SLC10A4 is a seven-transmembrane integral membrane protein of the SLC10 / bile acid:sodium symporter (BASS, TC 2.A.28) family, the family whose characterized members are the hepatic bile salt carrier NTCP (SLC10A1) and the ileal bile salt carrier ASBT (SLC10A2). Despite that close phylogenetic relationship, SLC10A4 is an orphan carrier whose substrate has not been identified; it has no measurable transport activity for bile salts, steroid sulfates, choline, acetylcholine, monoamines, amino-acid neurotransmitters or ATP in CHO, HEK293, CAD or Xenopus oocyte systems, and an engineered chimera that restores its delivery to the plasma membrane remains inactive [PMID:17106928, PMID:18355966, PMID:26084360]. Rather than residing in the bulk plasma membrane like NTCP, the protein is concentrated in the membranes of synaptic vesicles in cholinergic and monoaminergic neurons of the central and peripheral nervous system, where it co-localizes with synaptophysin, the vesicular acetylcholine transporter VAChT and the vesicular monoamine transporter VMAT2, and in the secretory granules of mast cells [PMID:18355966, PMID:21742018, PMID:25176177]; this distribution gives it its alternative name VAAT, vesicular aminergic-associated transporter. In the human brain it is most prominent in cholinergic and monoaminergic neurons and the lateral geniculate body [PMID:23948907]. Its reported biology is modulatory rather than directly transport-defining; synaptic vesicles lacking it load dopamine less efficiently while vesicles from overexpressing mice are more acidic, null mice have reduced striatal monoamine content, elevated dopamine turnover and slowed dopamine clearance, hippocampal slices show spontaneous gamma oscillations and cholinergic hypersensitivity, neuromuscular junctions are structurally and functionally remodelled, and mast cells release fewer granule-associated mediators after IgE/antigen stimulation [PMID:25176177, PMID:23022458, PMID:25410831, PMID:28439090]. It also heterodimerizes with NTCP in co-expression systems and reduces NTCP plasma-membrane abundance and taurocholate uptake, a negative effect on a partner's trafficking rather than bile salt transport of its own [PMID:22029531, PMID:31256060]. The protein carries an unusually long extracellular N-terminus relative to SLC10A1/A2/A6 that can be cleaved by thrombin [PMID:23589386].
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | REMOVE | Summary: Binary interaction with TMEM218 (A2RU14) detected in the HuRI systematic yeast two-hybrid interactome map [PMID:32296183]. The interaction is also listed in the UniProt INTERACTION block for Q96EP9 with NbExp=3. Reason: `GO:0005515 protein binding` conveys no functional information; it records that an interaction was detected without saying what the interaction accomplishes. HuRI is an unbiased proteome-scale Y2H screen and supports no more specific molecular function for SLC10A4 — the partner is another small membrane protein, consistent with SLC10A4's membrane residency but not diagnostic of any activity. Removal follows the repository policy that generic protein binding is uninformative rather than over-reaching, and does not imply the reported interaction is false; the IntAct record remains the appropriate home for it. Supporting Evidence: PMID:32296183 Specifically, yeast two-hybrid (Y2H) represents the only binary PPI assay that can be operated at sufficient throughput to systematically screen the human proteome for binary PPIs. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | REMOVE | Summary: Binary interaction with isoform 2 of COL4A5 (P29400-2) detected in the HuRI systematic yeast two-hybrid interactome map [PMID:32296183], also listed in the UniProt INTERACTION block with NbExp=3. Reason: Uninformative generic binding term. A high-throughput Y2H hit against a collagen IV isoform says nothing about what SLC10A4 does, and the pairing of a synaptic-vesicle membrane protein with a basement-membrane collagen is exactly the kind of result that needs orthogonal validation before any function could be read from it. No more informative molecular function is supported by this reference, so the generic term is removed rather than modified; the underlying interaction record is unaffected. Supporting Evidence: PMID:32296183 Specifically, yeast two-hybrid (Y2H) represents the only binary PPI assay that can be operated at sufficient throughput to systematically screen the human proteome for binary PPIs. