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Glycosylation of asparagines 136 and 184 is necessary for the α 2 δ subunit‐mediated regulation of voltage‐gated Ca 2+ channels
Author(s) -
Sandoval Alejandro,
Oviedo Norma,
Andrade Arturo,
Felix Ricardo
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2004.08.054
Subject(s) - glycosylation , protein subunit , hek 293 cells , extracellular , chemistry , stimulation , heterologous expression , biochemistry , mutagenesis , microbiology and biotechnology , mutant , biology , biophysics , recombinant dna , receptor , gene , neuroscience
The Ca V α 2 δ auxiliary subunit is a glycosylated protein that regulates the trafficking and function of voltage‐gated Ca 2+ channels. One of the most prominent roles of Ca V α 2 δ is to increase whole‐cell Ca 2+ current amplitude. Using N ‐glycosidase F and truncated forms of Ca V α 2 δ, 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 α 2 δ subunit on the regulation of Ba 2+ currents through recombinant Ca 2+ channels. We found two N ‐glycosylation consensus sites (NX(S/T)) in the extracellular α 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 ‐glycosylation is required for the Ca V α 2 δ 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.