Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

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 th...

Full description

Saved in:
Bibliographic Details
Published in:The Journal of biological chemistry 2016-02, Vol.291 (9), p.4826-4843
Main Authors: Tétreault, Marie-Philippe, Bourdin, Benoîte, Briot, Julie, Segura, Emilie, Lesage, Sylvie, Fiset, Céline, Parent, Lucie
Format: Article
Language:English
Subjects:
Amino Acid Substitution
Animals
Animals, Newborn
calcium channel
Calcium Channels, L-Type - genetics
Calcium Channels, L-Type - metabolism
Cell Membrane - chemistry
Cell Membrane - metabolism
Cells, Cultured
confocal microscopy
electrophysiology
flow cytometry
Glycosylation
HEK293 Cells
Humans
ion channel
Luminescent Proteins - genetics
Luminescent Proteins - metabolism
Membrane Biology
molecular imaging
Molecular Weight
Mutagenesis, Site-Directed
Myocytes, Cardiac - cytology
Myocytes, Cardiac - metabolism
N-linked glycosylation
Point Mutation
Protein Processing, Post-Translational
Protein Stability
Rabbits
Rats
Recombinant Fusion Proteins - metabolism
Recombinant Proteins - metabolism
Surface Properties
trafficking
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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 Ca2+ channel complexes. CaVα2δ1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type CaV1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant CaVα2δ1 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 CaVα2δ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the CaVα2δ1-mediated increase in the peak current density and voltage-dependent gating of CaV1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored CaVα2δ1 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 CaVα2δ1 as well as the CaVα2δ1-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 CaVα2δ1, and furthermore that N-glycosylation of CaVα2δ1 is essential to produce functional L-type Ca2+ channels.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M115.692178