenol and forskolin in Tg hearts were significantly reduced. Likewise, Ca 2؉ current augmentation induced by isoproterenol and forskolin was markedly depressed in Tg cardiomyocytes. Despite no change in ␤AR number, isoproterenol-stimulated adenylyl cyclase activity was absent in Tg membranes and NaF and forskolin responses were reduced. We postulate an important pathway for regulation of the ␤AR by Ca 2؉ channels.