Abstract
Determining the functional significance of post-translational modifications advances our understanding of many broadly-expressed proteins, and particularly ion channels. The enzymes that catalyze these modifications are often expressed in a cell-type specific manner, resulting in considerable structural diversity among post-translationally modified proteins that are expressed across a variety of cell types. TRP channels exhibit notably variable behavior between cell types in vitro and in vivo , and they are frequently modified with N-glycans that contribute to protein function. TRPA1 possesses two putative N-linked glycosylation sites at N747 and N753 that have not yet been studied in detail. Here, we show that both of these sites can be modified with an N-glycan and that the glycan at position N747 modulates agonist-sensitivity of TRPA1 in vitro Additionally, we found that N-glycosylation also modulates cooperative effects of temperature and the agonist cinnamaldehyde on TRPA1 channel activation. Collectively, these findings suggest a dynamic role played by the N-glycosylation of human TRPA1. They also provide further evidence of the versatility of N-glycans and will assist in efforts to fully understand the complex regulation of TRPA1 activity.
Highlights
Transient receptor potential (TRP) channels are a family of nonselective, cation-permeable integral membrane proteins that commonly act as receptors in various sensory processes
HTRPA1 is glycosylated in vitro To investigate N-glycosylation at both Asn747 and Asn753, we applied three strategies: (1) site-directed mutagenesis of cDNA to replace the critical asparagine residues with structurally similar glutamine [10,12,13,14,15,16]; (2) glycosidase digestion of hTRPA1FLAG purified from (whole)-cell lysate (CL) [10,11,14] and (3) lectin-mediated detection of glycosylated human TRPA1 (hTRPA1)-FLAG [15]
Expression of hTRPA1 is affected by N-Q mutation
Summary
Transient receptor potential (TRP) channels (reviewed in [1]–[7]) are a family of nonselective, cation-permeable integral membrane proteins that commonly act as receptors in various sensory processes. In the case of TRP channels, N-glycans have been shown to influence cell-surface (CS) expression [9,11,12,14,15], sensitivity to agonists [13], activity regulation [9,16] and temperature sensitivity [12].
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