{"gene":"PIRT","run_date":"2026-04-28T19:45:44","timeline":{"discoveries":[{"year":2008,"finding":"PIRT functions as a regulatory subunit of TRPV1: the C-terminus of PIRT directly binds TRPV1 and phosphoinositides including PIP2 (via a cluster of basic residues), and PIP2-dependent enhancement of TRPV1 activity requires PIRT. Pirt null mice show impaired noxious heat and capsaicin responsiveness, and heterologous expression of PIRT strongly enhances TRPV1-mediated currents.","method":"Co-immunoprecipitation/pulldown (PIRT C-terminus binds TRPV1 and PIP2), whole-cell electrophysiology in heterologous cells and DRG neurons, Pirt knockout mice with behavioral and electrophysiological phenotyping, mutagenesis of basic residues in C-terminus","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (binding assay, mutagenesis, electrophysiology, KO mice), foundational paper with 178 citations","pmids":["18455988"],"is_preprint":false},{"year":2013,"finding":"PIRT is an endogenous positive regulator of TRPM8: Pirt-/- mice show decreased behavioral responses to cold/cool temperatures, and PIRT increases TRPM8 sensitivity to menthol and cool temperature in heterologous expression systems.","method":"Pirt knockout mouse behavioral assays (cold plate, acetone evaporation), whole-cell electrophysiology in heterologous cells co-expressing PIRT and TRPM8","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — clean KO with defined behavioral phenotype plus electrophysiological validation in heterologous system","pmids":["23863968"],"is_preprint":false},{"year":2015,"finding":"PIRT negatively regulates P2X3 receptor activity in bladder DRG neurons through a direct interaction mediated by the N-terminal 14 amino acid residues of PIRT; PIRT deficiency causes bladder overactivity, and a TAT-conjugated Pirt(N14) peptide is sufficient to inhibit P2X3 activation and alleviate bladder overactivity.","method":"Co-localization (immunofluorescence), co-immunoprecipitation (PIRT with P2X3), whole-cell electrophysiology in DRG neurons and heterologous cells, Pirt-/- mouse bladder function assays, TAT-peptide rescue experiment","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 — reciprocal Co-IP, domain mapping with peptide rescue, KO phenotype, electrophysiology","pmids":["26151598"],"is_preprint":false},{"year":2015,"finding":"PIRT and PIP2 synergistically enhance TRPM8 channel activity; the mechanism involves PIRT increasing the single-channel conductance of TRPM8 as shown by cell-attached single-channel recordings.","method":"Whole-cell patch-clamp electrophysiology with intracellular PIP2 application, cell-attached single-channel recordings in CHO cells transfected with TRPM8 ± PIRT","journal":"Acta pharmacologica Sinica","confidence":"Medium","confidence_rationale":"Tier 1 — single-channel electrophysiology with direct mechanistic readout, single lab","pmids":["26657057"],"is_preprint":false},{"year":2011,"finding":"PIRT is required for both histamine-dependent and -independent itch, including forms of itch that are TRPV1-independent, demonstrating that PIRT's function extends beyond TRPV1 modulation to multiple itch signaling pathways.","method":"Pirt-/- mouse behavioral assays with multiple pruritogens, DRG neuron calcium imaging","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — KO mice with defined behavioral and cellular phenotypes across multiple pruritogens","pmids":["21655234"],"is_preprint":false},{"year":2018,"finding":"Human PIRT attenuates human TRPM8 conductance (opposite to mouse PIRT which enhances mouse TRPM8), and PIRT binds directly and specifically to the TRPM8 S1-S4 transmembrane domain with approximately 1:1 stoichiometry. This species-specific difference maps to the pore domain of TRPM8.","method":"Comparative electrophysiology (human vs mouse TRPM8 ± PIRT in heterologous cells), chimeric TRPM8 channels, quantitative Western blot for surface trafficking, NMR spectroscopy and pulldown assay (recombinant purified human TRPM8 S1-S4 domain and full-length human PIRT)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — NMR binding with pulldown confirmation, chimeric channel electrophysiology, multiple orthogonal methods in single study","pmids":["29724821"],"is_preprint":false},{"year":2019,"finding":"PIRT, TRPM8, and PIP2 form a regulatory complex in which PIRT competes with TRPM8 for PIP2 binding; PIRT modulation of TRPM8 arises at least in part by regulating local concentrations of PIP2 accessible to TRPM8. NMR backbone assignments of full-length human PIRT were obtained, and competitive interactions between PIRT and TRPM8 S1-S4 domain for PIP2 were demonstrated.","method":"Solution NMR spectroscopy (backbone resonance assignment of full-length human PIRT, binding studies), microscale thermophoresis (MST) binding