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

Schmidt S, Moncada M, Burger S, Geyer J - BMC Neurosci (2015)

Bottom Line: Differentiation of these cells to the neuronal phenotype altered neither SLC10A4 gene expression nor its vesicular expression pattern.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and Toxicology, Justus Liebig University of Giessen, Schubertstr. 81, 35392, Giessen, Germany. stephanie.schmidt@vetmed.uni-giessen.de.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus

Transport measurements in digitonin permeabilized SLC10A4-HEK293 and VMAT2-HEK293 cells. Prior to transport measurements, stably transfected human SLC10A4-HEK293 cells and human VMAT2-HEK293 cells were pre-incubated with 15 µM digitonin for 10 min for permeabilization. Then the uptake of 400 nM [3H]serotonin, [3H]norepinephrine, or [3H]dopamine was measured over 10 min in the presence and absence of 5 mM ATP in the transport buffer. In addition to ATP, 2 µM of the potent VMAT2 inhibitor tetrabenazine (TBZ) or 5 µM of the proton ionophore carbonylcyanide-p-trifluoromethoxyphenylhydrazon (FCCP) were added to the transport buffer as indicated. After 10 min, the cells were washed with ice-cold PBS, lysed, and subjected to scintillation counting. The values represent mean ± SD of one representative experiment (for dopamine, n = 4) or two independent experiments (for serotonin and norepinephrine, n = 8). *Significantly different from control with p < 0.05. #Significantly different from positive uptake, p < 0.05.
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Fig3: Transport measurements in digitonin permeabilized SLC10A4-HEK293 and VMAT2-HEK293 cells. Prior to transport measurements, stably transfected human SLC10A4-HEK293 cells and human VMAT2-HEK293 cells were pre-incubated with 15 µM digitonin for 10 min for permeabilization. Then the uptake of 400 nM [3H]serotonin, [3H]norepinephrine, or [3H]dopamine was measured over 10 min in the presence and absence of 5 mM ATP in the transport buffer. In addition to ATP, 2 µM of the potent VMAT2 inhibitor tetrabenazine (TBZ) or 5 µM of the proton ionophore carbonylcyanide-p-trifluoromethoxyphenylhydrazon (FCCP) were added to the transport buffer as indicated. After 10 min, the cells were washed with ice-cold PBS, lysed, and subjected to scintillation counting. The values represent mean ± SD of one representative experiment (for dopamine, n = 4) or two independent experiments (for serotonin and norepinephrine, n = 8). *Significantly different from control with p < 0.05. #Significantly different from positive uptake, p < 0.05.

Mentions: In our previous study, we reported co-expression of the SLC10A4 protein with VMAT2 in synaptic vesicles of rat brain preparations [14], so we next intended to analyze the SLC10A4 transport in direct comparison with VMAT2. In this approach, we used HEK293 cells stably transfected with human SLC10A4 or human VMAT2, which were permeabilized with digitonin in order to facilitate access of the transport substrate to the site of carrier expression in intracellular vesicular structures, as described before [23]. As shown in Figure 3, VMAT2 showed significant transport activity for [3H]serotonin, [3H]norepinephrine, and [3H]dopamine which was dependent on the presence of adenosine triphosphate (ATP) in the transport buffer as well as sensitive to tetrabenazin (TBZ) and carbonylcyanide-p-trifluoromethoxyphenylhydrazon (FCCP). However, in contrast, SLC10A4 showed no transport activity for serotonin, norepinephrine, and dopamine at all in this vesicular transport assay. We also could not demonstrate a transport of acetylcholine, neither for SLC10A4 nor for VAChT in permeabilized HEK293 cell lines transfected with the respective construct (data not shown).Figure 3


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

Schmidt S, Moncada M, Burger S, Geyer J - BMC Neurosci (2015)

Transport measurements in digitonin permeabilized SLC10A4-HEK293 and VMAT2-HEK293 cells. Prior to transport measurements, stably transfected human SLC10A4-HEK293 cells and human VMAT2-HEK293 cells were pre-incubated with 15 µM digitonin for 10 min for permeabilization. Then the uptake of 400 nM [3H]serotonin, [3H]norepinephrine, or [3H]dopamine was measured over 10 min in the presence and absence of 5 mM ATP in the transport buffer. In addition to ATP, 2 µM of the potent VMAT2 inhibitor tetrabenazine (TBZ) or 5 µM of the proton ionophore carbonylcyanide-p-trifluoromethoxyphenylhydrazon (FCCP) were added to the transport buffer as indicated. After 10 min, the cells were washed with ice-cold PBS, lysed, and subjected to scintillation counting. The values represent mean ± SD of one representative experiment (for dopamine, n = 4) or two independent experiments (for serotonin and norepinephrine, n = 8). *Significantly different from control with p < 0.05. #Significantly different from positive uptake, p < 0.05.
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Related In: Results  -  Collection

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Fig3: Transport measurements in digitonin permeabilized SLC10A4-HEK293 and VMAT2-HEK293 cells. Prior to transport measurements, stably transfected human SLC10A4-HEK293 cells and human VMAT2-HEK293 cells were pre-incubated with 15 µM digitonin for 10 min for permeabilization. Then the uptake of 400 nM [3H]serotonin, [3H]norepinephrine, or [3H]dopamine was measured over 10 min in the presence and absence of 5 mM ATP in the transport buffer. In addition to ATP, 2 µM of the potent VMAT2 inhibitor tetrabenazine (TBZ) or 5 µM of the proton ionophore carbonylcyanide-p-trifluoromethoxyphenylhydrazon (FCCP) were added to the transport buffer as indicated. After 10 min, the cells were washed with ice-cold PBS, lysed, and subjected to scintillation counting. The values represent mean ± SD of one representative experiment (for dopamine, n = 4) or two independent experiments (for serotonin and norepinephrine, n = 8). *Significantly different from control with p < 0.05. #Significantly different from positive uptake, p < 0.05.
Mentions: In our previous study, we reported co-expression of the SLC10A4 protein with VMAT2 in synaptic vesicles of rat brain preparations [14], so we next intended to analyze the SLC10A4 transport in direct comparison with VMAT2. In this approach, we used HEK293 cells stably transfected with human SLC10A4 or human VMAT2, which were permeabilized with digitonin in order to facilitate access of the transport substrate to the site of carrier expression in intracellular vesicular structures, as described before [23]. As shown in Figure 3, VMAT2 showed significant transport activity for [3H]serotonin, [3H]norepinephrine, and [3H]dopamine which was dependent on the presence of adenosine triphosphate (ATP) in the transport buffer as well as sensitive to tetrabenazin (TBZ) and carbonylcyanide-p-trifluoromethoxyphenylhydrazon (FCCP). However, in contrast, SLC10A4 showed no transport activity for serotonin, norepinephrine, and dopamine at all in this vesicular transport assay. We also could not demonstrate a transport of acetylcholine, neither for SLC10A4 nor for VAChT in permeabilized HEK293 cell lines transfected with the respective construct (data not shown).Figure 3

Bottom Line: Differentiation of these cells to the neuronal phenotype altered neither SLC10A4 gene expression nor its vesicular expression pattern.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and Toxicology, Justus Liebig University of Giessen, Schubertstr. 81, 35392, Giessen, Germany. stephanie.schmidt@vetmed.uni-giessen.de.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus