Current interpretation: ANO10 is an ER phospholipid scramblase, with a separately observed, condition-dependent plasma-membrane pool. Channel and NOT assertions retain their source-specific assay limits. The entries below preserve the research chronology; later dated follow-ups supersede earlier pending-access and draft-status statements.
The verified Seed8 input contains 16 annotations and five alternative products. The original YAML, UniProt record and GOA bytes are preserved in tmp/ANO10-initial/. Fresh primary GitHub preflight at 025b22a6a49de665ffb482eb51803e3b1c387c3f found no ANO10/TMEM16K canonical or history paths, task branch, or matching open PR. Existing raw data and publication caches remain read-only.
One properly configured installed-client Falcon attempt failed authentication; its Perplexity fallback failed DNS. No provider report was produced. One additional normal four-PMID batch timed out after 45 seconds with zero outputs. This is one interrupted batch, not four completed individual failures. The terminal receipts are in tmp/ANO10-initial/. These notes are manual research, not provider output.
PMID:31477691(https://www.nature.com/articles/s41467-019-11753-1), The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K, directly studies purified full-length human Q9NW15/NM_018075 protein. Sf9 and mammalian cells are expression hosts, not the reagent species. Reconstituted protein scrambles phospholipids; calcium and shorter-chain lipids favor activity. The same paper measures nonselective ion flux, so lipid scrambling does not exclude ion permeability. Negative whole-cell currents concern the tested plasma-membrane setting. Tagged-protein colocalization and endogenous U2OS staining support ER residence. Actual abstract, functional/localization Results, Discussion and relevant cloning, electrophysiology, staining and reconstitution Methods were read; all supplementary pixels and structural-processing details were not inspected. The normal cache is requested. A new scramblase MF and ER-membrane localization are candidates; disease severity alone will not supply a new process.
PMID:32620747(https://www.nature.com/articles/s41467-020-17016-8), TMEM16K is an interorganelle regulator of endosomal sorting, reports ER/endosome contact and sorting experiments. PubMed identity and abstract, publisher Introduction, targeted Results and Discussion were read; full construct Methods remain pending. Contact proximity is not automatically binary Rab7 binding. The mechanistic connection between scrambling and sorting remains unresolved. Knockout/rescue necessity does not by itself justify new sorting participation, a tether MF, or an adaptor MF.
The three seeded publication records are abstract-only: PMID:19946888 is a membrane-proteome survey; PMID:21984732 reports the family channel comparison; PMID:22946059 reports positive currents and localization. This cache limitation is distinct from external article access.
PMID:22946059(https://journals.biologists.com/jcs/article/125/21/4991/32556/Anoctamins-are-a-family-of-Ca2-activated-Cl) is now accessible in the original publisher body. The relevant Results/Discussion, Figures 1–5/8 legends and cDNA/immunostaining Methods were read. The constructs are explicitly human. ANO10 expression produces stimulation-associated currents, with anion substitutions assessed for ANO1/4/6/10. Cation substitution experiments include ANO10, but the authors leave direct cation permeability versus activation of a separate conductance unresolved. They detect weak surface staining and extracellular FLAG accessibility for ANO10, while cautioning that native protein may predominantly act intracellularly. This supports a conditional surface pool, not universal surface residence. The full supplemental image panel has not been inspected. The abstract-only cache is preserved. Positive and negative channel claims require assay-specific adjudication; a scramblase label alone does not negate channels.
The normal abstract of PMID:20056604 reports FRT localization and channel measurements; full Methods remain uninspected. PMID:22075693 explicitly studies ANO3–7, but only its abstract was read here; its title alone cannot establish whether ANO10 appears elsewhere in that paper. Neither is being used to declare a source misattributed. Reactome R-HSA-2684901 is a historical family-level channel event, and R-HSA-983712 is the broader ion-transport pathway. Their cached summaries do not add independent target experiments.
PMID:32517157(https://pmc.ncbi.nlm.nih.gov/articles/PMC7312528/) uses CAAX targeting and terminal-domain chimeras to examine intracellular anoctamins at the surface. Indexed original Introduction, Results 2.1, Discussion and Conclusions were read; full Methods and construct-species checks remain pending. Engineered targeting cannot establish native localization. Its anion-permeability and surface-scrambling assays will be interpreted with the actual constructs after normal recovery.