assays, computational PIP2 docking to TRPM8 model","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 — NMR structural characterization plus MST quantitative binding, competitive interaction mechanistically defined","pmids":["31575973"],"is_preprint":false},{"year":2020,"finding":"PIRT binds calmodulin at its C-terminal α-helix, and also contains a cholesterol-recognition amino acid consensus (CRAC) domain in its first transmembrane helix through which it specifically binds cholesterol derivatives, cholecalciferol, and oxytocin, suggesting broader ligand-binding capacity beyond TRP channels and PIP2.","method":"Microscale thermophoresis (MST), pulldown assay, NMR-detected binding, Rosetta-based computational modeling","journal":"Biomolecules","confidence":"Medium","confidence_rationale":"Tier 2–3 — multiple methods (MST, pulldown, NMR) from single lab, novel binding partners","pmids":["32245175"],"is_preprint":false},{"year":2016,"finding":"PIRT co-localizes with P2X2 receptors in neurons of the mouse enteric nervous system (myenteric and submucosal plexuses), and co-immunoprecipitation shows PIRT and P2X2 are in the same complex, suggesting PIRT may regulate P2X2 receptor function in the gut.","method":"Immunofluorescence co-localization, co-immunoprecipitation","journal":"Purinergic signalling","confidence":"Low","confidence_rationale":"Tier 3 — single Co-IP and co-localization without functional electrophysiology validation","pmids":["27105971"],"is_preprint":false},{"year":2018,"finding":"PIRT together with TRPV1 contributes to neuropathic pain (CCI model): Pirt-/- mice show reduced mechanical allodynia and thermal hyperalgesia, and loss of both Pirt and TRPV1 produces greater pain attenuation than loss of either alone, placing PIRT in the TRPV1 signaling pathway for neuropathic pain.","method":"Pirt knockout and double knockdown (Pirt/TRPV1) mouse CCI model, behavioral assays, DRG neuron calcium imaging, immunofluorescence","journal":"Neural plasticity","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with defined behavioral phenotype and cellular readout","pmids":["29808083"],"is_preprint":false}],"current_model":"PIRT is a two-transmembrane domain membrane protein expressed in peripheral sensory neurons that functions as a regulatory subunit of multiple TRP channels and purinergic receptors: it directly binds TRPV1 (via its C-terminus) and PIP2 (via basic residues in its C-terminus) to positively regulate TRPV1 activity in nociception and itch; it positively modulates mouse TRPM8 (but attenuates human TRPM8) by binding the S1-S4 domain in competition with PIP2, thereby regulating cold sensing; it inhibits P2X3 receptor activity through its N-terminal 14 residues to suppress bladder overactivity; and it additionally binds calmodulin and cholesterol derivatives, suggesting broad modulatory roles in sensory neuron physiology."},"narrative":{"teleology":[{"year":2008,"claim":"The discovery that PIRT directly binds TRPV1 and PIP2 via its C-terminus and is required for normal noxious heat and capsaicin sensitivity established PIRT as a regulatory subunit of TRPV1 and a critical component of nociceptive signaling.","evidence":"Co-immunoprecipitation, mutagenesis of C-terminal basic residues, whole-cell electrophysiology, and Pirt knockout mice with behavioral phenotyping","pmids":["18455988"],"confidence":"High","gaps":["Stoichiometry of the PIRT–TRPV1 complex not determined","Whether PIRT modulates TRPV1 trafficking versus gating not resolved","Structural basis of the PIRT C-terminus–TRPV1 interaction unknown"]},{"year":2011,"claim":"Demonstrating that Pirt-null mice have defective itch responses to both histamine-dependent and histamine-independent pruritogens—including TRPV1-independent itch—revealed that PIRT's sensory modulatory role extends beyond TRPV1 to additional itch pathways.","evidence":"Pirt knockout mouse behavioral assays with multiple pruritogens and DRG neuron calcium imaging","pmids":["21655234"],"confidence":"Medium","gaps":["The specific channel or receptor targets mediating PIRT-dependent itch signaling beyond TRPV1 were not identified","Mechanism by which PIRT modulates TRPV1-independent itch unknown"]},{"year":2013,"claim":"Identifying PIRT as a positive regulator of TRPM8 that is required for normal cold/cool behavioral responses expanded PIRT's role from nociception to thermosensation.","evidence":"Pirt knockout mouse behavioral assays (cold plate, acetone) and heterologous co-expression electrophysiology with TRPM8","pmids":["23863968"],"confidence":"High","gaps":["Binding interface between PIRT and TRPM8 not yet mapped","Whether PIP2 mediates PIRT's effect on TRPM8 not tested"]},{"year":2015,"claim":"Mapping the N-terminal 14 residues of PIRT as sufficient for P2X3 receptor