title: Glycosylation of TRPM4 and TRPM5 channels: molecular determinants and functional aspects.
abstract: The transient receptor potential channel, TRPM4, and its closest homolog, TRPM5, are non-selective cation channels that are activated by an increase in intracellular calcium. They are expressed in many cell types, including neurons and myocytes. Although the electrophysiological and pharmacological properties of these two channels have been previously studied, less is known about their regulation, in particular their post-translational modifications. We, and others, have reported that wild-type (WT) TRPM4 channels expressed in HEK293 cells, migrated on SDS-PAGE gel as doublets, similar to other ion channels and membrane proteins. In the present study, we provide evidence that TRPM4 and TRPM5 are each N-linked glycosylated at a unique residue, Asn(992) and Asn(932), respectively. N-linked glycosylated TRPM4 is also found in native cardiac cells. Biochemical experiments using HEK293 cells over-expressing WT TRPM4/5 or N992Q/N932Q mutants demonstrated that the abolishment of N-linked glycosylation did not alter the number of channels at the plasma membrane. In parallel, electrophysiological experiments demonstrated a decrease in the current density of both mutant channels, as compared to their respective controls, either due to the Asn to Gln mutations themselves or abolition of glycosylation. To discriminate between these possibilities, HEK293 cells expressing TRPM4 WT were treated with tunicamycin, an inhibitor of glycosylation. In contrast to N-glycosylation signal abolishment by mutagenesis, tunicamycin treatment led to an increase in the TRPM4-mediated current. Altogether, these results demonstrate that TRPM4 and TRPM5 are both N-linked glycosylated at a unique site and also suggest that TRPM4/5 glycosylation seems not to be involved in channel trafficking, but mainly in their functional regulation.
authors.affiliation: Department of Clinical Research, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern Bern, Switzerland.
authors.forename: Ninda
authors.initials: N
authors.lastname: Syam
authors.affiliation: Department of Clinical Research, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern Bern, Switzerland.
authors.forename: Jean-Sébastien
authors.initials: JS
authors.lastname: Rougier
authors.affiliation: Department of Clinical Research, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern Bern, Switzerland.
authors.forename: Hugues
authors.initials: H
authors.lastname: Abriel
keywords: N-linked glycosylation; TRPM4; TRPM5; ion channels; regulation