{"gene":"CNNM4","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":2009,"finding":"CNNM4 (encoding a putative metal transporter) was identified as the causative gene for Jalili syndrome; expression was confirmed in neural retina and ameloblasts of developing teeth, establishing its role in metal transport linked to visual function and biomineralization.","method":"Positional-candidate gene sequencing in seven families; expression confirmed in retina and ameloblasts","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 — replicated across two independent groups in the same year with multiple families and expression data","pmids":["19200525","19200527"],"is_preprint":false},{"year":2014,"finding":"CNNM4 functions as a Mg2+ efflux transporter at the plasma membrane; the oncogenic phosphatase PRL binds CNNM4 and inhibits CNNM4-dependent Mg2+ efflux, thereby elevating intracellular Mg2+ and activating AMPK/mTOR energy-metabolism signaling to promote tumor progression.","method":"Biochemical binding assay (co-immunoprecipitation of PRL and CNNM4), cellular Mg2+ flux measurements, Cnnm4-knockout mouse model (ApcΔ14/+ background), mTOR inhibitor (rapamycin) rescue","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (Co-IP, Mg2+ flux assay, genetic KO mouse, pharmacological rescue) in a single well-cited study","pmids":["25347473"],"is_preprint":false},{"year":2014,"finding":"Basolateral sorting of CNNM4 in polarized epithelial cells requires three conserved dileucine motifs in CNNM4 that mediate interaction with clathrin adaptor protein complexes AP-1A (μ1A subunit) and AP-1B (μ1B subunit); simultaneous knockdown of both μ1A and μ1B, but not either alone, disrupts basolateral localization.","method":"siRNA knockdown of μ1A and μ1B in MDCK cells, mutational analysis of dileucine motifs, co-immunoprecipitation with μ1A/μ1B","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP plus mutagenesis plus KD with defined localization phenotype in same study","pmids":["25449265"],"is_preprint":false},{"year":2016,"finding":"CNNM4 is required for Ca2+ influx during sperm capacitation; Cnnm4-deficient sperm show abrogated hyperactivation and perturbed Ca2+ influx phenotypically similar to CatSper-null sperm, and forced Ca2+ entry into Cnnm4-deficient sperm normalizes tyrosine phosphorylation levels.","method":"Cnnm4 germline and germ-cell-specific knockout mice, Ca2+ imaging in sperm, sperm motility analysis, tyrosine phosphorylation western blot, forced Ca2+ entry rescue","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with Ca2+ imaging, functional rescue, and two independent KO strategies","pmids":["27006114"],"is_preprint":false},{"year":2011,"finding":"The CBS-pair regulatory domain of human CNNM4 was purified and crystallized, yielding diffracting crystals in orthorhombic space group C222 with two molecules per asymmetric unit, providing structural characterization of this domain.","method":"Protein overexpression, purification, crystallization, and preliminary X-ray crystallographic analysis (synchrotron, 3.6 Å)","journal":"Acta crystallographica. Section F","confidence":"Medium","confidence_rationale":"Tier 1 — crystal structure at preliminary resolution; functional validation not yet performed in this report","pmids":["21393841"],"is_preprint":false},{"year":2005,"finding":"CNNM4 (as ACDP4) physically interacts with the intracellular metal ion chaperone COX11 in a yeast two-hybrid screen; co-expression of ACDP4 and COX11 in HEK293 cells enhanced toxicity to copper, manganese, and cobalt, suggesting functional coupling in metal ion homeostasis.","method":"Yeast two-hybrid screen of human fetal brain cDNA library; ectopic co-expression in HEK293 cells with metal ion toxicity assay","journal":"Molecular pain","confidence":"Medium","confidence_rationale":"Tier 3 — yeast two-hybrid plus cell-based toxicity assay, single lab, no direct transport assay","pmids":["15840172"],"is_preprint":false},{"year":2019,"finding":"CNNM4 deficiency in colon epithelium suppresses TRPV1-mediated Ca2+ influx and constitutively activates EGF receptor signaling, promoting cell proliferation; gefitinib (EGFR inhibitor) rescues the hyperproliferation phenotype, establishing a Mg2+–Ca2+ functional interplay in colonic homeostasis.","method":"Cnnm4-knockout mice, organoid Ca2+ imaging, EGFR phosphorylation western blot, gefitinib pharmacological rescue","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with Ca2+ imaging, defined signaling pathway, pharmacological rescue","pmids":["30670776"],"is_preprint":false},{"year":2018,"finding":"CNNM4 physically interacts with IQCB1 (a Leber congenital amaurosis gene product); a truncated CNNM4 protein (p.R605X) increases apoptosis and enhances CNNM4–IQCB1 interaction, functionally linking Jalili syndrome to LCA pathology.","method":"Co-immunoprecipitation of CNNM4 and IQCB1, apoptosis assay with truncated CNNM4 construct","journal":"Molecular genetics and genomics","confidence":"Medium","confidence_rationale":"Tier 3 — single Co-IP plus apoptosis assay, single lab","pmids":["29322253"],"is_preprint":false},{"year":2019,"finding":"Missense variants in the CBS domain of CNNM4 (p.Gly492Cys and p.Gly492Asp) disrupt