inhibition and showing that PIRT deficiency causes bladder overactivity demonstrated a distinct, non-TRP channel regulatory function with therapeutic relevance.","evidence":"Co-immunoprecipitation, domain-mapping electrophysiology, Pirt knockout bladder function assays, TAT-peptide rescue in vivo","pmids":["26151598"],"confidence":"High","gaps":["Structural basis of PIRT N-terminus–P2X3 interaction unknown","Whether PIRT modulates other P2X family members not tested"]},{"year":2015,"claim":"Single-channel recordings showing PIRT increases TRPM8 single-channel conductance, synergistically with PIP2, provided a biophysical mechanism for PIRT's enhancement of TRPM8.","evidence":"Cell-attached single-channel recordings in CHO cells co-expressing TRPM8 and PIRT, with intracellular PIP2 application","pmids":["26657057"],"confidence":"Medium","gaps":["Whether PIRT changes TRPM8 open probability versus conductance not fully dissected","Mechanism by which PIRT alters conductance at the structural level unknown"]},{"year":2018,"claim":"Discovering that human PIRT attenuates human TRPM8 (opposite to mouse) and that the species difference maps to the TRPM8 pore domain revealed a species-specific regulatory mechanism; NMR and pulldown demonstrated ~1:1 stoichiometric binding of PIRT to the TRPM8 S1-S4 domain.","evidence":"Comparative electrophysiology with chimeric TRPM8 channels, NMR spectroscopy and pulldown with purified proteins, quantitative Western blot for surface expression","pmids":["29724821"],"confidence":"High","gaps":["Atomic-resolution structure of PIRT–TRPM8 complex not available","Physiological consequence of species-specific modulation in vivo not tested"]},{"year":2018,"claim":"Epistasis experiments in Pirt/TRPV1 double-knockout mice showed additive pain attenuation in a neuropathic pain model, formally placing PIRT within but not exclusively through the TRPV1 pathway in chronic pain.","evidence":"Chronic constriction injury model in Pirt knockout and Pirt/TRPV1 double-knockdown mice, behavioral and calcium imaging assays","pmids":["29808083"],"confidence":"Medium","gaps":["Additional PIRT targets contributing to neuropathic pain not identified","Mechanism of PIRT upregulation or sensitization after nerve injury unknown"]},{"year":2019,"claim":"NMR backbone assignments of full-length human PIRT and competitive binding assays established that PIRT and TRPM8 compete for PIP2, providing a mechanistic model in which PIRT modulates TRPM8 by controlling local PIP2 availability.","evidence":"Solution NMR spectroscopy (backbone assignment), microscale thermophoresis binding assays, computational PIP2 docking","pmids":["31575973"],"confidence":"High","gaps":["Full three-dimensional structure of PIRT not resolved","In vivo evidence for PIP2 competition model lacking"]},{"year":2020,"claim":"Identification of calmodulin binding at the PIRT C-terminal helix and cholesterol/cholecalciferol binding at a CRAC motif in the first transmembrane helix broadened PIRT's known ligand repertoire beyond PIP2 and channel partners.","evidence":"Microscale thermophoresis, pulldown, NMR-detected binding, Rosetta modeling","pmids":["32245175"],"confidence":"Medium","gaps":["Functional consequence of calmodulin and cholesterol binding on channel modulation not tested","Findings from a single lab without independent replication","Whether oxytocin binding is physiologically relevant is untested"]},{"year":null,"claim":"A high-resolution structure of PIRT alone or in complex with its channel partners is lacking, the full set of ion channels regulated by PIRT remains undefined, and the in vivo relevance of PIRT's calmodulin and cholesterol binding is unknown.","evidence":"","pmids":[],"confidence":"High","gaps":["No high-resolution structure of PIRT or PIRT–channel complex","Full repertoire of PIRT-regulated channels not mapped","In vivo relevance of calmodulin and cholesterol binding untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,2,5]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0,6,7]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,2,5]}],"pathway":[{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[0,1,2,9]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,5]}],"complexes":[],"partners":["TRPV1","TRPM8","P2X3","PIP2","CALMODULIN"],"other_free_text":[]},"mechanistic_narrative":"PIRT is a two-transmembrane domain membrane protein expressed in peripheral sensory neurons that functions as a broad modulatory subunit of ion channels and purinergic receptors to regulate nociception, thermosensation, and itch. Its C-terminus directly binds both TRPV1 and PIP2 via a cluster of basic residues, and PIP2-dependent enhancement of TRPV1 activity requires PIRT, as demonstrated by impaired noxious heat and capsaicin responses in Pirt-null mice [PMID:18455988]. PIRT also modulates TRPM8 by binding the S1-S4 transmembrane domain and competing with TRPM8 for PIP2, with species-specific effects—enhancing mouse TRPM8 but attenuating human TRPM8—mapped to the pore domain [PMID:29724821, PMID:31575973]. Through its N-terminal 14 residues, PIRT inhibits P2X3 receptor activity to suppress bladder overactivity, and it additionally binds calmodulin and cholesterol derivatives, indicating ligand-binding functions beyond TRP channel regulation [PMID:26151598, PMID:32245175]."