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | REMOVE | Summary: Binary interaction with VMA12/TMEM199 (Q8N511) detected in the HuRI systematic yeast two-hybrid interactome map [PMID:32296183], also listed in the UniProt INTERACTION block with NbExp=3. Reason: Uninformative generic binding term. TMEM199 is an ER membrane protein of the V-ATPase assembly machinery, which is tantalizing given that SLC10A4 dose affects synaptic-vesicle acidification [PMID:25176177], but a single proteome-scale Y2H hit is nowhere near sufficient to assert a role in V-ATPase assembly or vesicle acidification, and inventing a specific function from interaction evidence alone is precisely what the curation guidelines forbid. The generic term is therefore removed, and the possible link is raised instead under suggested_questions and suggested_experiments. Supporting Evidence: PMID:32296183 Specifically, yeast two-hybrid (Y2H) represents the only binary PPI assay that can be operated at sufficient throughput to systematically screen the human proteome for binary PPIs. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | REMOVE | Summary: Binary interaction with SLC35A4 (Q96G79) detected in the HuRI systematic yeast two-hybrid interactome map [PMID:32296183], also listed in the UniProt INTERACTION block with NbExp=3. Reason: Uninformative generic binding term. SLC35A4 is another orphan multi-pass membrane carrier, so the hit is consistent with SLC10A4 being an integral membrane protein but establishes no activity for it. No informative replacement molecular function is supported by this reference, so the generic annotation is removed as uninformative rather than modified. Supporting Evidence: PMID:32296183 Specifically, yeast two-hybrid (Y2H) represents the only binary PPI assay that can be operated at sufficient throughput to systematically screen the human proteome for binary PPIs. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | REMOVE | Summary: Binary interaction with DNAJC30 (Q96LL9) detected in the HuRI systematic yeast two-hybrid interactome map [PMID:32296183], also listed in the UniProt INTERACTION block with NbExp=3. Reason: Uninformative generic binding term. DNAJC30 is a small mitochondrial J-domain protein; the pairing supports no molecular function for SLC10A4 and no colocalization of the two proteins has been reported in native tissue. As with the other four HuRI rows, removal reflects the uninformativeness of `GO:0005515` rather than a judgement that the interaction was not observed. Supporting Evidence: PMID:32296183 Specifically, yeast two-hybrid (Y2H) represents the only binary PPI assay that can be operated at sufficient throughput to systematically screen the human proteome for binary PPIs. |
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | MODIFY | Summary: PAINT places plasma-membrane localization at ancestral node PANTHER:PTN000040759 in the SLC10/BASS family, and SLC10A4 inherits it as an `is_active_in` assertion. The node is sound for the family's characterized members — NTCP (UniProtKB:Q14973) and the rodent bile salt carriers are bona fide plasma-membrane transporters — but SLC10A4 diverged to a vesicular compartment. Its steady-state pool is in the membranes of synaptic vesicles of cholinergic and monoaminergic neurons and in mast cell granules [PMID:21742018, PMID:25176177], and every functional effect ever measured for it (vesicular dopamine loading efficiency, vesicle acidification, granule mediator release) was measured on those vesicles, not at the cell surface. Reason: The quarrel here is with the compartment, not with the node placement or the donor set; this is a genuine case of functional and compartmental divergence within a family rather than a weak inference. `is_active_in` is the strong form of a localization assertion — it claims the gene product executes its molecular function at that location — and for SLC10A4 no activity has been demonstrated at the plasma membrane or anywhere else. What is positively established is vesicular residence, by immunoprecipitation from rat brain vesicle preparations with synaptophysin, VAChT and VMAT2 [PMID:21742018], co-localization on VAChT- or VMAT2-positive synaptic