ATP-binding mode within the CBS domain as shown by molecular dynamics simulations; analogous CBS-domain variants cause conformational shifts predicted to impair metal transport regulation.","method":"Molecular dynamics simulations and docking analysis of CBS-domain wild-type vs. mutant CNNM4","journal":"Molecular genetics & genomic medicine","confidence":"Low","confidence_rationale":"Tier 4 — computational prediction only, no experimental validation of ATP binding","pmids":["31347285"],"is_preprint":false},{"year":2024,"finding":"CNNM4 missense variants p.Gly492Cys and p.Gly492Asp (CBS domain) reduce protein stability and increase mRNA decay but do not mislocalize the transporter; the mutant proteins show significantly reduced Mg2+ extrusion activity, indicating that reduced stability/expression rather than mislocalization underlies pathogenicity.","method":"Mg2+ efflux assay, protein stability assay, mRNA decay measurement, localization by fluorescence microscopy in transfected cells","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1-2 — direct Mg2+ transport assay combined with protein/mRNA stability and localization data","pmids":["39580587"],"is_preprint":false},{"year":2024,"finding":"In thermogenic adipocytes, CNNM4 is induced by ADRB3-PKA-CREB signaling during cold exposure and mediates Mg2+ efflux; secreted Mg2+ then binds the DFG motif of mTOR in macrophages to activate mTORC2 and drive M2 polarization, establishing a paracrine adipocyte–macrophage Mg2+ signaling axis.","method":"ADRB3 agonist stimulation, PKA/CREB pathway inhibitors, CNNM4 overexpression/knockdown in adipocytes, mTORC2 activity assay, macrophage polarization assay, Mg2+ supplementation/chelation experiments","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 — multiple pathway inhibitors and gain/loss-of-function with defined signaling readout, single lab","pmids":["39517124"],"is_preprint":false},{"year":2024,"finding":"In alcohol-associated liver disease, CNNM4 upregulation disrupts Mg2+ homeostasis; silencing Cnnm4 via GalNAc-siRNA restores PCMT1 (protein isoaspartyl methyltransferase) activity by increasing S-adenosylmethionine levels through improved mitochondrial function, linking CNNM4-dependent Mg2+ efflux to protein damage repair.","method":"GalNAc-siRNA silencing of Cnnm4 in mice, PCMT1 activity assay, SAM level measurement, mitochondrial function assay","journal":"Hepatology","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo siRNA with biochemical activity assays, single lab","pmids":["39641635"],"is_preprint":false},{"year":2025,"finding":"microRNAs directly bind the 3'UTR of CNNM4 mRNA to regulate CNNM4 expression and thereby control cellular Mg2+ concentrations in hepatocytes; both upregulatory and downregulatory miRNA activities were detected in a high-throughput 3'UTR assay.","method":"High-throughput miRNA-3'UTR binding assay, direct miRNA:3'UTR interaction validation, cellular Mg2+ concentration measurement in hepatocytes","journal":"ACS chemical biology","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding assay plus functional cellular Mg2+ readout, but single lab and newly published","pmids":["40862638"],"is_preprint":false},{"year":2026,"finding":"In cholangiocarcinoma, CNNM4 silencing attenuates ferroptosis via a pathway involving nuclear protein 1 (NUPR1) as an upstream regulator; iron chelation with deferiprone reversed the antiproliferative effect of CNNM4 silencing, while HO-1 inhibition did not, establishing CNNM4-linked ferroptosis as a distinct cell-death mechanism.","method":"siRNA/shRNA knockdown of CNNM4 in CCA cell lines and GalNAc-siRNA in transposon-based mouse CCA model, proteomic analysis, ferroptosis rescue with deferiprone/zinc protoporphyrin IX, chicken chorioallantoic membrane invasion assay","journal":"Gut","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro and in vivo KD with proteomic pathway identification and pharmacological rescue, single lab","pmids":["40764063"],"is_preprint":false}],"current_model":"CNNM4 is a plasma membrane Mg2+ efflux transporter whose CBS domain binds ATP and interacts with the oncogenic phosphatase PRL (which inhibits Mg2+ efflux), is sorted to the basolateral membrane of polarized epithelia via dileucine motifs recognized by AP-1A/AP-1B adaptors, regulates intracellular Mg2+ to control AMPK/mTOR energy metabolism and Ca2+ homeostasis in sperm and colon epithelia, and whose loss-of-function mutations (causing reduced protein/mRNA stability or truncation) underlie Jalili syndrome by impairing Mg2+ transport in retinal photoreceptors and ameloblasts."