},"prefetch_data":{"uniprot":{"accession":"P0C851","full_name":"Phosphoinositide-interacting protein","aliases":[],"length_aa":137,"mass_kda":15.3,"function":"Regulatory subunit of TRPV1, a molecular sensor of noxious heat and capsaicin. Positively regulates TRPV1 channel activity via phosphatidylinositol 4,5-bisphosphate (PIP2). Binds various phosphoinositide, including phosphatidylinositol 4,5-bisphosphate (PIP2), but not phosphatidylinositol (PI) (By similarity)","subcellular_location":"Membrane","url":"https://www.uniprot.org/uniprotkb/P0C851/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PIRT","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PIRT","total_profiled":1310},"omim":[{"mim_id":"612068","title":"PHOSPHOINOSITIDE-INTERACTING REGULATOR OF TRANSIENT RECEPTOR POTENTIAL CHANNELS; PIRT","url":"https://www.omim.org/entry/612068"},{"mim_id":"602076","title":"TRANSIENT RECEPTOR POTENTIAL CATION CHANNEL, SUBFAMILY V, MEMBER 1; TRPV1","url":"https://www.omim.org/entry/602076"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Cytosol","reliability":"Uncertain"},{"location":"Plasma membrane","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"brain","ntpm":11.9},{"tissue":"intestine","ntpm":4.1}],"url":"https://www.proteinatlas.org/search/PIRT"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P0C851","domains":[{"cath_id":"1.10.287","chopping":"49-118","consensus_level":"high","plddt":75.5446,"start":49,"end":118}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P0C851","model_url":"https://alphafold.ebi.ac.uk/files/AF-P0C851-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P0C851-F1-predicted_aligned_error_v6.png","plddt_mean":63.97},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PIRT","jax_strain_url":"https://www.jax.org/strain/search?query=PIRT"},"sequence":{"accession":"P0C851","fasta_url":"https://rest.uniprot.org/uniprotkb/P0C851.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P0C851/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P0C851"}},"corpus_meta":[{"pmid":"18455988","id":"PMC_18455988","title":"Pirt, a phosphoinositide-binding protein, functions as a regulatory subunit of TRPV1.","date":"2008","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/18455988","citation_count":178,"is_preprint":false},{"pmid":"32060036","id":"PMC_32060036","title":"Mapping of Sensory Nerve Subsets within the Vagal Ganglia and the Brainstem Using Reporter Mice for Pirt, TRPV1, 5-HT3, and Tac1 Expression.","date":"2020","source":"eNeuro","url":"https://pubmed.ncbi.nlm.nih.gov/32060036","citation_count":67,"is_preprint":false},{"pmid":"21655234","id":"PMC_21655234","title":"Pirt, a TRPV1 modulator, is required for histamine-dependent and -independent itch.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21655234","citation_count":46,"is_preprint":false},{"pmid":"23863968","id":"PMC_23863968","title":"Pirt functions as an endogenous regulator of TRPM8.","date":"2013","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/23863968","citation_count":46,"is_preprint":false},{"pmid":"36264609","id":"PMC_36264609","title":"scRNA-sequencing reveals subtype-specific transcriptomic perturbations in DRG neurons of Pirt mice in neuropathic pain condition.","date":"2022","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/36264609","citation_count":43,"is_preprint":false},{"pmid":"29808083","id":"PMC_29808083","title":"Pirt Together with TRPV1 Is Involved in the Regulation of Neuropathic Pain.","date":"2018","source":"Neural plasticity","url":"https://pubmed.ncbi.nlm.nih.gov/29808083","citation_count":26,"is_preprint":false},{"pmid":"26151598","id":"PMC_26151598","title":"Pirt reduces bladder overactivity by inhibiting purinergic receptor P2X3.","date":"2015","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/26151598","citation_count":20,"is_preprint":false},{"pmid":"26376721","id":"PMC_26376721","title":"Pirt contributes to uterine contraction-induced pain in mice.","date":"2015","source":"Molecular pain","url":"https://pubmed.ncbi.nlm.nih.gov/26376721","citation_count":18,"is_preprint":false},{"pmid":"29724821","id":"PMC_29724821","title":"Phosphoinositide-interacting regulator of TRP (PIRT) has opposing effects on human and mouse TRPM8 ion