PMID:39337485(https://epub.uni-regensburg.de/59227/1/ijms-25-09998-v2.pdf) compares parental and ANO6-knockout HEK293 cells. The actual PDF title/DOI, abstract, Results 2.2 and relevant Discussion passages were read. Expression background affects observed currents. ANO5 retains scrambling without ANO6; ANO10 does not show surface scrambling in the tested setting. This does not refute scrambling in ER-like membranes or by purified protein. Supplemental image pixels and exact construct-species Methods remain to be inspected. This normal source is shared with the ANO5 review and requested only once.
Official GO:0005254 requires chloride passage through a pore, not exclusive anion selectivity. GO:0005227 specifically requires calcium-dependent opening for cation passage; increased cellular calcium plus an induced current is not a resolved molecular gating mechanism. The GO:0017128 definition and parent describe ATP-independent transbilayer lipid movement.
The current Human Protein Atlas ANO10 summary reports plasma membrane plus additional vesicle/cilium localization. This is an actual target page, separate from a gene-name inference; individual image/antibody validation remains to be checked before final source-specific rationale. No new vesicle or cilium row is proposed from this summary alone.
PTHR12308 membership records can identify family members but do not establish equivalent functions or PAINT ancestral placement. The Seed8 family metadata record with conflicting timestamps remains quarantined. Exact PAINT nodes and source mappings still require inspection. ANO10 appearing in its own IBA donor list is legitimate descendant experimental evidence, not circularity. No new BP is presently proposed.
Independent all-16-row consultation supported three UNDECIDED assertions, twelve secondary retained assertions and one ACCEPT membrane assertion. The drafted review adds one directly demonstrated scramblase MF. ER membrane is recorded as the precisely supported core location rather than another NEW row alongside the existing broad membrane annotation. No new BP or scrambling-to-ion-transport/sorting causal edge is proposed. All sixteen original source objects and five products are unchanged.
The actual HPA subcellular table was inspected: antibody HPA016624 supports plasma membrane in BJ, HUVEC/TERT2 and U2OS cells. HUVEC/TERT2 is one cell line. Individual image pixels were not inspected. This antibody differs from HPA051569 used for endogenous U2OS ER staining in the 2019 primary study. HPA's additional vesicle/cilium annotations are not promoted to new rows here.
The actual 2019 published PDF clarifies the ion endpoint: chloride retained in liposomes is measured after a KCl gradient dissipates without ionophores. Cation movement is an inference from salt flux, not a directly recorded potassium-selective current. The narrow calcium-gated cation MF and its automatic inference premise remain unresolved while broad cation movement is retained as secondary.
No local GO-CAM index record matched Q9NW15, ANO10 or TMEM16K. No exact local PAINT record resolved PTN002642655/PTN000267334; family membership does not substitute for that tree. No OLS MCP is available, so official GO definition/parent pages were used. The four Source32 normal records are still pending. PMID references without exact snippets or availability flags are deliberate draft attachments pending actual cache reconciliation, not claims that source closure is complete.
PMID:20056604, original PMC2844227 now exposes indexed cDNA, culture, immunostaining, iodide-quenching and patch-clamp Methods, plus targeted ANO10 Results/Discussion. The ANO10 construct is human OriGene SC113757 with a C-terminal V5/His tag in rat FRT cells. These hosts express endogenous ANO6, ANO8 and ANO10. The paper distinguishes weak surface/intracellular staining and slow whole-cell current development from iodide-quenching results. Its context qualifies pore attribution; it does not disprove conditional chloride conductance. Supplementary pixels were not inspected, and the normal abstract cache stays unchanged.
PMID:22075693, original publisher Methods/Discussion independently confirms mouse ANO10 NM_133979.2 in the construct series. The indexed targeted body describes intracellular localization and ANO7 coexpression findings; these are ortholog/context evidence, not the seeded PMID:21984732 assay. No whole-article or supplementary-image read is claimed. The direct publisher page returned 403; actual indexed original passages were accessible. PMID:21984732 remains abstract-only after official PubMed confirmation.
These access extensions revise the two reference read scopes; they do not change any annotation action, source assertion or compact core. Schema-only validation of the initial authored draft passed without fetching missing references. Full source validation remains pending the four actual normal Source32 records.
This entry records the state at that time. Its unread-body rationale and one-ACCEPT count were superseded by the following PR3347 follow-up, which read PMID:21984732 and refined the broad membrane annotation.