},"narrative":{"teleology":[{"year":2005,"claim":"Before CNNM4's transport function was known, a yeast two-hybrid screen identified the metal chaperone COX11 as a physical interactor and showed that co-expression with CNNM4 sensitized cells to copper, manganese, and cobalt, providing the first evidence that CNNM4 participates in metal ion homeostasis.","evidence":"Yeast two-hybrid screen plus metal toxicity assay in HEK293 cells","pmids":["15840172"],"confidence":"Medium","gaps":["No direct metal transport assay was performed","COX11 interaction not validated by reciprocal Co-IP","Relevance to Mg²⁺ versus other divalent cations unclear"]},{"year":2009,"claim":"Positional cloning in multiple consanguineous families established CNNM4 as the causative gene for Jalili syndrome (cone-rod dystrophy with amelogenesis imperfecta), linking a putative metal transporter to both retinal and dental mineralization biology.","evidence":"Homozygosity mapping and candidate-gene sequencing in seven independent families by two groups, with expression confirmed in neural retina and ameloblasts","pmids":["19200525","19200527"],"confidence":"High","gaps":["The mechanism by which CNNM4 loss causes photoreceptor degeneration was not resolved","Which metal ion CNNM4 transports was still unknown"]},{"year":2011,"claim":"Crystallization of the CBS-pair regulatory domain provided the first structural information on CNNM4, establishing the feasibility of atomic-resolution studies of its regulatory module.","evidence":"Protein purification, crystallization, and preliminary X-ray diffraction at 3.6 Å","pmids":["21393841"],"confidence":"Medium","gaps":["Resolution too low for detailed atomic model","No ligand-bound structure obtained","Functional consequences of CBS domain conformation not tested"]},{"year":2014,"claim":"Two concurrent studies resolved the core molecular function and trafficking mechanism of CNNM4: it mediates Mg²⁺ efflux at the plasma membrane, the PRL phosphatase binds and inhibits this efflux to activate AMPK/mTOR signaling in colorectal tumors, and basolateral sorting in polarized epithelia requires three dileucine motifs recognized by AP-1A/AP-1B adaptors.","evidence":"Co-IP of PRL–CNNM4, cellular Mg²⁺ flux assays, Cnnm4-KO mice on Apc-mutant background with rapamycin rescue; dileucine mutagenesis with μ1A/μ1B siRNA in MDCK cells","pmids":["25347473","25449265"],"confidence":"High","gaps":["Direct structural basis of PRL–CNNM4 inhibition was not determined","Whether AP-1A and AP-1B are fully redundant or context-dependent remained unresolved","Whether Mg²⁺ transport is electrogenic or coupled to a counter-ion was not established"]},{"year":2016,"claim":"CNNM4 was shown to be required for Ca²⁺ influx during sperm capacitation, revealing that its Mg²⁺ efflux activity indirectly regulates Ca²⁺ signaling; Cnnm4-deficient sperm phenocopied CatSper-null sperm, and forced Ca²⁺ entry rescued tyrosine phosphorylation.","evidence":"Germline and germ-cell-specific Cnnm4 KO mice with Ca²⁺ imaging, motility analysis, and forced Ca²⁺ entry rescue","pmids":["27006114"],"confidence":"High","gaps":["Molecular mechanism linking Mg²⁺ efflux to CatSper-dependent Ca²⁺ influx was not identified","Whether CNNM4 loss contributes to human male infertility was not tested"]},{"year":2018,"claim":"A physical interaction between CNNM4 and IQCB1 (a Leber congenital amaurosis gene product) was identified, and a Jalili-associated truncation (p.R605X) enhanced this interaction and increased apoptosis, suggesting a functional bridge between the two retinal-disease pathways.","evidence":"Co-IP of CNNM4 and IQCB1, apoptosis assay with truncated construct","pmids":["29322253"],"confidence":"Medium","gaps":["Single Co-IP without reciprocal validation","Physiological relevance of enhanced interaction to retinal degeneration not demonstrated in vivo","IQCB1 interaction not confirmed by an independent group"]},{"year":2019,"claim":"In colonic epithelium, CNNM4 deficiency was shown to suppress TRPV1-mediated Ca²⁺ influx and constitutively activate EGFR signaling, directly linking CNNM4-dependent Mg²⁺ homeostasis to proliferative control; gefitinib rescue proved EGFR as the downstream effector.","evidence":"Cnnm4-KO mice, organoid Ca²⁺ imaging, EGFR phosphorylation assay, gefitinib pharmacological rescue","pmids":["30670776"],"confidence":"High","gaps":["How altered intracellular Mg²⁺ suppresses TRPV1 activity at the channel level is unknown","Whether this EGFR axis operates in non-colonic epithelia was not tested"]},{"year":2024,"claim":"Multiple studies expanded CNNM4's physiological scope: pathogenic CBS-domain missense variants were shown to reduce protein/mRNA stability (not localization) and impair Mg²⁺ extrusion, cold-induced CNNM4 expression in adipocytes established a paracrine Mg²⁺ signal that activates mTORC2 in macrophages, and hepatic CNNM4 silencing restored PCMT1 activity by improving mitochondrial SAM metabolism.","evidence":"Mg²⁺ efflux assay and stability measurements for CBS-domain variants; ADRB3-PKA-CREB pathway dissection with CNNM4 gain/loss-of-function in adipocytes and mTORC2 activity in macrophages; GalNAc-siRNA Cnnm4 silencing in alcohol-fed mice with PCMT1/SAM assays","pmids":["39580587","39517124","39641635"],"confidence":"High","gaps":["Whether CNNM4 is the sole Mg²⁺ efflux pathway in adipocytes is unclear","The DFG-motif Mg²⁺ sensor model on mTOR lacks structural validation","Hepatic CNNM4 silencing effects on Mg²⁺-dependent enzymes beyond PCMT1 are unexplored"]},{"year":2025,"claim":"Post-transcriptional regulation of CNNM4 by miRNAs was demonstrated through direct binding to its 3′UTR in hepatocytes, showing that cellular Mg²⁺ levels can be tuned at the mRNA level.","evidence":"High-throughput miRNA–3′UTR binding assay with cellular