Background: Missense mutations in Ca V ␣2␦1, an auxiliary subunit of cardiac L-type Ca V 1.2 channels, are associated with arrhythmias. Results:The reduction in the cell surface density of Ca V ␣2␦1 D550Y/Q917H was sufficient to impair Ca V 1.2 currents. Conclusion: Defects in the cell surface trafficking of Ca V ␣2␦1 mutants down-regulate L-type currents. Significance: CACNA2D1 genetic variants may trigger arrhythmias by reducing L-type Ca 2ϩ currents.

Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca 2؉ channel complexes. Ca V ␣2␦1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type Ca V 1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant Ca V ␣2␦1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of Ca V ␣2␦1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the Ca V ␣2␦1-mediated increase in the peak current density and voltage-dependent gating of Ca V 1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored Ca V ␣2␦1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of Ca V ␣2␦1 as well as the Ca V ␣2␦1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of Ca V ␣2␦1, and furthermore that N-glycosylation of Ca V ␣2␦1 is essential to produce functional L-type Ca 2؉ channels.The regulation of Ca 2ϩ influx in cardiac cells is critical to the generation of the force necessary for the myocardium to meet the physiological needs of the body (1). In resting cells, intracellular free ionized Ca 2ϩ is maintained at a low concentration (high nanomolar range) by the concerted action of mechanisms that prevent Ca 2ϩ entry, promote its extrusion (mostly via the Na ϩ /Ca 2ϩ exchanger), and ensure its storage in the sarcoplasmic reticulum (2). Ca 2ϩ entry is mediated mainly by the cardiac L-type Ca 2ϩ channel, which is central to the initiation of excitation-contraction coupling via Ca 2ϩ -induced Ca 2ϩ release from the sarcoplasmic reticulum. Regulation of the L-type Ca 2ϩ current has profound physiological significance. Indeed, alterations in density or the activation/inactivation gating of L-type Ca 2ϩ channels have been implicated in a variety of cardiovascular diseases (3, 4), including cardiac arrhythmias such as atrial fibrillation (5-8), heart failure (9, 10), and ischemic heart disease (10). The molecular mechanisms underlying changes in the activity of the L-type Ca 2ϩ channel remain under study for most pathologies.The L-type Ca V 1.2 channel belongs to the molecular family of high voltage-activated Ca V channels. High voltage-activated Ca V 1.2 channels are hetero-oligo...

Voltage-dependent Ca 2ϩ channels (Ca V ) are membrane proteins that play a key role in promoting Ca 2ϩ influx in response to membrane depolarization in excitable cells. To this date, molecular cloning has identified the primary structures for 10 distinct calcium channel Ca V ␣ 1 subunits (1-7) that are classified into three main subfamilies according to their high voltageactivated (HVA) 2 gating (Ca V 1 and Ca V 2) or low voltage-activated gating (Ca V 3). In addition to the transmembrane poreforming Ca V ␣1 subunit, Ca V 1 and Ca V 2 channels arise from the multimerization of three other proteins (7): a cytoplasmic Ca V ␤ subunit, a mostly extracellular Ca V ␣2␦ subunit, and calmodulin constitutively bound to the C terminus of Ca V ␣1 (8 -12).A considerable body of work documents the interaction and modulation of the Ca V ␣1 subunit of Ca V 1 and Ca V 2 channels (13-18) by the auxiliary Ca V ␤. The high affinity Ca V ␣1-Ca V ␤ interaction site on the pore-forming Ca V ␣1 subunit is a conserved 18-residue sequence in the I-II linker called the ␣ interaction domain (AID) (19,20) that has been structurally resolved by high resolution x-ray crystallography (21-23). Structural work showed that the AID forms a ␣-helix that binds to the ␣ binding pocket (ABP) in the Ca V ␤ nucleotide kinase (NK) domain. It has been proposed that the MMQKAL cluster of residues within the latter determines the high affinity nanomolar interaction between the two proteins (24 -29). Numerous mutational analyses of the AID residues have correlated the Ca V ␤-induced biophysical modulation with the high affinity binding of Ca V ␤ to the AID peptide in a variety of Ca V ␣1 isoforms for Ca V 1 and Ca V 2 channels (25, 29 -32).The association of Ca V ␣1 and Ca V ␤ subunits is also critical for proper channel maturation and cell surface expression of Ca V 2.2 (17), Ca V 1.2 (33, 34), and Ca V 2.3 (35). In Ca V 2.2, the I-II linker is presumed to play a role in this process (17,18), and mutations within the AID motif eliminated its cell surface expression and biophysical modulation by Ca V ␤1b and Ca V ␤3 (32). In addition, the Ca V ␤2-induced increase in Ca V 1.2 whole cell currents was abolished with the AID-defective YWI/AAA mutant (29), suggesting that high affinity binding of Ca V ␤ onto AID is required to traffic Ca V ␣1 to the plasma membrane. Nonetheless, the unique character of the high affinity AID-ABP interface in the membrane targeting of Ca V ␣1 has been questioned (27, 36 -40). In particular, it has been suggested that Ca V ␤-mediated plasma membrane targeting could be uncoupled from Ca V ␤-mediated modulation of channel gating (26, 41) with important contributions from other intracellular regions (33, 39,(42)(43)(44).In addition to Ca V ␤, the ancillary subunit Ca V ␣2␦ and the ubiquitous calmodulin (CaM) protein have also been proposed to modulate HVA channel maturation and targeting (9). For instance, co-expression of Ca V ␣2␦ promoted the trafficking of the Ca V ␣1 subunit of Ca V 2.2 in COS-7 cells (45), suggesting that Ca V ␣2...

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