The four requested normal caches were imported by the project owner without overwriting existing records. All four imported byte hashes match the assessed exact archive and carry XML full-text flags. This flag describes the cached format, not a claim that every structural analysis, supplement or image was inspected. The original five abstract-only caches and two Reactome records remain unchanged; external original-paper observations recorded above remain separate from cached availability.
PMID:31477691 now supplies literal result attachments for the new scramblase annotation and ER core location. Its human NM_018075/Q9NW15 construct, purified in either insect or mammalian expression hosts, directly moves phospholipids in reconstituted vesicles. Lipid scrambling depends on calcium and membrane composition. Its endpoint chloride-release assay measures dissipation of a KCl gradient without ionophores: broad cation movement is an inference from salt flux, not a potassium-selective current measurement. The channel-active fraction depends only minimally on calcium. Thus the exact calcium-gated cation annotations remain UNDECIDED. Endogenous U2OS staining and tagged human construct colocalization with ER markers support the ER membrane core.
PMID:32620747 Methods explicitly identifies mouse TMEM16K cDNA OMu10422D in pCDNA3.1-TMEM16K-FLAG. Human/monkey cell hosts and mutations corresponding to human disease variants do not change that construct identity. I read its targeted BioID, retrograde-transport, Rab7-contact, N-terminal lipid-overlay, scrambling-domain chimera and disease-variant Results, Discussion, and corresponding construct/culture/purification/overlay/split-GFP Methods. The mouse knockout and rescue work supports the conserved endosomal role described in the biological summary. BioID and split-GFP demonstrate proximity/contact, while the chimera results suggest a functional link; no new binary Rab7-binding activity, tether activity or sorting-process annotation is manufactured from these results. Supplementary Table 1 and image pixels were not independently inspected.
PMID:32517157 actual Methods specifies TMEM16K NM_001346468.1 with a C-terminal CFP-CAAX fusion. The official Broad GPP human ANO10 transcript inventory includes that accession (source); the direct NCBI nuccore page was inaccessible. The targeting, iodide-quenching and whole-cell Results and construct/patch-clamp Methods were read. Artificial plasma-membrane targeting cannot establish native predominant localization, and surface annexin staining is not an ER scrambling assay.
PMID:39337485 actual Results, Discussion, cell-culture, patch-clamp and flow-cytometry Methods were read. Its ANO6-deficient human cell context changes observed anoctamin currents. The paper explicitly restricts its ANO10 scrambling negative to the plasma membrane and allows intracellular activity. Reagent species is not assigned from the human host; it remains unresolved from the inspected Methods. Supplementary image panels were not inspected. This contextual evidence qualifies channel claims without contradicting the purified human ER scramblase evidence.
All 16 original GOA source objects, the original NOT and five alternative products are preserved. All prior draft actions remain unchanged: one ACCEPT, twelve KEEP_AS_NON_CORE, three UNDECIDED, plus one NEW molecular function. No NEW biological process or redundant ER annotation was added. The sole core is ER phospholipid scrambling; uncertain exact PAINT/rule derivations remain explicitly unresolved. The unread PMID:21984732 assay body still requires UNDECIDED under the user instructions. All annotation decisions and source-cache closure are complete; final validation and independent consultation are recorded in the matching history.
This entry supersedes the earlier access statements for PMID:21984732; the cached abstract is unchanged. The original publisher main body and Figures 1/2 captions are now read. Although labelled Symposium Report, it includes original negative experiments, not just a literature summary. The TMEM16K-negative iodide-quenching result is from ionomycin-stimulated transfected HEK293 cells. The Figure 2C patch-clamp caption names other family members, so it does not establish K-specific recording conditions. There is no separate detailed Methods section establishing the K construct species. These limits are distinct from unread evidence; the preserved NOT remains uncertain because assay contexts conflict.
The original PMID:20056604 body was read for construct/culture and electrophysiology Methods and targeted ANO10 Results. Human tagged ANO10 was expressed in rat FRT hosts. Negative quenching and reduced baseline conductance coexist with slow calcium-dependent patch-clamp currents. ANO1 coexpression and knockdown affect different current phases; results do not support a universal inhibitor mechanism. The unchanged UniProt statement that ANO10 lacks CaCC activity and can inhibit ANO1 is explicitly considered against these assay-specific results, rather than treated as a new experiment.