Mg²⁺ concentration measurement in hepatocytes","pmids":["40862638"],"confidence":"Medium","gaps":["Specific miRNAs that are physiologically dominant in regulating CNNM4 in vivo are not identified","Whether miRNA regulation is tissue-specific was not addressed"]},{"year":2026,"claim":"CNNM4 silencing in cholangiocarcinoma was shown to attenuate ferroptosis via a NUPR1-dependent pathway; iron chelation reversed the effect while HO-1 inhibition did not, establishing a Mg²⁺-linked ferroptosis axis distinct from canonical heme oxygenase pathways.","evidence":"siRNA/shRNA KD in CCA cell lines, GalNAc-siRNA in transposon-based mouse CCA model, proteomics, deferiprone rescue","pmids":["40764063"],"confidence":"Medium","gaps":["Mechanism connecting Mg²⁺ efflux to NUPR1 expression is not resolved","Single-lab finding awaiting independent replication","Whether this ferroptosis link generalizes beyond cholangiocarcinoma is unknown"]},{"year":null,"claim":"Key open questions include the transport mechanism (electrogenic vs. coupled), a high-resolution full-length structure, the molecular basis of PRL-mediated inhibition, and whether CNNM4-dependent Mg²⁺ efflux directly or indirectly regulates CatSper and TRPV1 channel gating.","evidence":"","pmids":[],"confidence":"High","gaps":["No full-length CNNM4 structure exists","Ion coupling stoichiometry and electrogenicity unresolved","PRL inhibition mechanism at atomic detail unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[1,3,6,9,10,11]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,2,9]}],"pathway":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[1]}],"complexes":[],"partners":["PRL","COX11","IQCB1","AP1M1","AP1M2"],"other_free_text":[]},"mechanistic_narrative":"CNNM4 is a plasma-membrane Mg²⁺ efflux transporter that couples intracellular magnesium homeostasis to diverse signaling outputs including AMPK/mTOR energy metabolism, Ca²⁺ influx, and EGFR activation in epithelia, sperm, adipocytes, and hepatocytes [PMID:25347473, PMID:27006114, PMID:30670776, PMID:39517124]. Its CBS-pair domain binds ATP and is the site of pathogenic missense variants that reduce protein and mRNA stability and impair Mg²⁺ extrusion without mislocalizing the transporter [PMID:39580587]. Basolateral sorting in polarized epithelia depends on three dileucine motifs recognized by AP-1A and AP-1B clathrin adaptor complexes, and the oncogenic phosphatase PRL directly binds CNNM4 to inhibit Mg²⁺ efflux, thereby promoting tumor-associated metabolic reprogramming [PMID:25449265, PMID:25347473]. Loss-of-function mutations in CNNM4 cause Jalili syndrome, a recessive disorder of cone-rod dystrophy and amelogenesis imperfecta, reflecting the transporter's essential roles in retinal photoreceptors and ameloblasts [PMID:19200525, PMID:19200527]."},"prefetch_data":{"uniprot":{"accession":"Q6P4Q7","full_name":"Metal transporter CNNM4","aliases":["Ancient conserved domain-containing protein 4","Cyclin-M4"],"length_aa":775,"mass_kda":86.6,"function":"Probable metal transporter. The interaction with the metal ion chaperone COX11 suggests that it may play a role in sensory neuron functions (By similarity). May play a role in biomineralization and retinal function","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q6P4Q7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CNNM4","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CNNM4","total_profiled":1310},"omim":[{"mim_id":"607805","title":"CYCLIN M4; CNNM4","url":"https://www.omim.org/entry/607805"},{"mim_id":"607804","title":"CYCLIN M3; CNNM3","url":"https://www.omim.org/entry/607804"},{"mim_id":"217080","title":"JALILI SYNDROME","url":"https://www.omim.org/entry/217080"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Actin filaments","reliability":"Uncertain"},{"location":"Plasma membrane","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"retina","ntpm":36.0}],"url":"https://www.proteinatlas.org/search/CNNM4"},"hgnc":{"alias_symbol":["SLC70A4","KIAA1592"],"prev_symbol":["ACDP4"]},"alphafold":{"accession":"Q6P4Q7","domains":[{"cath_id":"2.60.40,2.60.40","chopping":"46-174","consensus_level":"high","plddt":81.1621,"start":46,"end":174},{"cath_id":"-","chopping":"183-329","consensus_level":"high","plddt":88.6172,"start":183,"end":329},{"cath_id":"3.10.580.10","chopping":"370-518","consensus_level":"high","plddt":90.0452,"start":370,"end":518},{"cath_id":"2.60.120.10","chopping":"544-647_695-727","consensus_level":"high","plddt":89.1387,"start":544,"end":727}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6P4Q7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6P4Q7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6P4Q7-F1-predicted_aligned_error_v6.png","plddt_mean":76.