Glycosylation of asparagines 136 and 184 is necessary for the α2δ subunit-mediated regulation of voltage-gated Ca2+ channels

The Ca(V)alpha(2)delta auxiliary subunit is a glycosylated protein that regulates the trafficking and function of voltagegated Ca2+ channels. One of the most prominent roles of Ca(V)alpha(2)delta is to increase whole-cell Ca2+ current amplitude. Using N-glycosidase F and truncated forms of Ca(v)alpha(2)delta, earlier studies suggested an important role for N-linked glycosylation in current stimulation. Here, we used site-directed mutagenesis and heterologous expression in HEK-293 cells to examine the impact of individual glycosylation sites within the Ca(V)alpha(2)delta subunit on the regulation of Ca2+ currents through recombinant Ca2+ channels. We found two N-glycosylation consensus sites (NX(S/T)) in the extracellular alpha(2) domain of the protein that are functional. Substitution of asparagines for glutamines at amino acid positions 136 and 184 rendered these sites non-functional as shown by patch-clamp experiments. These results corroborate that N-glyeosylation is required for the Ca(V)alpha(2)delta subunit-induced current stimulation and suggest that sites N136 and N184 are directly involved in this action. Likewise, N136Q and N184Q mutations prevented whole-cell current stimulation without altering its kinetic properties, suggesting a regulation on the number of functional channels at the plasma membrane. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.