The original PMID:22946059 Results and Methods report positive stimulated currents and anion substitutions with human ANO10 in HEK293 cells. Its cation experiments leave direct ANO10 permeation versus activation of a separate conductance unresolved. This cation-attribution uncertainty, rather than weak calcium dependence in another assay, distinguishes the two channel decisions. Only ANO9 suppressed ANO1 in its ATP/coexpression comparison. The 2024 PMID:39337485 Discussion specifically associates ANO5/ANO10 currents with ANO6 absence, and ANO9 currents with ANO6 presence; this is not a blanket refutation of ANO10 currents.
The positive calcium-gated chloride assertion is retained as a conditional secondary activity with its original experimental source. Its evidence is the human cellular channel study, not the later minimally calcium-dependent purified salt-flux assay. The separate NOT remains UNDECIDED on the conflicting conditions. No exclusive chloride selectivity is required by the checked GO:0005229 definition. No new channel-inhibitor function or process is inferred.
The broad membrane HDA is refined to ER membrane, GO:0005789 using independent human PMID:31477691 localization. This does not attribute ER-specific evidence to the NK-cell proteome survey, whose target peptide entry remains uninspected. HPA plasma-membrane observations and tagged-human surface staining remain separately retained as secondary pools. The exact PAINT tree/IBD was not recovered: no matching local node data, and the official PTHR12308 family page was inaccessible during the bounded follow-up. Other species' annotation displays and a different node identifier cannot establish the target's ancestral placement. No donor-count or self-donor objection is made; ordinary source qualifiers remain unchanged under the annotation-review instructions.
Independent historical consultation reviewed the original 21984732 main body, indexed 20056604 assays and the author thesis version of 22946059; my own 22946059 read used the published journal body. These scopes are kept separate. All sixteen original source objects, the original NOT, five alternative products, prior NEW scramblase assertion and published history are preserved. The only action change is membrane ACCEPT to MODIFY ER membrane. New findings expose the mixed historical results and the established scramblase/contact results. Final checks and consultation are recorded in the new follow-up history.
The published PMID:22946059 localization Results, under the section on plasma-membrane localization immediately before Figure 8, report an extracellular FLAG insertion and labeling of nonpermeabilized cells: "The results show a spotted membrane expression of Ano10". Figure 8 and the cDNA Methods identify human anoctamins expressed in HEK293 cells. The adjacent Discussion cautions that native abundance may be insufficient for substantial surface accumulation. I reread those Results, Figure 8 legend and construct/immunocytochemistry Methods; supplementary Figure S3 pixels remain uninspected. The abstract-only normal cache is preserved. This original-body observation, rather than a truncated abstract or ER-localization quotation, supports the retained secondary surface pool.
The current HPA ANO10 subcellular summary and assay table state "Localized to the Plasma membrane (supported)" and list HPA016624 staining at the plasma membrane in BJ, HUVEC/TERT2 and U2OS cells. HUVEC/TERT2 is a single cell line. The table was inspected; image pixels were not independently scored. HPA's secondary cilium and vesicle observations are not promoted to new annotations. This positive HPA evidence is distinct from the endogenous ER staining with HPA051569 in PMID:31477691.
All five plasma-membrane rows retain KEEP_AS_NON_CORE. Their support now points to the actual positive surface observations; ER localization remains contextual evidence and supports the ER core. Neither tagged surface access nor HPA staining establishes native plasma-membrane predominance or surface lipid scrambling. The historical Reactome and electronic-transfer source records remain intact; independent human support does not reconstruct their original source chains.
The precise PAINT placement remains unresolved. No exact local IBD/tree data resolve PTN000267334 or PTN002642655. The official current PAINT directory lists IBD.gaf, but web retrieval was inaccessible and one bounded direct attempt failed DNS with zero bytes. The project owner independently encountered the same access limit. No ancestral branch, loss or divergence is inferred from different node identifiers, donor counts or self-inclusion. The annotation-review instructions treat ordinary relationship qualifiers as inert in this review; original qualifiers remain unchanged. This is an explicit scope limitation, not a finding that the curated ancestral judgment is wrong.
The three previously empty findings sections now summarize the already inspected negative-quenching, engineered-targeting and expression-background experiments, with their respective read scopes. All 16 original assertions, NOT, five products, prior NEW scramblase row, all actions and the compact core are preserved. Earlier immutable history records retain their original filenames; this follow-up receives a newly scaffolded codex record. Validation and final independent consultation are recorded there.