Role of N‐glycosylation in the stability of Ca V α2δ1

L‐type Ca V 1.2 channels play a key role in the excitation‐contraction coupling in the heart. They are formed of a pore‐forming Ca V α1 subunit in complex with the intracellular Ca V β and the disulfur‐linked Ca V α2δ accessory subunits. Ca V α2δ significantly increases peak current densities of Ca V 1.2. The mechanism underlying this effect is still under study but requires that Ca V α2δ be trafficked at the cell surface. Ca V α2δ contains 18 putative N‐glycosylation sites. A study was carried out to i dentify the role of N‐glycosylation in the trafficking and protein stability of the subunit Ca V α2δ. Site‐directed mutagenesis was used to modify the asparagine residues (individually or in combination) to the conservative glutamine residue. Surface density and protein stability were evaluated using a flow cytometry assay using a constitutively fluorescent Ca V α2δ construct. Preliminary data show that the cell surface and total protein densities of Ca V α2δ decreased proportionally to the number of mutations. Functional analysis of the same constructs using whole‐cell patch clamp recordings in HEK cells, confirmed that the peak current density of Ca V 1.2 decreased in the presence of the Ca V α2δ mutants. These results confirm that the surface expression of Ca V α2δ plays a critical role in the function of L‐type Ca V 1.2 channels. Work is under way to identify the most critical residues in this process. Ultimately, our studies will shed light on the molecular mechanism accounting for the modulation of L‐type Ca 2+ currents by the auxiliary Ca V α2δ subunit.