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J. Biol. Chem., Vol. 281, Issue 49, 37353-37360, December 8, 2006

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The Cold and Menthol Receptor TRPM8 Contains a Functionally Important Double Cysteine Motif^*

Ilaria Dragoni, Elizabeth Guida, and Peter McIntyre¹

From the Department of Pharmacology, University of Melbourne, Grattan Street Carlton, Victoria 3010, Australia and Novartis Institutes for Biomedical Research, 5 Gower Place, London WC1E 6BS, United Kingdom

We have investigated the glycosylation, disulfide bonding, and subunit structure of mouse TRPM8. To do this, amino-terminal c-myc or hemagglutinin epitope-tagged proteins were incorporated and expressed in Chinese hamster ovary cells. These modifications had no obvious effects on channel function in intracellular calcium imaging assays upon application of agonists, icilin or menthol, and cold temperatures. Unmodified TRPM8 migrates with an apparent mass of 129 kDa and can be glycosylated in Chinese hamster ovary cells to give glycoproteins with apparent masses of 136 and 147 kDa. We identified two potential N-linked glycosylation sites in TRPM8 (Asn-821 and Asn-934) and mutated them to show that only the site in the putative pore region at position 934 is modified and that glycosylation of this site is not absolutely necessary for cell surface expression or responsiveness to icilin, menthol, and cool temperatures. Enzymatic cleavage of the carbohydrate chains indicated that they are complex carbohydrate. The glycosylation site is flanked in the pore by two cysteine residues that we mutated, to prove that they are involved in a conserved double cysteine motif, which is essential for channel function. Mutation of either of these cysteines abolishes function and forces the formation of a non-functional complex of the size of a homodimer. The double cysteine mutant is also non-functional. Finally, we showed in Perfluoro-octanoic acid-polyacrylamide gels that TRPM8 can form a tetramer (in addition to dimer and trimer forms), consistent with current thinking that functional TRP ion channels are tetrameric.

Received for publication, July 31, 2006 , and in revised form, September 27, 2006.

^* This work was supported by the Novartis Institutes for Biomedical Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ To whom correspondence should be addressed: Dept. of Pharmacology, Grattan St. Carlton, Victoria 3010, Australia. Tel.: 61-383445745; Fax: 61-83445743; E-mail: pmci{at}unimelb.edu.au.

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				C. B. Phelps and R. Gaudet The Role of the N Terminus and Transmembrane Domain of TRPM8 in Channel Localization and Tetramerization J. Biol. Chem., December 14, 2007; 282(50): 36474 - 36480. [Abstract] [Full Text] [PDF]