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

Abstract

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, cross-linking 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. Europe PMC Funders GroupAuthor Manuscript FASEB J. Author manuscript; available in PMC 2015 July 15. The human organic cation transporter 2 (hOCT2), together with its paralogs hOCT1 and hOCT3, belongs to the SLC22 family of membrane transporters. OCT1, OCT2, and OCT3 are mainly expressed in epithelia of intestine, liver, brain, and kidney in a species-and paralog-specific fashion (1). These transporters have important physiological, pharmacological, and toxicological implications because of their role in the transport of endogenous and exogenous organic cations. Specifically, hOCT2 transports the dopaminergic neuromodulators histidyl-proline diketopiperazine and salsolinol (2), histamine (3), and xenobiotica, such as metformin (4,5), platin derivatives (6-9), ifosfamide (10), and paraquat (11). The high expression of hOCT2 in the dopaminergic brain region of the substantia nigra (2), and in the proximal tubules (PTs) of the kidney (12), where renal dopamine is produced (13), suggests that this transporter has a crucial importance in modulating cerebral and renal dopaminergic activity. Dopamine is one of the major natriuretic factors and plays a pivotal role in Na + homeostasis and blood pressure regulation (13). This fact, together with the notion that OCT2 is also part of t...

Functional significance of conserved cysteines in the human organic cation transporter 2

“…Consistent with the near loss of membrane expression, the J max value associated with MPP transport by the C89A and C103A mutants, both of which supported modest MPP transport, was reduced ϳ15-to 25-fold. These data are in agreement with those of Brast et al (2), who used a green fluorescent protein-tagged construct of hOCT2 to show by immunocytochemistry that single loop cysteine mutants are largely retained in the cytosol of HeLa SS6 cells. Plasma membrane expression of mouse Oat1 at the plasma membrane of HeLa cells was also reduced following mutation of conserved cysteine residues in its long extracellular loop (24).…”

“…OCT2 belongs to a larger family of solute carriers (OCT family), which include other OCTs (OCT1 and OCT3), the "novel" organic cation transporters 1-3 (OCTN1-3), and the organic anion transporters [1][2][3][4][5]. OCT family members have several structural features in common, including 12 putative transmembrane spanning helices (TMHs), intracellular COOH and NH 2 termini, a large extracellular loop between TMHs 1 and 2, and a large intracellular loop between TMHs 6 and 7.…”

“…Homology models of the three-dimensional structures of OAT1, OCT1, and OCT2 have been generated (20,21,27). Centrally located within these models is a large hydrophilic cleft that is formed by TMHs 1,2,4,5,7,8,10,and 11. The hydrophilic cleft is proposed to contain the substrate binding surface(s) since mutation of several residues lining the cleft has been shown to influence substrate selectivity (6,7,20,21,27).…”

Interplay between Disulfide Bonding and N-Glycosylation Defines SLC4 Na+-coupled Transporter Extracellular Topography

“…Disulfide bonding on the surface of the human organic cation transporter 2 and the Cys-loop ligand-gated ion channel receptors was shown to have critical functional roles (20,21). Our functional assay showed that presence or absence of the two disulfide bonds in EL-3 had no demonstrable effect on NBCe1-A base transport, indicating that in the heterologous expression system the two disulfide bonds in EL-3 do not affect the baseline NBCe1-A base transport activity.…”

“…The N-glycosylation of EL-3 is predicted to be important for NBCe1-A plasma membrane trafficking, whereas the role of the 4 highly conserved cysteines in EL-3 has remained unclear. In the human organic cation transporter 2, cysteines in the extracellular loops play a crucial role in protein plasma membrane trafficking and are involved in transporter oligomerization (20). Moreover, in Cys-loop ligand-gated ion channel receptors, the disulfide bonds in a large extracellular loop are involved in forming the substrate interaction site (21).…”