vesicles [PMID:25176177], and a vesicle-like pattern in neuronal cell lines [PMID:26084360]. Indeed PMID:26084360 shows the wild-type protein does NOT reach the plasma membrane efficiently in CAD/HEK293 cells — plasma-membrane expression had to be engineered by swapping in the NTCP C-terminus. The term is therefore replaced with `GO:0030672 synaptic vesicle membrane`, the compartment for which the evidence is direct. Note that the surface pool reported in heterologous overexpression is not being denied; it is retained by the two separate `located_in plasma membrane` rows (IDA and IEA), which this review keeps as non-core — the differing actions across the three GO:0005886 rows are deliberate and track the difference between claiming the protein is merely present at the surface and claiming it acts there. Note also that SLC10A4 appears in its own WITH/FROM list (UniProtKB:Q96EP9); that is correct and expected, because its own IDA plasma-membrane annotation is one of the descendant evidences the PAINT curator used to place the IBD, and it is not circular. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: COMPARTMENT OR COMPLEX MISMATCH FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000040759 SUPPORTS SOURCE BUT NOT TARGET Ancestral SLC10/BASS node; plasma-membrane localization is right for the carrier members but SLC10A4 relocated to synaptic vesicle and secretory granule membranes. UniProtKB:Q14973 · SLC10A1 SUPPORTS SOURCE BUT NOT TARGET NTCP is a genuine basolateral plasma-membrane bile salt symporter; the compartment does not transfer to its vesicular paralog. UniProtKB:Q96EP9 · SLC10A4 SUPPORTS TRANSFER Self-reference is the target's own IDA plasma-membrane annotation feeding the IBD; it grounds a surface pool and is not circular, but it supports located_in rather than is_active_in. Proposed replacements: synaptic vesicle membrane Supporting Evidence: PMID:21742018 Western blot and immunoprecipitation experiments with rat brain vesicle preparations revealed that the Slc10a4 protein was expressed in synaptic vesicles where it co-localized with synaptophysin, VAChT and VMAT2. PMID:25176177 We show that SLC10A4 is localized on the same synaptic vesicles as either vesicular acetylcholine transporter or vesicular monoamine transporter 2. PMID:26084360 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. |
| GO:0005886 plasma membrane | IDA PMID:23589386 SLC10A4 is a protease-activated transporter that transports ... | KEEP AS NON CORE | Summary: Direct observation of SLC10A4 at the cell membrane in PMID:23589386, the study in which thrombin cleaves the protein's extracellular N-terminus — an experiment that requires at least some of the protein to be surface-exposed. UniProt records the same conclusion in its SUBCELLULAR LOCATION field as Cell membrane with ECO:0000269|PubMed:23589386. An earlier overexpression study reached a similar split conclusion in CHO cells [PMID:17106928]. Reason: The annotation is retained. The cached record for PMID:23589386 is abstract-only (`full_text_available: false`), so the full images and fractionations the UniProt curator saw are not available to this review, and the annotation is not second-guessed; the thrombin-cleavage design independently implies surface exposure, and an independent group reports plasma-membrane plus intracellular staining in transfected CHO cells [PMID:17106928]. It is marked non-core rather than accepted because the plasma membrane is not where this protein does its known biology. Its principal steady-state location in native tissue is the synaptic vesicle membrane and the mast cell granule membrane [PMID:21742018, PMID:25176177], the surface signal is reported from heterologous overexpression systems, and PMID:26084360 found that wild-type SLC10A4 required an NTCP C-terminal swap to reach the plasma membrane at all in neuronal and HEK293 cells. A minority surface pool is a real but peripheral localization for this carrier, which is why this `located_in` row is kept while the `is_active_in` IBA row for the same term is modified. Supporting Evidence: PMID:23589386 After thrombin treatment, western blotting and immunofluorescence staining demonstrated that the N-terminus of SLC10A4 might be cleaved. PMID:17106928 immunoblotting analysis and immunofluorescence staining demonstrated a 49-kDa protein that