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CNNM4","jax_strain_url":"https://www.jax.org/strain/search?query=CNNM4"},"sequence":{"accession":"Q6P4Q7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6P4Q7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6P4Q7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6P4Q7"}},"corpus_meta":[{"pmid":"19200525","id":"PMC_19200525","title":"Mutations in CNNM4 cause Jalili syndrome, consisting of autosomal-recessive cone-rod dystrophy and amelogenesis imperfecta.","date":"2009","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/19200525","citation_count":130,"is_preprint":false},{"pmid":"25347473","id":"PMC_25347473","title":"Membrane protein CNNM4-dependent Mg2+ efflux suppresses tumor progression.","date":"2014","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/25347473","citation_count":98,"is_preprint":false},{"pmid":"19200527","id":"PMC_19200527","title":"Mutations in CNNM4 cause recessive cone-rod dystrophy with amelogenesis imperfecta.","date":"2009","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/19200527","citation_count":81,"is_preprint":false},{"pmid":"27006114","id":"PMC_27006114","title":"The Mg2+ transporter CNNM4 regulates sperm Ca2+ homeostasis and is essential for reproduction.","date":"2016","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/27006114","citation_count":34,"is_preprint":false},{"pmid":"24194943","id":"PMC_24194943","title":"Dental phenotype in Jalili syndrome due to a c.1312 dupC homozygous mutation in the CNNM4 gene.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24194943","citation_count":30,"is_preprint":false},{"pmid":"15840172","id":"PMC_15840172","title":"Physical interaction and functional coupling between ACDP4 and the intracellular ion chaperone COX11, an implication of the role of ACDP4 in essential metal ion transport and homeostasis.","date":"2005","source":"Molecular pain","url":"https://pubmed.ncbi.nlm.nih.gov/15840172","citation_count":23,"is_preprint":false},{"pmid":"30670776","id":"PMC_30670776","title":"Cnnm4 deficiency suppresses Ca2+ signaling and promotes cell proliferation in the colon epithelia.","date":"2019","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/30670776","citation_count":19,"is_preprint":false},{"pmid":"27070327","id":"PMC_27070327","title":"A new familial case of Jalili syndrome caused by a novel mutation in CNNM4.","date":"2016","source":"Ophthalmic genetics","url":"https://pubmed.ncbi.nlm.nih.gov/27070327","citation_count":15,"is_preprint":false},{"pmid":"29322253","id":"PMC_29322253","title":"Identification of a mutation in CNNM4 by whole exome sequencing in an Amish family and functional link between CNNM4 and IQCB1.","date":"2018","source":"Molecular genetics and genomics : MGG","url":"https://pubmed.ncbi.nlm.nih.gov/29322253","citation_count":15,"is_preprint":false},{"pmid":"30278312","id":"PMC_30278312","title":"Molecular expression of Mg2+ regulator TRPM7 and CNNM4 in rat odontoblasts.","date":"2018","source":"Archives of oral biology","url":"https://pubmed.ncbi.nlm.nih.gov/30278312","citation_count":14,"is_preprint":false},{"pmid":"31347285","id":"PMC_31347285","title":"A novel pathogenic missense variant in CNNM4 underlying Jalili syndrome: Insights from molecular dynamics simulations.","date":"2019","source":"Molecular genetics & genomic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/31347285","citation_count":13,"is_preprint":false},{"pmid":"29421602","id":"PMC_29421602","title":"Report of two unrelated families with Jalili syndrome and a novel nonsense heterozygous mutation in CNNM4 gene.","date":"2018","source":"European journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29421602","citation_count":13,"is_preprint":false},{"pmid":"25449265","id":"PMC_25449265","title":"Basolateral sorting of the Mg²⁺ transporter CNNM4 requires interaction with AP-1A and AP-1B.","date":"2014","source":"Biochemical and biophysical research 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Section F, Structural biology and crystallization communications","url":"https://pubmed.ncbi.nlm.nih.gov/21393841","citation_count":11,"is_preprint":false},{"pmid":"39641635","id":"PMC_39641635","title":"Modulatory effects of CNNM4 on protein- l -isoaspartyl- O -methyltransferase repair function during alcohol-induced hepatic damage.","date":"2024","source":"Hepatology (Baltimore, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/39641635","citation_count":9,"is_preprint":false},{"pmid":"28246031","id":"PMC_28246031","title":"Novel splice site mutation in CNNM4 gene in a family with Jalili syndrome.","date":"2017","source":"European journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/28246031","citation_count":9,"is_preprint":false},{"pmid":"32022389","id":"PMC_32022389","title":"Expanding the genotypic spectrum of Jalili syndrome: Novel CNNM4 variants and uniparental isodisomy in a north American patient cohort.","date":"2020","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/32022389","citation_count":9,"is_preprint":false},{"pmid":"39517124","id":"PMC_39517124","title":"Thermogenic Adipocytes Promote M2 Macrophage Polarization through CNNM4-Mediated Mg Secretion.","date":"2024","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/39517124","citation_count":6,"is_preprint":false},{"pmid":"34875963","id":"PMC_34875963","title":"Cone pathway dysfunction in Jalili syndrome due to a novel familial variant of CNNM4 revealed by pupillometry and electrophysiologic investigations.","date":"2021","source":"Ophthalmic genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34875963","citation_count":6,"is_preprint":false},{"pmid":"35150469","id":"PMC_35150469","title":"Novel