Glycosylation of asparagines 136 and 184 is necessary for the alpha2delta subunit-mediated regulation of voltage-gated Ca2+ channels.
The CaValpha2delta auxiliary subunit is a glycosylated protein that regulates the trafficking and function of voltage-gated Ca2+ channels. One of the most prominent roles of CaValpha2delta is to increase whole-cell Ca2+ current amplitude. Using N-glycosidase F and truncated forms of CaValpha2delta, 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 CaValpha2delta subunit on the regulation of Ba2+ currents through recombinant Ca2+ channels. We found two N-glycosylation consensus sites (NX(S/T)) in the extracellular alpha2 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 CaValpha2delta 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.
Sandoval
Alejandro
A
Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav-IPN), Avenida IPN #2508, Colonia Zacatenco, Mexico City CP 07300, Mexico.
Oviedo
Norma
N
Andrade
Arturo
A
Felix
Ricardo
R
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001216:Asparagine / Q000378:metabolism*; D001464:Barium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D016384:Consensus Sequence; D017930:Genes, Reporter; D006031:Glycosylation; D006801:Humans; D015640:Ion Channel Gating; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015854:Up-Regulation
D015220:Calcium Channels; D010446:Peptide Fragments; D011994:Recombinant Proteins; D001464:Barium; D001216:Asparagine