is expressed at the plasma membrane and intracellular compartments PMID:26084360 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. |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Mechanical mapping of the UniProt subcellular location keyword SL-0039 ("Cell membrane") on the Q96EP9 record to `GO:0005886`. That UniProt statement is itself annotated ECO:0000269|PubMed:23589386, so this row carries the same underlying evidence as the IDA row above, arriving by a different route. Reason: The mapping is faithful — UniProt does assert Cell membrane for Q96EP9, and SL-0039 maps correctly to `GO:0005886` — so there is no propagation failure to correct. It is marked non-core for the same reason as the IDA row; a surface pool observed largely in overexpression systems is peripheral to a protein whose native residence and whose every measured functional effect belong to synaptic vesicle and secretory granule membranes [PMID:21742018, PMID:25176177]. The row adds no evidence beyond the IDA, but duplicate annotations arriving by different routes are acceptable and no preference between them is required. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: UniProtKB-SubCell:SL-0039 · Cell membrane SUPPORTS TRANSFER Keyword is present on Q96EP9 with ECO:0000269|PubMed:23589386 and maps correctly to GO:0005886; it reproduces the IDA row's evidence rather than adding new support. Supporting Evidence: PMID:23589386 After thrombin treatment, western blotting and immunofluorescence staining demonstrated that the N-terminus of SLC10A4 might be cleaved. |
| GO:0008508 bile acid:sodium symporter activity | IMP NOT PMID:23589386 SLC10A4 is a protease-activated transporter that transports ... | ACCEPT | Summary: This is a **negated** annotation (`NOT|enables`, ECO:0000315) — GOA asserts that SLC10A4 does NOT have bile acid:sodium symporter activity, on the basis of PMID:23589386. The negation is the correct reading of that paper and of the wider literature. The paper's own opening statement is that the protein has no bile acid transport activity, and its positive conclusion is heavily hedged and conditional on proteolytic activation. Reason: The annotation is accepted exactly as asserted — as a negation. PMID:23589386 is abstract-only in the cache, and its title ("SLC10A4 is a protease-activated transporter that transports bile acids") reads as a positive claim, so it is worth being precise about what the study shows. It reports that thrombin treatment of TE671 cells increases lithocholic and taurocholic acid accumulation and LCA cytotoxicity, and that siRNA knockdown of SLC10A4 abolishes that increment — an SLC10A4-dependent, protease-dependent uptake phenotype in whole cells. It does not report sodium dependence, transport stoichiometry, saturation kinetics, or activity in a reconstituted or defined heterologous system, which is what `GO:0008508` requires, namely "bile acid(out) + Na+(out) = bile acid(in) + Na+(in)". The baseline the authors themselves state is no activity, and their conclusion is phrased as "may have low activity but becomes activated". The UniProt curator, who read the full text, drew the same conclusion and recorded it twice — as this negated GO row and as the FUNCTION comment "No significant bile acid transporter activity could be measured despite its similarity to bile acid:sodium symporters" (ECO:0000269|PubMed:23589386). Four independent studies agree, with no taurocholate uptake in CHO cells [PMID:17106928], none for any NTCP substrate in HEK293 or Xenopus oocytes [PMID:18355966], none across a fourteen-substrate screen including a plasma-membrane-targeted chimera [PMID:26084360], and the field's own summary that the substrate remains unknown [PMID:28439090]. Accepting the negation is therefore both faithful to the curator and the best-supported position; the GOA record's remaining problem is the `GO:0015721` IBA that contradicts this negation, which is handled on that row. Supporting Evidence: PMID:23589386 SLC10A4 belongs to the sodium bile acid cotransporter family, but has no transport activity for bile acids. PMID:23589386 Therefore, SLC10A4 may have low activity but becomes activated by proteases, including thrombin, following cleavage. PMID:17106928 Functional analysis of SLC10A4 showed no significant taurocholate uptake in the presence of sodium when compared to untransfected CHO cells. PMID:18355966 Despite its close phylogenetic relationship to Ntcp, Slc10a4 showed no transport activity for the Ntcp substrates taurocholate, estrone-3-sulfate, dehydroepiandrosterone sulfate, and pregnenolone sulfate when expressed in HEK293 cells or Xenopus laevis oocytes. PMID:28439090 In spite of significant efforts, the substrate(s) of SLC10A4 still essentially remains unknown |
