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

Abstract

BackgroundSLC10A4 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.ResultsWe 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.ConclusionsAlthough 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.

Characterization of cognitive impairments and neurotransmitter changes in a novel transgenic mouse lacking Slc10a4

“…Despite being found to co-localize with the vesicular transporters VMAT2 and VAChT, SLC10A4 has not been found to have any capacity for neurotransmitter transport itself (Schmidt et al, 2015). Our observation of increased dopamine metabolism in the striatum of Slc10a4 Δ/Δ animals is in agreement with previous studies (Larhammar et al, 2015) and suggests that, rather than act as a solute carrier, as suggested by its homology, SLC10A4 instead somehow regulates neurotransmitter turnover in the synapse.…”

Recent advances in understanding hepatic drug transport

“…SLC10A4 is, to the best of our knowledge, a transporter whose substrates have not been identified conclusively yet. While one group reported that protease activation of SLC10A4 makes this protein a bile acid transporter 64 , another group could not reproduce this finding and also did not find evidence for a transport activity of neuromodulators 65 . One possible explanation for these different findings is the use of different experimental systems.…”

The Pathological Effects of Circulating Hydrophobic Bile Acids in Alzheimer’s Disease

“…SLC10A1 and SLC10A2 carry bile acids. SLC10A4 is another member that is expressed in the brain [94][95][96]. It is located in the synaptic vesicles as either vesicular acetylcholinesterase or vesicular monoamine transporter 2 [92].…”