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The cysteines of the extracellular loop are crucial for trafficking of human organic cation transporter 2 to the plasma membrane and are involved in oligomerization
Author(s) -
Brast Sabine,
Grabner Alexander,
Sucic Sonja,
Sitte Harald H.,
Hermann Edwin,
Pavenstädt Hermann,
Schlatter Eberhard,
Ciarimboli Giuliano
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.11-180679
Subject(s) - hek 293 cells , transporter , extracellular , chemistry , organic cation transport proteins , transmembrane protein , transfection , cysteine , förster resonance energy transfer , membrane , mutant , biochemistry , microbiology and biotechnology , biophysics , fluorescence , biology , gene , receptor , enzyme , physics , quantum mechanics
Human organic cation transporter 2 (hOCT2) is involved in transport of many endogenous and exogenous organic cations, mainly in kidney and brain cells. Because the quaternary structure of transmembrane proteins plays an essential role for their cellular trafficking and function, we investigated whether hOCT2 forms oligomeric complexes, and if so, which part of the transporter is involved in the oligomerization. A yeast 2‐hybrid mating‐based split‐ubiquitin system (mbSUS), fluorescence resonance energy transfer, Western blot analysis, crosslinking experiments, immunofluorescence, and uptake measurements of the fluorescent organic cation 4‐(4‐(dimethylamino) styryl)‐ N ‐methylpyridinium were applied to human embryonic kidney 293 (HEK293) cells transfected with hOCT2 and partly also to freshly isolated human proximal tubules. The role of cysteines for oligomerization and trafficking of the transporter to the plasma membranes was investigated in cysteine mutants of hOCT2. hOCT2 formed oligomers both in the HEK293 expression system and in native human kidneys. The cysteines of the large extracellular loop are important to enable correct folding, oligomeric assembly, and plasma membrane insertion of hOCT2. Mutation of the first and the last cysteines of the loop at positions 51 and 143 abolished oligomer formation. Thus, the cysteines of the extracellular loop are important for correct trafficking of the transporter to the plasma membrane and for its oligomerization.—Brast, S., Grabner, A., Sucic, S., Sitte, H. H., Hermann, E., Pavenstädt, H., Schlatter, E., and Ciarimboli, G. The cysteines of the extracellular loop are crucial for trafficking of human organic cation transporter 2 to the plasma membrane and are involved in oligomerization. FASEB J. 26, 976‐986 (2012). www.fasebj.org