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...

In this work, regulation of organic cation transporter type 2 from rat (rOCT2) stably transfected in HEK293 cells was investigated by microfluorimetry with 4-(4-(dimethylamino)styryl)-N-methylpyridinium as substrate. The transport mediated by rOCT2 was specifically stimulated by PKA, phosphatidylinositol-3-kinase, p56(lck) tyrosine kinase, mitogen-extracellular-signal-regulated-kinase-1/2, calmodulin (CaM), and CaM-kinase-II. The regulatory pattern of rOCT2 differs markedly quantitatively and qualitatively from that of other OCT isoforms. Only CaM-dependent upregulation is conserved throughout the OCT family. For this reason, CaM regulation of rOCT2 was also investigated in isolated S3-segments (known to express only rOCT2) of male and female rat proximal tubules. Inhibition of CaM by calmidazolium significantly decreased rOCT2 activity (-49.0 +/- 13.6%, n = 4) in male but not female (9.0 +/- 13.0%, n = 4) rats. Real-time PCR and Western blot investigations of CaM expression in rat kidneys showed that male animals have significantly higher CaM expression. This is the first study describing post-translational gender-dependent rOCT2 regulation.

CD63 is a ubiquitously expressed member of the tetraspanin superfamily. Using a mating-based split-ubiquitin-yeast 2-hybrid system, pull-down experiments, total internal reflection fluorescence microscopy, Förster resonance energy transfer, and biotinylation assays, we found that CD63 interacts with human organic cation transporter 2 (hOCT2), which transports endogenous and exogenous substrates, such as neurotransmitters and drugs in several epithelial cells. CD63 overexpression affects cellular localization of hOCT2 expressed in human embryonic kidney (HEK)293 cells. Studies with CD63-knockout mice indicate that in renal proximal tubules, CD63 determines the insertion of the mouse ortholog of the transporter into the proper membrane domain and mediates transporter regulation by trafficking processes. In polarized Madin-Darby kidney canine kidney (MDCK) epithelial cells, CD63 and hOCT2 colocalize with the small GTPase Rab4, which controls the rapid recycling from sorting endosomes back to the cell surface. Suitable negative and positive control experiments were performed for each experimental approach. Empty vector transfected cells and wild-type mice were used as control. CD63 seems to play a role in the recycling of hOCT2 from endosomes to the basolateral membrane in polarized epithelia. These data indicate that CD63 has a previously uncharacterized function in regulating trafficking of specific membrane proteins in polarized cells.-Schulze, U., Brast, S., Grabner, A., Albiker, C., Snieder, B., Holle, S., Schlatter, E., Schröter, R., Pavenstädt, H., Herrmann, E., Lambert, C., Spoden, G. A., Florin, L., Saftig, P., Ciarimboli, G. Tetraspanin CD63 controls basolateral sorting of organic cation transporter 2 in renal proximal tubules.

It was previously shown that a1-antitrypsin (AAT) interacts with the type III secreted (T3S) EspB and EspD proteins of enteropathogenic Escherichia coli (EPEC), resulting in reduced functionality of the proteins. To determine if AAT is also able to interact with T3S proteins of other pathogens, the binding of AAT to Yop proteins of Yersinia enterocolitica was analysed. AAT did not interact with YopB or YopD, which have functions in type III translocation similar to EspB and EspD in EPEC, but specifically interacts with YopM, a member of the leucine-rich repeat (LRR) family of proteins, in overlay and pull-down assays. To determine regions of YopM involved in AAT binding, various N-and C-terminally truncated versions of YopM were recombinantly expressed, and their ability to interact with AAT analysed. All versions tested were able to bind AAT, indicating that at least eight LRR of YopM are sufficient for AAT interaction. The main physiological role of AAT is to inhibit neutrophil elastase; however, elastase was efficiently inhibited by AAT in the presence and absence of YopM, indicating that YopM does not interfere with the anti-protease inhibition activity of AAT, and that the domain of AAT interacting with YopM is not identical to AAT's protease interaction domain. Furthermore, it was shown that elastase efficiently degrades YopM and other Yop proteins. The data suggest that AAT has additional functions in the host response against bacterial infections that are not related to its anti-protease activity.

Human organic cation transporter 2 (hOCT2) is involved in the transport of endogenous and exogenous organic cations mainly in cells of the kidney and the brain. Here, we focus on the regulation of hOCT2 by direct protein-protein interaction. Screening within a mating-based split-ubiquitin-yeast-two-hybrid system (mBSUS) revealed the lysosomal-associated protein transmembrane 4 alpha (LAPTM4A) as a potential interacting protein. Interaction of LAPTM4A and hOCT2 was confirmed by pulldown assays, FRET microscopy analysis and immunofluorescence microscopy. Functionally, overexpression of LAPTM4A significantly decreased ASP(+) uptake in HEK293 cells stably transfected with hOCT2, suggesting a negative regulation of hOCT2-mediated transport. Furthermore, overexpression of LAPTM4A leads to a significantly decreased hOCT2 plasma membrane expression in surface biotinylation experiments. In addition, significant expression of LAPTM4A in human kidney was demonstrated by immunoblotting and immunofluorescence.In this work, LAPTM4A has been identified as interaction partner of hOCT2. LAPTM4A regulates the function of hOCT2 by influencing its trafficking to/from the cell membrane and processing it via the intracellular sorting machinery.

Enteropathogenic Escherichia coli (EPEC), atypical enteropathogenic E. coli, and Shiga toxin-producing E. coli differ in their virulence factor profiles, clinical manifestations, and prognosis, and they require different therapeutic measures. We developed and evaluated a robust multiplex PCR to identify these pathogroups based on sequences complementary to escV, bfpB, stx 1 , and stx 2 .Pathogenic Escherichia coli strains are responsible for a broad spectrum of intestinal and extraintestinal diseases, including diarrhea, urinary tract infections, septicemia, and neonatal meningitis (11). Enteropathogenic E. coli (EPEC) and the majority of clinical isolates of Shiga toxin (Stx)-producing E. coli (STEC) harbor the "locus of enterocyte effacement" (LEE), a pathogenicity island that is responsible for the phenotype of attaching-and-effacing (A/E) lesions (6, 11).EPEC are a major cause of human infantile diarrhea predominantly in less-developed countries but are also identified with increasing frequency in industrialized areas (1,11,17). These pathogens colonize the small intestine, induce the degeneration of epithelial microvilli, and intimately adhere to the host cell. Comparable to a "molecular syringe," the chromosomally encoded type III secretion system injects "effector" proteins into the host cell, inducing a characteristic rearrangement of the actin cytoskeleton resulting in the formation of "pedestals." These characteristic histopathological alterations are summarized as "A/E lesions."The genes responsible for the A/E lesions are located on an ϳ35-kb pathogenicity island, known as the locus of enterocyte effacement (LEE). Typical EPEC harbor an additional 60-MDa plasmid, the EPEC adherence factor (EAF) plasmid (16), that is not present in atypical EPEC (here abbreviated as ATEC) strains (3, 28). The EAF plasmid harbors the bundleforming pilus (bfp) operon, encoding the type IV pili responsible for localized adherence and the formation of microcolonies on host cells. ATEC strains harbor homologues of the LEE pathogenicity island but, due to the lack of the EAF plasmid (3, 8), they mostly adhere in a diffuse pattern to epithelial cells. Recent epidemiological evidence indicates an increasing prevalence of ATEC particularly in developed countries (see, for example, references 1, 17, 21, and 28) but also in developing countries (see, for example, references 9 and 27). This also indicates that in the field the EAF plasmid is not essential to cause disease.Like ATEC strains, the closely related STEC responsible for sporadic infections as well as serious outbreaks worldwide, mostly harbor the LEE pathogenicity island and lack the BFP-encoding EAF plasmid. STEC strains differ genoand phenotypically from ATEC by their production of Stx. These pathogens cause an acute inflammation of the colon, resulting in hemorrhagic colitis with rare but serious sequelae including neurological disorders and the hemolyticuremic syndrome (HUS), the leading cause of acute renal failure in children (11,12).Identification of EPEC, ATEC,...

Organic cation transporters (OCTs) are membrane proteins with relevant physiological (because they accept neurotransmitters as substrate) and pharmacological (because of their interaction with drugs) roles. The human OCTs hOCT1 ( SLC22A1/hOCT1) and hOCT2 ( SLC22A2/hOCT2) are highly expressed in hepatic (hOCT1) and in renal and neuronal tissue (hOCT2), suggesting a possible role in modulating neurotransmitter activity in the liver, kidney, and brain, and their clearance from the blood. Even though there are several data demonstrating that OCTs are regulated under various patho-physiological conditions, it remains largely unknown which proteins directly interact with OCTs and thereby influence their cellular processing, localization, and function. In this work, using a mating-based split-ubiquitin yeast two-hybrid system, we characterized the potential interactome of hOCT1 and 2. It became evident that these OCTs share some potential interaction partners, such as the tetraspanins CD63 and CD9. Moreover, we confirmed interaction of hOCT2 with CD9 by fluorescence-activated cell sorting coupled with Förster resonance energy transfer analysis. Together with other proteins, tetraspanins build “tetraspanins webs” in the plasma membrane, which are able to regulate cellular trafficking and compartmentalization of interacting partners. While CD63 was demonstrated to mediate the localization of the hOCT2 to the endosomal system, we show here that co-expression of hOCT2 and CD9 led to strong cell surface localization of the transporter. These data suggest that tetraspanins regulate the cellular localization and function of OCTs. Co-localization of CD9 and hOCT was confirmed in tissues endogenously expressing proteins, highlighting the potential biological relevance of this interaction.

Human organic cation transporters (hOCT1 to 3) are involved in the transport of endogenous and exogenous organic cations in the liver, kidney and brain. Here we analyse the regulation of hOCT1 to hOCT3 by direct protein‐protein interaction using a mating based Split‐Ubiquitin‐Yeast‐Two‐Hybrid System and pull‐down assays. Specifically, we found that the tetraspanin CD63 is an interacting partner of hOCT2 and hOCT3. A strong co‐localisation of CD63 and hOCT2 was shown on the plasma membrane and in vesicular compartments of HEK293 cells by TIRF‐microscopy and in tissue sections of human kidneys by immunofluorescence. Further immunofluorescence and surface biotinylation experiments after CD63 overexpression revealed a loss of cell surface hOCT2 and its relocalisation into intracellular vesicles. In HEK293 cells stably transfected with hOCT2, CD63‐overexpression caused a decrease, CD63‐knockdown an increase of cation‐uptake. As functional consequences of CD63 knockout in mice we observed that OCT2 lost its specific basolateral localization in the kidney, appearing to be widespread distributed in the cell and that basophil‐mediated histamine secretion, which apparently involves OCT3, was increased. Our data strongly suggest that CD63 regulates the function of hOCT2 and −3 by influencing their trafficking to/from the cell membrane and processing via the intracellular sorting machinery. Supported by the Deutsche Forschungsgemeinschaft (DFG CI 107/4‐1 to 2).