channels.","date":"2018","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29724821","citation_count":12,"is_preprint":false},{"pmid":"26657057","id":"PMC_26657057","title":"Phosphoinositide interacting regulator of TRP (Pirt) enhances TRPM8 channel activity in vitro via increasing channel conductance.","date":"2015","source":"Acta pharmacologica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/26657057","citation_count":10,"is_preprint":false},{"pmid":"33806699","id":"PMC_33806699","title":"Ca2+ Signalling Induced by NGF Identifies a Subset of Capsaicin-Excitable Neurons Displaying Enhanced Chemo-Nociception in Dorsal Root Ganglion Explants from Adult pirt-GCaMP3 Mouse.","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33806699","citation_count":9,"is_preprint":false},{"pmid":"27105971","id":"PMC_27105971","title":"Co-localization of Pirt protein and P2X2 receptors in the mouse enteric nervous system.","date":"2016","source":"Purinergic signalling","url":"https://pubmed.ncbi.nlm.nih.gov/27105971","citation_count":8,"is_preprint":false},{"pmid":"31575973","id":"PMC_31575973","title":"Competitive Interactions between PIRT, the Cold Sensing Ion Channel TRPM8, and PIP2 Suggest a Mechanism for Regulation.","date":"2019","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/31575973","citation_count":8,"is_preprint":false},{"pmid":"33931991","id":"PMC_33931991","title":"[Involvement of Pirt /TRPV1 signaling in acupuncture-induced reduction of visceral hypersensitivity in diarrhea-predominant irritable bowel syndrome rats].","date":"2021","source":"Zhen ci yan jiu = Acupuncture research","url":"https://pubmed.ncbi.nlm.nih.gov/33931991","citation_count":8,"is_preprint":false},{"pmid":"37303829","id":"PMC_37303829","title":"piRT-IFC: Physics-informed real-time impedance flow cytometry for the characterization of cellular intrinsic electrical properties.","date":"2023","source":"Microsystems & nanoengineering","url":"https://pubmed.ncbi.nlm.nih.gov/37303829","citation_count":8,"is_preprint":false},{"pmid":"32245175","id":"PMC_32245175","title":"PIRT the TRP Channel Regulating Protein Binds Calmodulin and Cholesterol-Like Ligands.","date":"2020","source":"Biomolecules","url":"https://pubmed.ncbi.nlm.nih.gov/32245175","citation_count":5,"is_preprint":false},{"pmid":"34242501","id":"PMC_34242501","title":"Population Coding of Capsaicin Concentration by Sensory Neurons Revealed Using Ca2+ Imaging of Dorsal Root Ganglia Explants from Adult pirt-GCaMP3 Mouse.","date":"2021","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/34242501","citation_count":5,"is_preprint":false},{"pmid":"30902502","id":"PMC_30902502","title":"Pirt deficiency has subtle female-specific effects on energy and glucose metabolism in mice.","date":"2019","source":"Molecular metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/30902502","citation_count":4,"is_preprint":false},{"pmid":"40938705","id":"PMC_40938705","title":"In vivo Pirt-Marina voltage sensor imaging detects primary sensory neuron-specific voltage dynamics and neuronal plasticity changes.","date":"2025","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/40938705","citation_count":0,"is_preprint":false},{"pmid":"40808296","id":"PMC_40808296","title":"PIRT-Seq: a high-resolution whole-genome assay to identify protein-coding genes.","date":"2025","source":"Nucleic acids 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standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2008,\n      \"finding\": \"PIRT functions as a regulatory subunit of TRPV1: the C-terminus of PIRT directly binds TRPV1 and phosphoinositides including PIP2 (via a cluster of basic residues), and PIP2-dependent enhancement of TRPV1 activity requires PIRT. Pirt null mice show impaired noxious heat and capsaicin responsiveness, and heterologous expression of PIRT strongly enhances TRPV1-mediated currents.\",\n      \"method\": \"Co-immunoprecipitation/pulldown (PIRT C-terminus binds TRPV1 and PIP2), whole-cell electrophysiology in heterologous cells and DRG neurons, Pirt knockout mice with behavioral and electrophysiological phenotyping, mutagenesis of basic residues in C-terminus\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (binding assay, mutagenesis, electrophysiology, KO mice), foundational paper with 178 citations\",\n      \"pmids\": [\"18455988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PIRT is an endogenous positive regulator of TRPM8: Pirt-/- mice show decreased behavioral responses to cold/cool temperatures, and PIRT increases TRPM8 sensitivity to menthol and cool temperature in heterologous expression systems.