| GO:0015721 bile acid and bile salt transport | IBA GO_REF:0000033 | REMOVE | Summary: PAINT places participation in bile acid and bile salt transport at node PANTHER:PTN000040761, seeded by the experimentally characterized rat SLC10 bile salt carriers, and propagates it to SLC10A4. The assertion is directly contradicted by experiment on SLC10A4 itself — including by a negated annotation in this same GOA record — and by four independent transport studies in four expression systems. Reason: This is an over-propagation to a family member that lost (or never had) the family activity, and it creates an internal contradiction in the GOA record, because `NOT|enables GO:0008508 bile acid:sodium symporter activity` (IMP) and `involved_in GO:0015721 bile acid and bile salt transport` (IBA) cannot both stand. The experimental side wins, and it is not a single study. No taurocholate uptake in CHO cells [PMID:17106928]; none for taurocholate, estrone-3-sulfate, DHEA-sulfate or pregnenolone sulfate in HEK293 cells or Xenopus oocytes [PMID:18355966]; a systematic fourteen-substrate screen with positive controls found nothing, and — crucially closing the trafficking escape hatch — an SLC10A4-NTCP chimera that does reach the plasma membrane was still inactive, with or without the N-terminal domain [PMID:26084360]; and reviews of the family place SLC10A4 among the orphan carriers [PMID:31256060]. The challenge here is to the node placement and not to donor strength. The rat donors are well-characterized bile salt carriers and a short donor list is not weak support, but there is strong target-specific evidence of divergence. SLC10A4 kept the BASS fold and seven-TM topology while losing the bile-acid sub-activity, and relocated from the plasma membrane to synaptic vesicles and secretory granules [PMID:21742018]. Its only reported connection to bile salt handling is indirect and of the opposite sign, since heterodimerization with NTCP in forced co-expression reduces NTCP surface abundance and taurocholate uptake [PMID:22029531, PMID:31256060] — a negative effect on a partner's trafficking, in systems where the two proteins are artificially co-expressed, and in native tissue SLC10A4 (brain, intestine) and NTCP (hepatocyte) barely overlap. That is not participation in bile acid transport, and it is far too indirect to justify a regulation-of-transport term either; it is raised under suggested_questions instead. Recorded as explicit RETAINED residue claims against PANTHER:PTHR10361#na_coordination so that this removal cannot be misread as a claim of residue degeneration: the sodium-coordinating side chains are all present in SLC10A4. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE PSEUDO OR SUBACTIVITY LOSS Sources checked: PANTHER:PTN000040761 SUPPORTS SOURCE BUT NOT TARGET Ancestral SLC10 bile salt transport node is well founded for the NTCP/ASBT clade, but SLC10A4 is an orphan branch with direct experimental negation of the activity. RGD:3681 SUPPORTS SOURCE BUT NOT TARGET Rat SLC10 bile salt carrier donor; the donor annotation is sound and the transfer is what fails. RGD:3682 SUPPORTS SOURCE BUT NOT TARGET Rat SLC10 bile salt carrier donor; the donor annotation is sound and the transfer is what fails. Supporting Evidence: PMID:26084360 But this chimera also did not show any transport activity, even when the N-terminal domain of SLC10A4 was deleted by mutagenesis. PMID:18355966 Despite its close phylogenetic relationship to Ntcp, Slc10a4 showed no transport activity for the Ntcp substrates taurocholate, estrone-3-sulfate, dehydroepiandrosterone sulfate, and pregnenolone sulfate when expressed in HEK293 cells or Xenopus laevis oocytes. PMID:17106928 To date, we have shown that this protein has no capacity to transport taurocholate relative to SLC10A1 PMID:23589386 SLC10A4 belongs to the sodium bile acid cotransporter family, but has no transport activity for bile acids. PMID:31256060 the steroid sulfate transporter SOAT as well as four orphan carriers (SLC10A3, SLC10A4, SLC10A5 and SLC10A7) |
