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Summary:
Over 10 years the solute carrier family SLC10 comprised two well established sodium-dependent bile acid transporters, i.e. the Na+/taurocholate cotransporting polypeptide NTCP (SLC10A1) and the apical sodium-dependent bile acid transporter ASBT (SLC10A2). These carriers are essentially involved in the maintenance of the enterohepatic circulation of bile acids mediating the first step of active bile acid transport through the membrane barriers in the liver (NTCP) and the intestine (ASBT). Recently, two further members of this transporter family were identified in our group; they are referred to as sodium-dependent organic anion transporter SOAT (SLC10A6) and SLC10A7. While SOAT transports sulfoconjugated steroid hormones and sulfoconjugated bile acids, SLC10A7 remains an orphan carrier with yet unknown substrates. In the present study, two novel members were discovered and referred to as Slc10a4 and SLC10A5 (GenBank accession nos. AY825924, AY825925, and DQ074435). This work describes molecular cloning, expression and molecular analysis as well as functional testing for these novel putative membrane transporters. Sequence lengths of the SLC10A4 and SLC10A5 proteins are between 434-438 amino acids in man, rat, and mouse which is much longer compared with other members this carrier family (i.e. 340-377 amino acids). However, sequence identity/similarity of SLC10A4 is quite high to NTCP, ASBT, and SOAT, being 29%/54%. In contrast, identity/similarity values of SLC10A5 are highest to SLC10A3 which is an orphan and hitherto uncharacterized member of the SLC10 family. Slc10a4 exhibits a seven transmembrane domain topology with Nexo/Ccyt trans-orientation of the N- and C-terminal ends, whereas SLC10A5/Slc10a5 shows nine putative TMDs. Based on real time quantitative PCR and northern blot analyses SLC10A4 expression is predominant in the brain, whereas SLC10A5 is highest expressed in the liver and kidney. A polyclonal rabbit antibody was generated against the rat Slc10a4 protein and used for western blot and immunohistochemical analyses. Slc10a4-immunoreactivity was detected in cholinergic regions throughout the rat central nervous system. Co-localization studies with the cholinergic markers VAChT, ChAT, and CHT1 confirmed the presence of Slc10a4 in cholinergic neurons. Furthermore, the Slc10a4 protein is expressed in two dopaminergic regions: the substantia nigra and ventral tegmental area. Here, this protein was co-localized with ChAT and the catecholaminergic marker tyrosine hydroxylase. Slc10a5 expression was localized by in situ hybridization to hepatocytes and renal proximal tubules in rat liver and kidney sections, respectively. For the functional characterization of Slc10a4 and SLC10A5/Slc10a5, these proteins were expressed in X. laevis oocytes and HEK293 cells. Thereby, the C-terminal end of the proteins was tagged by the FLAG epitope and plasma membrane expression was confirmed by immunofluorescence microscopy. However, up to now transport studies failed to show transport activity of Slc10a4 and SLC10A5/Slc10a5 for bile acids and steroid sulfates, which are the typical substrates for NTCP, ASBT, and SOAT. Regarding SLC10A5/Slc10a5, because similar expression patterns to NTCP and ASBT and as these bile acid carriers are the most related carriers to SLC10A5 though, it's strongly supposed that SLC10A5 also has carrier function. Regarding Slc10a4, this carrier did not transport choline neither, which is a substrate of CHT1. Thus, in this study, the functional properties of Slc10a4 could not be completely elucidated, but Slc10a4 is assumed to be a new marker protein for cholinergic neurons. This unexpected finding for a novel member of the sodium/bile acid cotransporter family SLC10 sets Slc10a4 apart and needs further clarification.

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