Targeted disruption of the voltage-dependent calcium channel α₂/δ-1-subunit

Abstract

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

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

“…L-type Channel Modulation by Ca V ␣2␦1-Up-regulation of L-type currents requires robust cell surface density of Ca V ␣2␦1 in cardiomyocytes (19) and in HEKT cells (22). Alterations in the cell surface density of Ca V ␣2␦1 caused by mutating glycosylation sites (our study), arrhythmogenic mutations (22), mutations within the "von Willebrand factor" structural domain (76,77), or following pharmacological modulation (e.g.…”

The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential

“…For the Ca V 1.1 channel complex in skeletal muscle, a 2 d-1 was also found to have a functional role in excitation-coupled entry of Ca 2+ into skeletal myotubes, although not in the formation of the Ca V 1.1 tetrad structure or in excitation-contraction coupling (Obermair et al, 2005;Gach et al, 2008). In mice in which a 2 d subunits were depleted, there was a reduction of calcium currents in relevant cell types (Barclay et al, 2001;Fuller-Bicer et al, 2009;Patel et al, 2013;Pirone et al, 2014).…”

“…Human mutations in CAC-NA2D1 have been associated with cardiac dysfunction in a small number of patients, including Brugada (Burashnikov et al, 2010) and short QT (Templin et al, 2011;Bourdin et al, 2015) syndromes. Furthermore, knockout of cacna2d1 in mice resulted in a cardiac phenotype; the mice had compromised cardiac function and smaller cardiac calcium channel current density (Fuller-Bicer et al, 2009). …”

Domain cooperativity in the β1asubunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle

“…Among these subunits, the α 1S -and β 1a subunit are indispensable for skeletal muscle EC coupling (9)(10)(11)(12)(13). Although α 1S is understood to transduce the opening signal to RyR1, essentially via its intracellular loop connecting homologous repeats II and III (14)(15)(16), the intracellular β 1a subunit has multiple fundamental roles in ultrastructural membrane targeting and modulating the biophysical properties of the α 1S subunit (17,18).…”