Expression, sorting and transport studies for the orphan carrier SLC10A4 in neuronal and non-neuronal cell lines and in Xenopus laevis oocytes.

SLC10A4 belongs to the solute carrier family SLC10 whose founding members are the Na(+)/taurocholate co-transporting polypeptide (NTCP, SLC10A1) and the apical sodium-dependent bile acid transporter (ASBT, SLC10A2). These carriers maintain the enterohepatic circulation of bile acids between the liver and the gut. SLC10A4 was identified as a novel member of the SLC10 carrier family with the highest phylogenetic relationship to NTCP. The SLC10A4 protein was detected in synaptic vesicles of cholinergic and monoaminergic neurons of the peripheral and central nervous system, suggesting a transport function for any kind of neurotransmitter. Therefore, in the present study, we performed systematic transport screenings for SLC10A4 and also aimed to identify the vesicular sorting domain of the SLC10A4 protein. We detected a vesicle-like expression pattern of the SLC10A4 protein in the neuronal cell lines SH-SY5Y and CAD. Differentiation of these cells to the neuronal phenotype altered neither SLC10A4 gene expression nor its vesicular expression pattern. Functional transport studies with different neurotransmitters, bile acids and steroid sulfates were performed in SLC10A4-transfected HEK293 cells, SLC10A4-transfected CAD cells and in Xenopus laevis oocytes. For these studies, transport by the dopamine transporter DAT, the serotonin transporter SERT, the choline transporter CHT1, the vesicular monoamine transporter VMAT2, the organic cation transporter Oct1, and NTCP were used as positive control. SLC10A4 failed to show transport activity for dopamine, serotonin, norepinephrine, histamine, acetylcholine, choline, acetate, aspartate, glutamate, gamma-aminobutyric acid, pregnenolone sulfate, dehydroepiandrosterone sulfate, estrone-3-sulfate, and adenosine triphosphate, at least in the transport assays used. When the C-terminus of SLC10A4 was replaced by the homologous sequence of NTCP, the SLC10A4-NTCP chimeric protein revealed clear plasma membrane expression in CAD and HEK293 cells. But this chimera also did not show any transport activity, even when the N-terminal domain of SLC10A4 was deleted by mutagenesis. Although different kinds of assays were used to screen for transport function, SLC10A4 failed to show transport activity for a series of neurotransmitters and neuromodulators, indicating that SLC10A4 does not seem to represent a typical neurotransmitter transporter such as DAT, SERT, CHT1 or VMAT2.