title: Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity.
abstract: 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 Ca(2+) 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 Ca(2+) channels.
authors.affiliation: From the Départment de Physiologie Moléculaire et Intégrative, Faculté de Médecine, and.
authors.forename: Marie-Philippe
authors.initials: MP
authors.lastname: Tétreault
authors.affiliation: From the Départment de Physiologie Moléculaire et Intégrative, Faculté de Médecine, and.
authors.forename: Benoîte
authors.initials: B
authors.lastname: Bourdin
authors.affiliation: From the Départment de Physiologie Moléculaire et Intégrative, Faculté de Médecine, and.
authors.forename: Julie
authors.initials: J
authors.lastname: Briot
authors.affiliation: From the Départment de Physiologie Moléculaire et Intégrative, Faculté de Médecine, and.
authors.forename: Emilie
authors.initials: E
authors.lastname: Segura
authors.affiliation: Départment de Microbiologie, Infectiologie, and Immunologie, Faculté de Médecine, Centre de Recherche de l'Hôpital Maisonneuve-Rosemont, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Lesage
authors.affiliation: Faculté de Pharmacie, Institut de Cardiologie de Montréal and.
authors.forename: Céline
authors.initials: C
authors.lastname: Fiset
authors.affiliation: From the Départment de Physiologie Moléculaire et Intégrative, Faculté de Médecine, and lucie.parent@umontreal.ca.
authors.forename: Lucie
authors.identifier: http://orcid.org/0000-0002-7932-4264
authors.initials: L
authors.lastname: Parent
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000831:Animals, Newborn; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013499:Surface Properties
chemical_list: C486060:Cacna2d1 protein, rat; D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins
keywords: N-linked glycosylation; calcium channel; confocal microscopy; electrophysiology; flow cytometry; ion channel; molecular imaging; trafficking