Dragoni Ilaria, Guida Elizabeth, McIntyre Peter
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.
J Biol Chem. 2006 Dec 8;281(49):37353-60. doi: 10.1074/jbc.M607227200. Epub 2006 Oct 2.
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.
我们研究了小鼠瞬时受体电位香草酸亚家族成员8（TRPM8）的糖基化、二硫键连接和亚基结构。为此，将氨基末端带有c-myc或血凝素表位标签的蛋白质导入中国仓鼠卵巢细胞并进行表达。在应用激动剂异丝氨酸内酯或薄荷醇以及低温进行细胞内钙成像分析时，这些修饰对通道功能没有明显影响。未修饰的TRPM8迁移时表观质量为129 kDa，在中国仓鼠卵巢细胞中可被糖基化，产生表观质量为136 kDa和147 kDa的糖蛋白。我们在TRPM8中鉴定出两个潜在的N-连接糖基化位点（天冬酰胺-821和天冬酰胺-934），并对它们进行突变，结果表明只有位于假定孔区域第934位的位点被修饰，且该位点的糖基化对于细胞表面表达或对异丝氨酸内酯、薄荷醇及低温的反应性并非绝对必要。碳水化合物链的酶切表明它们是复合碳水化合物。糖基化位点在孔的两侧有两个半胱氨酸残基，我们对其进行了突变，以证明它们参与了一个保守的双半胱氨酸基序，这对通道功能至关重要。这两个半胱氨酸中的任何一个发生突变都会使功能丧失，并促使形成一个同型二聚体大小的无功能复合物。双半胱氨酸突变体也无功能。最后，我们在全氟辛酸-聚丙烯酰胺凝胶中显示，TRPM8可以形成四聚体（除了二聚体和三聚体形式），这与目前认为功能性瞬时受体电位（TRP）离子通道是四聚体的观点一致。