| GO:0016020 membrane | IEA GO_REF:0000120 | ACCEPT | Summary: Automatic annotation from the InterPro bile acid:sodium symporter signature IPR002657 together with an ARBA rule, placing SLC10A4 in a membrane. SLC10A4 is a seven-transmembrane integral membrane protein, so this is correct irrespective of which membrane it occupies. Reason: The inference is sound and uncontroversial. SLC10A4 has a demonstrated seven-transmembrane topology with an extracellular N-terminus and cytoplasmic C-terminus [PMID:18355966], and UniProt records it as a multi-pass membrane protein. `GO:0016020 membrane` is deliberately unspecific, which is a virtue here rather than a defect, because the protein is distributed between synaptic vesicle and secretory granule membranes, intracellular compartments, and a minority plasma-membrane pool, and the specific compartments are captured by the other cellular-component rows and by the proposed `GO:0030672` replacement. The signature is the right one — IPR002657 is the BASS family domain, which SLC10A4 genuinely carries — so there is no propagation defect to correct. Propagation Review Root cause: NO FAILURE CORE Sources checked: InterPro:IPR002657 SUPPORTS TRANSFER SLC10A4 genuinely carries the BASS family signature and is a multi-pass membrane protein, so the membrane mapping holds even though the family's transport activity does not. ARBA:ARBA00028763 SUPPORTS TRANSFER Rule-based membrane assignment consistent with the experimentally determined seven-transmembrane topology. Supporting Evidence: PMID:18355966 The rat Slc10a4 protein consists of 437 amino acids and exhibits a seven transmembrane domain topology with N(exo)/C(cyt)trans-orientation of the N- and C-terminal ends. |
| GO:0022857 transmembrane transporter activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT places generic transmembrane transporter activity deep in the BASS family, at node PANTHER:PTN000040756, a node whose descendants include the Arabidopsis BASS carriers as well as the mammalian SLC10 bile salt transporters. The curator therefore judged that carrier activity arose at or before the divergence of plants and animals, and deliberately asserted it only at the uninformative parent level, without committing to a substrate. SLC10A4 inherits it. Reason: This is exactly the right annotation for this protein, and its shallowness is the point rather than a defect to be fixed. SLC10A4 is an orphan carrier. It retains the BASS fold and the seven-transmembrane carrier architecture [PMID:18355966], but no substrate has been found for it despite systematic effort across bile acids, steroid sulfates, monoamines, acetylcholine, choline, amino-acid transmitters and ATP, in four expression systems, and including a chimera engineered to reach the plasma membrane [PMID:17106928, PMID:18355966, PMID:26084360]. A parent term that asserts carrier-type activity without naming a cargo is the only honest molecular-function statement available, and it should NOT be deepened to `GO:0008508` or any other substrate-specific child unless and until a substrate is identified — note that this same GOA record already carries an explicit negation of `GO:0008508`. The alternative objection, that even the parent overstates matters because SLC10A4 may act as a vesicular regulator rather than a carrier, is not pursued here. Reviewing an IBA means arguing with the node placement, the fold and topology are demonstrably retained, and there is no target-specific evidence of structural loss of the transport pathway. Failure to identify a substrate in the assays tried is not evidence that no substrate exists, as the authors of the most systematic screen are careful to say ("at least in the transport assays used"). Structural check added after this review was first written: a family-wide comparison against the ligand-bound NTCP cryo-EM structures finds every side-chain sodium ligand conserved in SLC10A4 (the one substitution, C260 to S338, is at a main-chain-only contact) and all three polar anchors of the bile-salt pocket retained, with the hydrophobic lining remodelled (T203 to F281). That replaces the earlier assertion that the fold and pathway are retained with a coordinate-level observation, and argues against both deepening and further downgrading this term: the ion-ligand set is present, the cargo is unknown. The models are apo, so this is evidence against one mechanistic explanation of the orphan status, not evidence that the site functions. A single OpenScientist run on SLC10A4, blind to this analysis, independently returned the same REFUTED verdict for the degeneration hypothesis; it is cited directly here and is also the run summarized in [file:human/SLC10A4/SLC10A4-bioinformatics/RESULTS.md]. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000040756 SUPPORTS TRANSFER Deep BASS-family node asserting only substrate-agnostic carrier activity; this is the correct level of specificity for an orphan carrier and must not be deepened to a substrate-specific child. Supporting Evidence: PMID:18355966 The rat Slc10a4 protein consists of 437 amino acids and exhibits a seven transmembrane domain topology with N(exo)/C(cyt)trans-orientation of the N- and C-terminal ends. PMID:26084360 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. file:human/SLC10A4/SLC10A4-bioinformatics/RESULTS.md SLC10A4 **retains the sodium-coordinating machinery intact** — all 12 side-chain ligands conserved — so its orphan status is **not** explained by degeneration of the ion-coupling site. file:human/SLC10A4/SLC10A4-hypotheses/structure-na-site-retention/openscientist.md SLC10A4 **retains** the complete sodium-coordination machinery |
| GO:0055085 transmembrane transport | IEA GO_REF:0000108 | ACCEPT | Summary: Logical inference from the accepted `GO:0022857 transmembrane transporter activity` molecular function to the corresponding biological process, via the GOC inter-ontology link pipeline (GO_REF:0000108). Reason: The inference is mechanically correct and inherits the judgement made on the molecular function it derives from. Since `GO:0022857` is accepted as the appropriate substrate-agnostic parent for an orphan carrier, its process counterpart is accepted on the same basis. As with the molecular function, the deliberate uninformativeness is the right outcome here and not a granularity defect to repair, because SLC10A4 cannot be placed in any specific transport process while the substrate it moves is unknown and while the family's bile-acid process term has been removed from this record as an over-propagation. This row should likewise not be deepened until a substrate is identified. Propagation Review Root cause: NO FAILURE CORE Sources checked: GO:0022857 · transmembrane transporter activity SUPPORTS TRANSFER The source molecular function is itself accepted on this record; the MF-to-BP link is a deterministic inter-ontology inference with nothing target-specific to challenge. Supporting Evidence: PMID:26084360 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. |
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Download this section (compressed HTML)Q: GOA currently carries both a NOT|enables GO:0008508 bile acid:sodium symporter activity row (IMP, PMID:23589386) and an involved_in GO:0015721 bile acid and bile salt transport row (IBA, PANTHER:PTN000040761) for SLC10A4. These are mutually contradictory. Should PAINT revisit the placement of the bile acid transport assertion so that the orphan SLC10 branches (SLC10A3, SLC10A4, SLC10A5, SLC10A7) are excluded, and is there a mechanism for an experimental NOT annotation to block an incoming IBA automatically?
Q: Is there an appropriate way in GO to record "orphan carrier, substrate unidentified" as a positive curatorial statement rather than as the absence of a child term? At present the only honest molecular function for SLC10A4 is the bare parent GO:0022857, which is indistinguishable from an uncurated annotation, even though in this case the shallowness reflects an extensively documented negative result.
Q: SLC10A4 dose changes synaptic vesicle lumenal acidification and dopamine loading efficiency [PMID:25176177], and mast cell granule ATP retention and mediator release [PMID:28439090]. Should these be annotated as regulation of vesicle acidification or of vesicular transmitter loading, or does the participation test exclude them because the V-ATPase performs the acidification and VMAT2 performs the amine loading, with SLC10A4's own role not yet resolved into either a transport step or a regulatory interaction?
