Conserved cysteine residues in the pore region are obligatory for human TRPM2 channel function.
TRPM2 proteins belong to the melastatin-related transient receptor potential or TRPM subfamily and form Ca(2+)-permeable cationic channels activated by intracellular adenosine diphosphoribose (ADPR). The TRPM2 channel subunit, like all its close relatives, is structurally homologous to the well-characterized voltage-gated potassium channel subunits, each containing six transmembrane segments and a putative pore loop between the fifth and sixth segments. Nevertheless, the structural elements determining the TRPM2 channel functions are still not well understood. In this study, we investigated the functional role of two conserved cysteine residues (at positions 996 and 1008) in the putative pore region of the human TRPM2 by site-directed mutagenesis, combined with electrophysiological and biochemical approaches. Expression of wild-type hTRPM2 channels in human embryonic kidney (HEK-293) cells resulted in robust ADPR-evoked currents. Substitution of cysteine with alanine or serine generated mutant channels that failed to be activated by ADPR. Furthermore, experiments done by Western blot analysis, immunocytochemistry, biotin labeling, and coimmunoprecipitation techniques showed no obvious changes in protein expression, trafficking or membrane localization, and the ability to interact with neighboring subunits that is required for channel assembly. Coexpression of wild-type and mutant subunits significantly reduced the ADPR-evoked currents; for the combination of wild-type and C996S mutant subunits, the reduction was approximately 95%, indicating that incorporation of one or more nonfunctional C996S subunits leads to the loss of channel function. These results taken together suggest that the cysteine residues in the pore region are obligatory for TRPM2 channel function.
Mei
Zhu-Zhong
ZZ
Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Mao
Hong-Ju
HJ
Jiang
Lin-Hua
LH
D000246:Adenosine Diphosphate Ribose / Q000494:pharmacology; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000187:drug effects; D002478:Cells, Cultured; D017124:Conserved Sequence; D003545:Cysteine / Q000378:metabolism*; D006801:Humans; D007473:Ion Channels / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D013329:Structure-Activity Relationship; D050053:TRPM Cation Channels / Q000737:chemistry* / Q000378:metabolism*
D007473:Ion Channels; D050505:Mutant Proteins; D021122:Protein Subunits; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; D000246:Adenosine Diphosphate Ribose; D003545:Cysteine