First Author | Woo SK | Year | 2013 |
Journal | J Biol Chem | Volume | 288 |
Issue | 51 | Pages | 36409-17 |
PubMed ID | 24214984 | Mgi Jnum | J:207188 |
Mgi Id | MGI:5554641 | Doi | 10.1074/jbc.M113.530584 |
Citation | Woo SK, et al. (2013) Complex N-glycosylation stabilizes surface expression of transient receptor potential melastatin 4b protein. J Biol Chem 288(51):36409-17 |
abstractText | N-glycosylation is important for the function and regulation of ion channels. We examined the role of N-glycosylation of transient receptor potential melastatin (Trpm) 4b, a membrane glycoprotein that regulates calcium influx. Trpm4b was expressed in vivo in all rat tissues examined. In each tissue, Trpm4b had a different molecular mass, between approximately 129 and approximately 141 kDa, but all reverted to approximately 120 kDa following treatment with peptide:N-glycosidase F, consistent with N-glycosylation being the principal form of post-translational modification of Trpm4b in vivo. In six stable isogenic cell lines that express different levels of Trpm4b, two forms were found, high mannose, core-glycosylated and complex, highly glycosylated (HG), with HG-Trpm4b comprising 85% of the total Trpm4b expressed. For both forms, surface expression was directly proportional to the total Trpm4b expressed. Complex N-glycosylation doubled the percentage of Trpm4b at the surface, compared with high mannose N-glycosylation. Mutation of the single N-glycosylation consensus sequence at Asn-988 (Trpm4b-N988Q), located near the pore-forming loop between transmembrane helices 5 and 6, prevented glycosylation, but did not prevent surface expression, impair formation of functional membrane channels, or alter channel conductance. In transfection experiments, the time courses for appearance of HG-Trpm4b and Trpm4b-N988Q on the surface were similar. In experiments with cycloheximide inhibition of protein synthesis, the time course for disappearance of HG-Trpm4b from the surface was much slower than that for Trpm4b-N988Q. We conclude that N-glycosylation is not required for surface expression or channel function, but that complex N-glycosylation plays a crucial role in stabilizing surface expression of Trpm4b. |