Suggested experts: Klas Kullander, Jenny Hallgren
Q: SLC10A4 heterodimerizes with NTCP and reduces NTCP plasma-membrane abundance and taurocholate uptake [PMID:22029531, PMID:31256060]. The sign is negative, the system is forced co-expression, and native expression of the two genes barely overlaps (brain and intestine versus hepatocyte). Is there any tissue in which both are expressed, and if not, should this be treated purely as a heterologous-system artifact and left unannotated?
Suggested experts: Joachim Geyer, Stan F J van de Graaf
Q: A HuRI yeast two-hybrid hit pairs SLC10A4 with VMA12/TMEM199, an ER protein of the V-ATPase assembly pathway [PMID:32296183], which is suggestive given the vesicle acidification phenotype. Is this interaction reproducible in an orthogonal assay, and would it change how the acidification phenotype should be annotated?
Q: Does the proteolytic cleavage of the long extracellular N-terminus of SLC10A4 [PMID:23589386] occur in native neurons or mast cells, and if so should the cleaved form be treated as a distinct functional species for annotation purposes?
Experiment: Proteoliposome reconstitution of purified human SLC10A4, with and without N-terminal proteolytic cleavage, assayed against an unbiased metabolomic panel by LC-MS/MS rather than a candidate list. Every negative result so far comes from whole-cell or oocyte uptake, so a defined system with imposed ion and pH gradients is the test that could either identify the substrate or establish that the protein does not translocate anything.
Hypothesis: SLC10A4 translocates an unidentified solute across the synaptic vesicle membrane, and previous negative results reflect the wrong candidate list rather than the absence of transport.
Type: in vitro transport assay with untargeted metabolomic readout
Experiment: Comparative lumenal metabolomics and proteomics of immunoisolated synaptic vesicles from wild-type, Slc10a4-null and Slc10a4-overexpressing mice. If SLC10A4 moves a solute, the lumenal content of that solute should track its dose in the same way acidification and dopamine loading already do, which would convert the modulatory phenotype into a substrate identification.
Hypothesis: If SLC10A4 transports a solute into synaptic vesicles, the lumenal abundance of that solute will track SLC10A4 gene dose.
Type: comparative organelle metabolomics
Experiment: Separate the acidification phenotype from the loading phenotype by measuring V-ATPase-driven proton pumping, membrane potential and ATP-dependent dopamine uptake on the same immunoisolated vesicle preparations, with and without SLC10A4. This addresses whether SLC10A4 supplies a counter-ion or shunt conductance that the V-ATPase needs, which would make it a participant in vesicle acidification, or whether it acts upstream of vesicle physiology altogether.
Hypothesis: SLC10A4 provides a counter-ion or shunt conductance that the vesicular V-ATPase requires, making the acidification change primary and the monoamine loading change secondary to it.
Type: vesicle electrophysiology and uptake assay
Experiment: Test whether SLC10A4 physically interacts with V-ATPase assembly factors by orthogonal co-immunoprecipitation and proximity labelling in neurons and mast cells, following up the HuRI TMEM199 hit. A reproducible interaction would give a mechanistic route from the protein to the acidification phenotype that is currently only a correlation.
Hypothesis: SLC10A4 acts on vesicle acidification through a physical association with V-ATPase assembly machinery rather than by transporting a solute.
Type: interaction proteomics
Experiment: Determine the cryo-EM structure of human SLC10A4 and compare its substrate cavity with the solved NTCP structures. The BASS fold is retained, so a structure would show whether the translocation pathway is intact, occluded, or degenerate, which is the single most direct way to decide whether GO:0022857 is the right molecular function or an over-generous one.
Hypothesis: The BASS translocation pathway is structurally degenerate in SLC10A4, which would explain twenty years of negative transport assays and argue that even the generic transporter activity term is too generous.
Type: cryo-EM structure determination
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