homozygous nonsynonymous variant of CNNM4 gene in a Chinese family with Jalili syndrome.","date":"2022","source":"Molecular genetics & genomic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35150469","citation_count":6,"is_preprint":false},{"pmid":"40764063","id":"PMC_40764063","title":"Role of CNNM4 in the progression of cholangiocarcinoma: implications for ferroptosis and therapeutic potential.","date":"2026","source":"Gut","url":"https://pubmed.ncbi.nlm.nih.gov/40764063","citation_count":2,"is_preprint":false},{"pmid":"39580587","id":"PMC_39580587","title":"Functional and pathogenic insights into CNNM4 variants in Jalili syndrome.","date":"2024","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/39580587","citation_count":1,"is_preprint":false},{"pmid":"40862638","id":"PMC_40862638","title":"microRNAs Regulate Cellular Magnesium by Tuning Expression of the Plasma Membrane Protein CNNM4.","date":"2025","source":"ACS chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/40862638","citation_count":0,"is_preprint":false},{"pmid":"40232358","id":"PMC_40232358","title":"A novel mutation in CNNM4 is associated with a case of Jalili syndrome in Egypt.","date":"2025","source":"Documenta ophthalmologica. Advances in ophthalmology","url":"https://pubmed.ncbi.nlm.nih.gov/40232358","citation_count":0,"is_preprint":false},{"pmid":"41280631","id":"PMC_41280631","title":"Novel mutation in CNNM4 gene in a Chinese family with Jalili syndrome and literature review.","date":"2025","source":"International journal of ophthalmology","url":"https://pubmed.ncbi.nlm.nih.gov/41280631","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13359,"output_tokens":3417,"usd":0.045666},"stage2":{"model":"claude-opus-4-6","input_tokens":6828,"output_tokens":6076,"usd":0.27906},"total_usd":0.324726,"stage1_batch_id":"msgbatch_011kfMfE81o6CTcyhVUqumuW","stage2_batch_id":"msgbatch_01DTputS6WNkH745x1nVSoBo","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"CNNM4 (encoding a putative metal transporter) was identified as the causative gene for Jalili syndrome; expression was confirmed in neural retina and ameloblasts of developing teeth, establishing its role in metal transport linked to visual function and biomineralization.\",\n      \"method\": \"Positional-candidate gene sequencing in seven families; expression confirmed in retina and ameloblasts\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — replicated across two independent groups in the same year with multiple families and expression data\",\n      \"pmids\": [\"19200525\", \"19200527\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CNNM4 functions as a Mg2+ efflux transporter at the plasma membrane; the oncogenic phosphatase PRL binds CNNM4 and inhibits CNNM4-dependent Mg2+ efflux, thereby elevating intracellular Mg2+ and activating AMPK/mTOR energy-metabolism signaling to promote tumor progression.\",\n      \"method\": \"Biochemical binding assay (co-immunoprecipitation of PRL and CNNM4), cellular Mg2+ flux measurements, Cnnm4-knockout mouse model (ApcΔ14/+ background), mTOR inhibitor (rapamycin) rescue\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (Co-IP, Mg2+ flux assay, genetic KO mouse, pharmacological rescue) in a single well-cited study\",\n      \"pmids\": [\"25347473\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Basolateral sorting of CNNM4 in polarized epithelial cells requires three conserved dileucine motifs in CNNM4 that mediate interaction with clathrin adaptor protein complexes AP-1A (μ1A subunit) and AP-1B (μ1B subunit); simultaneous knockdown of both μ1A and μ1B, but not either alone, disrupts basolateral localization.\",\n      \"method\": \"siRNA knockdown of μ1A and μ1B in MDCK cells, mutational analysis of dileucine motifs, co-immunoprecipitation with μ1A/μ1B\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus mutagenesis plus KD with defined localization phenotype in same study\",\n      \"pmids\": [\"25449265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CNNM4 is required for Ca2+ influx during sperm capacitation; Cnnm4-deficient sperm show abrogated hyperactivation and perturbed Ca2+ influx phenotypically similar to CatSper-null sperm, and forced Ca2+ entry into Cnnm4-deficient sperm normalizes tyrosine phosphorylation levels.\",\n      \"method\": \"Cnnm4 germline and germ-cell-specific knockout mice, Ca2+ imaging in sperm, sperm motility analysis, tyrosine phosphorylation western blot, forced Ca2+ entry rescue\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with Ca2+ imaging, functional rescue, and two independent KO strategies\",\n      \"pmids\": [\"27006114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The CBS-pair regulatory domain of human CNNM4 was purified and crystallized, yielding diffracting crystals in orthorhombic space group C222 with two molecules per asymmetric unit, providing structural characterization of this domain.