\",\n      \"method\": \"Pirt knockout mouse behavioral assays (cold plate, acetone evaporation), whole-cell electrophysiology in heterologous cells co-expressing PIRT and TRPM8\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined behavioral phenotype plus electrophysiological validation in heterologous system\",\n      \"pmids\": [\"23863968\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PIRT negatively regulates P2X3 receptor activity in bladder DRG neurons through a direct interaction mediated by the N-terminal 14 amino acid residues of PIRT; PIRT deficiency causes bladder overactivity, and a TAT-conjugated Pirt(N14) peptide is sufficient to inhibit P2X3 activation and alleviate bladder overactivity.\",\n      \"method\": \"Co-localization (immunofluorescence), co-immunoprecipitation (PIRT with P2X3), whole-cell electrophysiology in DRG neurons and heterologous cells, Pirt-/- mouse bladder function assays, TAT-peptide rescue experiment\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — reciprocal Co-IP, domain mapping with peptide rescue, KO phenotype, electrophysiology\",\n      \"pmids\": [\"26151598\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PIRT and PIP2 synergistically enhance TRPM8 channel activity; the mechanism involves PIRT increasing the single-channel conductance of TRPM8 as shown by cell-attached single-channel recordings.\",\n      \"method\": \"Whole-cell patch-clamp electrophysiology with intracellular PIP2 application, cell-attached single-channel recordings in CHO cells transfected with TRPM8 ± PIRT\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — single-channel electrophysiology with direct mechanistic readout, single lab\",\n      \"pmids\": [\"26657057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PIRT is required for both histamine-dependent and -independent itch, including forms of itch that are TRPV1-independent, demonstrating that PIRT's function extends beyond TRPV1 modulation to multiple itch signaling pathways.\",\n      \"method\": \"Pirt-/- mouse behavioral assays with multiple pruritogens, DRG neuron calcium imaging\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mice with defined behavioral and cellular phenotypes across multiple pruritogens\",\n      \"pmids\": [\"21655234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Human PIRT attenuates human TRPM8 conductance (opposite to mouse PIRT which enhances mouse TRPM8), and PIRT binds directly and specifically to the TRPM8 S1-S4 transmembrane domain with approximately 1:1 stoichiometry. This species-specific difference maps to the pore domain of TRPM8.\",\n      \"method\": \"Comparative electrophysiology (human vs mouse TRPM8 ± PIRT in heterologous cells), chimeric TRPM8 channels, quantitative Western blot for surface trafficking, NMR spectroscopy and pulldown assay (recombinant purified human TRPM8 S1-S4 domain and full-length human PIRT)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR binding with pulldown confirmation, chimeric channel electrophysiology, multiple orthogonal methods in single study\",\n      \"pmids\": [\"29724821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PIRT, TRPM8, and PIP2 form a regulatory complex in which PIRT competes with TRPM8 for PIP2 binding; PIRT modulation of TRPM8 arises at least in part by regulating local concentrations of PIP2 accessible to TRPM8. NMR backbone assignments of full-length human PIRT were obtained, and competitive interactions between PIRT and TRPM8 S1-S4 domain for PIP2 were demonstrated.\",\n      \"method\": \"Solution NMR spectroscopy (backbone resonance assignment of full-length human PIRT, binding studies), microscale thermophoresis (MST) binding assays, computational PIP2 docking to TRPM8 model\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structural characterization plus MST quantitative binding, competitive interaction mechanistically defined\",\n      \"pmids\": [\"31575973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PIRT binds calmodulin at its C-terminal α-helix, and also contains a cholesterol-recognition amino acid consensus (CRAC) domain in its first transmembrane helix through which it specifically binds cholesterol derivatives, cholecalciferol, and oxytocin, suggesting broader ligand-binding capacity beyond TRP channels and PIP2.\",\n      \"method\": \"Microscale thermophoresis (MST), pulldown assay, NMR-detected binding, Rosetta-based computational modeling\",\n      \"journal\": \"Biomolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — multiple methods (MST, pulldown, NMR) from single lab, novel binding partners\",\n      \"pmids\": [\"32245175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PIRT co-localizes with P2X2 receptors in neurons of the mouse enteric nervous system (myenteric and submucosal plexuses), and co-immunoprecipitation shows PIRT and P2X2 are in the same complex, suggesting PIRT may regulate P2X2 receptor function in the gut.