Complex N-glycosylation stabilizes surface expression of transient receptor potential melastatin 4b protein.
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 ∼129 and ∼141 kDa, but all reverted to ∼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.
From the Departments of Neurosurgery.
Seung Kyoon
SK
Woo
Min Seong
MS
Kwon
Alexander
A
Ivanov
Zhihua
Z
Geng
Volodymyr
V
Gerzanich
J Marc
JM
Simard
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D006031:Glycosylation; D008358:Mannose / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D050053:TRPM Cation Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D050053:TRPM Cation Channels; C476766:TRPM4 protein, mouse; D008358:Mannose
Calcium Signaling; Glycoprotein; Ion Channels; Protein Stability; TRP Channels