Cysteine accessibility in the hydrophilic cleft of human organic cation transporter 2.
Organic cation transporters (OCTs) are involved in the renal elimination of many cationic drugs and toxins. A hypothetical three-dimensional structure of OCT2 based on a homology model that used the Escherichia coli glycerol 3-phosphate transporter as a template has been described (Zhang, X., Shirahatti, N. V., Mahadevan, D., and Wright, S. H. (2005) J. Biol. Chem. 280, 34813-34822). To further define OCT structure, the accessibility to hydrophilic thiol-reactive reagents of the 13 cysteine residues contained in the human ortholog of OCT2 was examined. Maleimide-PEO2-biotin precipitated (surface biotinylation followed by Western blotting) and reduced tetraethylammonium transport by OCT2 expressed in Chinese hamster ovary cells, effects that were largely reversed by co-exposure to substrates and transport inhibitors, suggesting interaction with cysteines that are near to or part of a substrate-binding surface. Cysteines at amino acid position 437, 451, 470, and 474 were identified from the model as being located in transmembrane helices that participate in forming the hydrophilic cleft, the proposed region of substrate-protein interaction. To determine which residues are exposed to the solvent, a mutant with all four of these cysteines converted to alanine, along with four variants of this mutant each with an individual cysteine restored, were created. Maleimide-PEO2-biotin was only effective at precipitating and reducing transport by wild-type OCT2 and the mutant with cysteine 474 restored. Additionally, the smaller thiol-reactive reagent, methanethiosulfonate ethylsulfonate, reduced transport by wild-type OCT2 and the mutant with cysteine 474 restored. These data demonstrate that cysteine 474 of OCT2 is exposed to the aqueous milieu of the cleft and contributes to forming a pathway for organic cation transport.
Pelis
Ryan M
RM
Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona 85743, USA. rpelis@email.arizona.edu
Zhang
Xiaohong
X
Dangprapai
Yodying
Y
Wright
Stephen H
SH
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008301:Maleimides / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D027701:Organic Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D000075102:Organic Cation Transporter 2; D011487:Protein Conformation; D021381:Protein Transport
D008301:Maleimides; D027701:Organic Cation Transport Proteins; D000075102:Organic Cation Transporter 2; C491013:SLC22A2 protein, human; D003545:Cysteine