\",\n      \"method\": \"Immunofluorescence co-localization, co-immunoprecipitation\",\n      \"journal\": \"Purinergic signalling\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP and co-localization without functional electrophysiology validation\",\n      \"pmids\": [\"27105971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PIRT together with TRPV1 contributes to neuropathic pain (CCI model): Pirt-/- mice show reduced mechanical allodynia and thermal hyperalgesia, and loss of both Pirt and TRPV1 produces greater pain attenuation than loss of either alone, placing PIRT in the TRPV1 signaling pathway for neuropathic pain.\",\n      \"method\": \"Pirt knockout and double knockdown (Pirt/TRPV1) mouse CCI model, behavioral assays, DRG neuron calcium imaging, immunofluorescence\",\n      \"journal\": \"Neural plasticity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with defined behavioral phenotype and cellular readout\",\n      \"pmids\": [\"29808083\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PIRT is a two-transmembrane domain membrane protein expressed in peripheral sensory neurons that functions as a regulatory subunit of multiple TRP channels and purinergic receptors: it directly binds TRPV1 (via its C-terminus) and PIP2 (via basic residues in its C-terminus) to positively regulate TRPV1 activity in nociception and itch; it positively modulates mouse TRPM8 (but attenuates human TRPM8) by binding the S1-S4 domain in competition with PIP2, thereby regulating cold sensing; it inhibits P2X3 receptor activity through its N-terminal 14 residues to suppress bladder overactivity; and it additionally binds calmodulin and cholesterol derivatives, suggesting broad modulatory roles in sensory neuron physiology.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PIRT is a two-transmembrane domain membrane protein expressed in peripheral sensory neurons that functions as a broad modulatory subunit of ion channels and purinergic receptors to regulate nociception, thermosensation, and itch. Its C-terminus directly binds both TRPV1 and PIP2 via a cluster of basic residues, and PIP2-dependent enhancement of TRPV1 activity requires PIRT, as demonstrated by impaired noxious heat and capsaicin responses in Pirt-null mice [PMID:18455988]. PIRT also modulates TRPM8 by binding the S1-S4 transmembrane domain and competing with TRPM8 for PIP2, with species-specific effects—enhancing mouse TRPM8 but attenuating human TRPM8—mapped to the pore domain [PMID:29724821, PMID:31575973]. Through its N-terminal 14 residues, PIRT inhibits P2X3 receptor activity to suppress bladder overactivity, and it additionally binds calmodulin and cholesterol derivatives, indicating ligand-binding functions beyond TRP channel regulation [PMID:26151598, PMID:32245175].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"The discovery that PIRT directly binds TRPV1 and PIP2 via its C-terminus and is required for normal noxious heat and capsaicin sensitivity established PIRT as a regulatory subunit of TRPV1 and a critical component of nociceptive signaling.\",\n      \"evidence\": \"Co-immunoprecipitation, mutagenesis of C-terminal basic residues, whole-cell electrophysiology, and Pirt knockout mice with behavioral phenotyping\",\n      \"pmids\": [\"18455988\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the PIRT–TRPV1 complex not determined\", \"Whether PIRT modulates TRPV1 trafficking versus gating not resolved\", \"Structural basis of the PIRT C-terminus–TRPV1 interaction unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrating that Pirt-null mice have defective itch responses to both histamine-dependent and histamine-independent pruritogens—including TRPV1-independent itch—revealed that PIRT's sensory modulatory role extends beyond TRPV1 to additional itch pathways.\",\n      \"evidence\": \"Pirt knockout mouse behavioral assays with multiple pruritogens and DRG neuron calcium imaging\",\n      \"pmids\": [\"21655234\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The specific channel or receptor targets mediating PIRT-dependent itch signaling beyond TRPV1 were not identified\", \"Mechanism by which PIRT modulates TRPV1-independent itch unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identifying PIRT as a positive regulator of TRPM8 that is required for normal cold/cool behavioral responses expanded PIRT's role from nociception to thermosensation.\",\n      \"evidence\": \"Pirt knockout mouse behavioral assays (cold plate, acetone) and heterologous co-expression electrophysiology with TRPM8\",\n      \"pmids\": [\"23863968\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding interface between PIRT and TRPM8 not yet mapped\", \"Whether PIP2 mediates PIRT's effect on TRPM8 not tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Mapping the N-terminal 14 residues of PIRT as sufficient for P2X3 receptor inhibition and showing that PIRT deficiency causes bladder overactivity demonstrated a distinct, non-TRP channel regulatory function with therapeutic relevance.