Served in the present study in untreated and differentiated CAD and SH-SY5Y cells. To more closely analyze the sorting domains of SLC10A4, a series of NTCP/SLC10A4 chimeras were generated and analyzed in HEK293 and CAD cells. These experiments clearly showed that the cytoplasmic C-terminus of SLC10A4 must contain dominant signals for its vesicular sorting. Previous studies with rat NTCP demonstrated that the tyrosine-based sorting motifs YXX?within its C-terminus (i.e. Y307-E-K-I and Y321-K-A-A) are involved in membrane delivery [29]. Although the C-terminus of SLC10A4 also contains these potential tyrosine-based sorting motifs (Y406-K -L und Y419-G-T-V), SLC10A4 is obviously not directed to the plasma membrane. In the same way, all chimeric constructs with the C-terminus of SLC10A4 (i.e. NTCP-CtSLC10A4 and NtSLC10A4NTCP-CtSLC10A4) showed a vesicular expression pattern and failed to transport typical NTCP substrates such as taurocholic acid and DHEAS. In contrast, both chimeras bearing the C-terminus of NTCP (i.e. SLC10A4CtNTCP and NtNTCP-SLC10A4-CtNCTP) were detected at the plasma membrane. Further mutagenesis experiments now have to localize the vesicular sorting motifs of SLC10A4 at the amino acid level.Conclusions SLC10A4 is expressed in vesicular structures not only in neurons of the central and peripheral nervous system, but also in neuronal cell lines such as SH-SY5Y and CAD. Although different kinds of assays were applied to screen for a transport function, SLC10A4 failed to show transport activity for dopamine, serotonin, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/20460822 norepinephrine, histamine, acetylcholine, choline, acetate, aspartate, glutamate, GABA, PREGS, 2-dioxaborolan-2-yl)benzene 2-Iodobenzo[b]thiophene 1 DHEAS, E-3-S, and ATP, indicating that SLC10A4 does not seem to be a typical neurotransmitter transporter. When the C-terminus of SLC10A4 was replaced by the homologous sequence of NTCP, the SLC10A4-CtNTCP chimera revealed clear plasma membrane expression in CAD and HEK293 cells. Vice versa, the C-terminus of SLC10A4 directed NTCP to intracellular vesicles, indicating that the sorting motifs of both carriers seem to be localized in the C-terminus. Further SLC10A4 transport studies should involve vesicle preparations from SLC10A4-deleted neuronal cell cultures or may use liposomal reconstitution of the SLC10A4 protein. Until then, the functional propertiesSchmidt et al. BMC Neurosci (2015) 16:Page 11 ofof the SLC10A4 orphan carrier protein still remain unknown.MethodsMaterials, chemicals, and radiochemicalsAll of the chemicals, unless otherwise stated, were from Sigma-Aldrich (Taufkirchen, Germany). Citalopram was purchased from Biotrend (Cologne, (R)-1-(3-Chlorophenyl)ethan-1-ol Germany) and collagenase D was from Serva (Heidelberg, Germany). [3H] DHEAS (70.5 Ci/mmol), [3H]E-3-S (45.6 Methyl 3-amino-2-chlorobenzoate Ci/mmol), [3H]aspartate (11.3 Ci/mmol), [3H]GABA (76.2 Ci/ mmol), [3H]histamine (13.4 Ci/mmol), [3H]choline chloride (66.7 Ci/mmol), [3H]norepinephrine (56.6 Ci/ mmol), [3H]serotonin (28.25 Ci/mmol), [3H]dopamine (38.7 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/10512904 Ci/mmol), [3H]glutamate (49.6 Ci/mmol), [3H]ATP (30.9 Ci/mmol), [35S]Adenosine 5-(-thio) triphosphate (12.5 mCi/mmol) and [3H]acetylcholine iodide (99.7 Ci/ mmol) were purchased from PerkinElmer Life Sciences (Boston, MA, USA). [3H]PREGS (20 Ci/mmol), [3H] taurocholic acid (10.0 Ci/mmol), [3H]acetate (150 mCi/ mmol), and [3H]lithocholic acid (50 Ci/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO, USA).Culture and differentiation of neuronal cell linesHuman neuroblastoma SH-SY5Y cells (obtained from DSMZ Braunschweig.

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