\",\n      \"method\": \"Protein overexpression, purification, crystallization, and preliminary X-ray crystallographic analysis (synchrotron, 3.6 Å)\",\n      \"journal\": \"Acta crystallographica. Section F\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure at preliminary resolution; functional validation not yet performed in this report\",\n      \"pmids\": [\"21393841\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CNNM4 (as ACDP4) physically interacts with the intracellular metal ion chaperone COX11 in a yeast two-hybrid screen; co-expression of ACDP4 and COX11 in HEK293 cells enhanced toxicity to copper, manganese, and cobalt, suggesting functional coupling in metal ion homeostasis.\",\n      \"method\": \"Yeast two-hybrid screen of human fetal brain cDNA library; ectopic co-expression in HEK293 cells with metal ion toxicity assay\",\n      \"journal\": \"Molecular pain\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — yeast two-hybrid plus cell-based toxicity assay, single lab, no direct transport assay\",\n      \"pmids\": [\"15840172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CNNM4 deficiency in colon epithelium suppresses TRPV1-mediated Ca2+ influx and constitutively activates EGF receptor signaling, promoting cell proliferation; gefitinib (EGFR inhibitor) rescues the hyperproliferation phenotype, establishing a Mg2+–Ca2+ functional interplay in colonic homeostasis.\",\n      \"method\": \"Cnnm4-knockout mice, organoid Ca2+ imaging, EGFR phosphorylation western blot, gefitinib pharmacological rescue\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with Ca2+ imaging, defined signaling pathway, pharmacological rescue\",\n      \"pmids\": [\"30670776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CNNM4 physically interacts with IQCB1 (a Leber congenital amaurosis gene product); a truncated CNNM4 protein (p.R605X) increases apoptosis and enhances CNNM4–IQCB1 interaction, functionally linking Jalili syndrome to LCA pathology.\",\n      \"method\": \"Co-immunoprecipitation of CNNM4 and IQCB1, apoptosis assay with truncated CNNM4 construct\",\n      \"journal\": \"Molecular genetics and genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP plus apoptosis assay, single lab\",\n      \"pmids\": [\"29322253\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Missense variants in the CBS domain of CNNM4 (p.Gly492Cys and p.Gly492Asp) disrupt ATP-binding mode within the CBS domain as shown by molecular dynamics simulations; analogous CBS-domain variants cause conformational shifts predicted to impair metal transport regulation.\",\n      \"method\": \"Molecular dynamics simulations and docking analysis of CBS-domain wild-type vs. mutant CNNM4\",\n      \"journal\": \"Molecular genetics & genomic medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 — computational prediction only, no experimental validation of ATP binding\",\n      \"pmids\": [\"31347285\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CNNM4 missense variants p.Gly492Cys and p.Gly492Asp (CBS domain) reduce protein stability and increase mRNA decay but do not mislocalize the transporter; the mutant proteins show significantly reduced Mg2+ extrusion activity, indicating that reduced stability/expression rather than mislocalization underlies pathogenicity.\",\n      \"method\": \"Mg2+ efflux assay, protein stability assay, mRNA decay measurement, localization by fluorescence microscopy in transfected cells\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct Mg2+ transport assay combined with protein/mRNA stability and localization data\",\n      \"pmids\": [\"39580587\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In thermogenic adipocytes, CNNM4 is induced by ADRB3-PKA-CREB signaling during cold exposure and mediates Mg2+ efflux; secreted Mg2+ then binds the DFG motif of mTOR in macrophages to activate mTORC2 and drive M2 polarization, establishing a paracrine adipocyte–macrophage Mg2+ signaling axis.\",\n      \"method\": \"ADRB3 agonist stimulation, PKA/CREB pathway inhibitors, CNNM4 overexpression/knockdown in adipocytes, mTORC2 activity assay, macrophage polarization assay, Mg2+ supplementation/chelation experiments\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple pathway inhibitors and gain/loss-of-function with defined signaling readout, single lab\",\n      \"pmids\": [\"39517124\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In alcohol-associated liver disease, CNNM4 upregulation disrupts Mg2+ homeostasis; silencing Cnnm4 via GalNAc-siRNA restores PCMT1 (protein isoaspartyl methyltransferase) activity by increasing S-adenosylmethionine levels through improved mitochondrial function, linking CNNM4-dependent Mg2+ efflux to protein damage repair.\",\n      \"method\": \"GalNAc-siRNA silencing of Cnnm4 in mice, PCMT1 activity assay, SAM level measurement, mitochondrial function assay\",\n      \"journal\": \"Hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo siRNA with biochemical activity assays, single lab\",\n      \"pmids\": [\"39641635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"microRNAs directly bind the 3'UTR of CNNM4 mRNA to regulate CNNM4 expression and thereby control cellular Mg2+ concentrations in hepatocytes; both upregulatory and downregulatory miRNA activities were detected in a high-throughput 3'UTR assay.