\",\n      \"evidence\": \"Co-immunoprecipitation, domain-mapping electrophysiology, Pirt knockout bladder function assays, TAT-peptide rescue in vivo\",\n      \"pmids\": [\"26151598\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of PIRT N-terminus–P2X3 interaction unknown\", \"Whether PIRT modulates other P2X family members not tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Single-channel recordings showing PIRT increases TRPM8 single-channel conductance, synergistically with PIP2, provided a biophysical mechanism for PIRT's enhancement of TRPM8.\",\n      \"evidence\": \"Cell-attached single-channel recordings in CHO cells co-expressing TRPM8 and PIRT, with intracellular PIP2 application\",\n      \"pmids\": [\"26657057\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PIRT changes TRPM8 open probability versus conductance not fully dissected\", \"Mechanism by which PIRT alters conductance at the structural level unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Discovering that human PIRT attenuates human TRPM8 (opposite to mouse) and that the species difference maps to the TRPM8 pore domain revealed a species-specific regulatory mechanism; NMR and pulldown demonstrated ~1:1 stoichiometric binding of PIRT to the TRPM8 S1-S4 domain.\",\n      \"evidence\": \"Comparative electrophysiology with chimeric TRPM8 channels, NMR spectroscopy and pulldown with purified proteins, quantitative Western blot for surface expression\",\n      \"pmids\": [\"29724821\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structure of PIRT–TRPM8 complex not available\", \"Physiological consequence of species-specific modulation in vivo not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Epistasis experiments in Pirt/TRPV1 double-knockout mice showed additive pain attenuation in a neuropathic pain model, formally placing PIRT within but not exclusively through the TRPV1 pathway in chronic pain.\",\n      \"evidence\": \"Chronic constriction injury model in Pirt knockout and Pirt/TRPV1 double-knockdown mice, behavioral and calcium imaging assays\",\n      \"pmids\": [\"29808083\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Additional PIRT targets contributing to neuropathic pain not identified\", \"Mechanism of PIRT upregulation or sensitization after nerve injury unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"NMR backbone assignments of full-length human PIRT and competitive binding assays established that PIRT and TRPM8 compete for PIP2, providing a mechanistic model in which PIRT modulates TRPM8 by controlling local PIP2 availability.\",\n      \"evidence\": \"Solution NMR spectroscopy (backbone assignment), microscale thermophoresis binding assays, computational PIP2 docking\",\n      \"pmids\": [\"31575973\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full three-dimensional structure of PIRT not resolved\", \"In vivo evidence for PIP2 competition model lacking\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identification of calmodulin binding at the PIRT C-terminal helix and cholesterol/cholecalciferol binding at a CRAC motif in the first transmembrane helix broadened PIRT's known ligand repertoire beyond PIP2 and channel partners.\",\n      \"evidence\": \"Microscale thermophoresis, pulldown, NMR-detected binding, Rosetta modeling\",\n      \"pmids\": [\"32245175\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of calmodulin and cholesterol binding on channel modulation not tested\", \"Findings from a single lab without independent replication\", \"Whether oxytocin binding is physiologically relevant is untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A high-resolution structure of PIRT alone or in complex with its channel partners is lacking, the full set of ion channels regulated by PIRT remains undefined, and the in vivo relevance of PIRT's calmodulin and cholesterol binding is unknown.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structure of PIRT or PIRT–channel complex\", \"Full repertoire of PIRT-regulated channels not mapped\", \"In vivo relevance of calmodulin and cholesterol binding untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 2, 5]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 6, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 2, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0050955\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [0, 1, 2, 9]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"TRPV1\", \"TRPM8\", \"P2X3\", \"PIP2\", \"calmodulin\"],\n    \"other_free_text\": []\n  }\n}\n```"}