\",\n      \"method\": \"High-throughput miRNA-3'UTR binding assay, direct miRNA:3'UTR interaction validation, cellular Mg2+ concentration measurement in hepatocytes\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding assay plus functional cellular Mg2+ readout, but single lab and newly published\",\n      \"pmids\": [\"40862638\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In cholangiocarcinoma, CNNM4 silencing attenuates ferroptosis via a pathway involving nuclear protein 1 (NUPR1) as an upstream regulator; iron chelation with deferiprone reversed the antiproliferative effect of CNNM4 silencing, while HO-1 inhibition did not, establishing CNNM4-linked ferroptosis as a distinct cell-death mechanism.\",\n      \"method\": \"siRNA/shRNA knockdown of CNNM4 in CCA cell lines and GalNAc-siRNA in transposon-based mouse CCA model, proteomic analysis, ferroptosis rescue with deferiprone/zinc protoporphyrin IX, chicken chorioallantoic membrane invasion assay\",\n      \"journal\": \"Gut\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro and in vivo KD with proteomic pathway identification and pharmacological rescue, single lab\",\n      \"pmids\": [\"40764063\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CNNM4 is a plasma membrane Mg2+ efflux transporter whose CBS domain binds ATP and interacts with the oncogenic phosphatase PRL (which inhibits Mg2+ efflux), is sorted to the basolateral membrane of polarized epithelia via dileucine motifs recognized by AP-1A/AP-1B adaptors, regulates intracellular Mg2+ to control AMPK/mTOR energy metabolism and Ca2+ homeostasis in sperm and colon epithelia, and whose loss-of-function mutations (causing reduced protein/mRNA stability or truncation) underlie Jalili syndrome by impairing Mg2+ transport in retinal photoreceptors and ameloblasts.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CNNM4 is a plasma-membrane Mg²⁺ efflux transporter that couples intracellular magnesium homeostasis to diverse signaling outputs including AMPK/mTOR energy metabolism, Ca²⁺ influx, and EGFR activation in epithelia, sperm, adipocytes, and hepatocytes [PMID:25347473, PMID:27006114, PMID:30670776, PMID:39517124]. Its CBS-pair domain binds ATP and is the site of pathogenic missense variants that reduce protein and mRNA stability and impair Mg²⁺ extrusion without mislocalizing the transporter [PMID:39580587]. Basolateral sorting in polarized epithelia depends on three dileucine motifs recognized by AP-1A and AP-1B clathrin adaptor complexes, and the oncogenic phosphatase PRL directly binds CNNM4 to inhibit Mg²⁺ efflux, thereby promoting tumor-associated metabolic reprogramming [PMID:25449265, PMID:25347473]. Loss-of-function mutations in CNNM4 cause Jalili syndrome, a recessive disorder of cone-rod dystrophy and amelogenesis imperfecta, reflecting the transporter's essential roles in retinal photoreceptors and ameloblasts [PMID:19200525, PMID:19200527].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Before CNNM4's transport function was known, a yeast two-hybrid screen identified the metal chaperone COX11 as a physical interactor and showed that co-expression with CNNM4 sensitized cells to copper, manganese, and cobalt, providing the first evidence that CNNM4 participates in metal ion homeostasis.\",\n      \"evidence\": \"Yeast two-hybrid screen plus metal toxicity assay in HEK293 cells\",\n      \"pmids\": [\"15840172\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct metal transport assay was performed\", \"COX11 interaction not validated by reciprocal Co-IP\", \"Relevance to Mg²⁺ versus other divalent cations unclear\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Positional cloning in multiple consanguineous families established CNNM4 as the causative gene for Jalili syndrome (cone-rod dystrophy with amelogenesis imperfecta), linking a putative metal transporter to both retinal and dental mineralization biology.\",\n      \"evidence\": \"Homozygosity mapping and candidate-gene sequencing in seven independent families by two groups, with expression confirmed in neural retina and ameloblasts\",\n      \"pmids\": [\"19200525\", \"19200527\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The mechanism by which CNNM4 loss causes photoreceptor degeneration was not resolved\", \"Which metal ion CNNM4 transports was still unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Crystallization of the CBS-pair regulatory domain provided the first structural information on CNNM4, establishing the feasibility of atomic-resolution studies of its regulatory module.\",\n      \"evidence\": \"Protein purification, crystallization, and preliminary X-ray diffraction at 3.6 Å\",\n      \"pmids\": [\"21393841\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Resolution too low for detailed atomic model\", \"No ligand-bound structure obtained\", \"Functional consequences of CBS domain conformation not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Two concurrent studies resolved the core molecular function and trafficking mechanism of CNNM4: it mediates Mg²⁺ efflux at the plasma membrane, the PRL phosphatase binds and inhibits this efflux to activate AMPK/mTOR signaling in colorectal tumors, and basolateral sorting in polarized epithelia requires three dileucine motifs recognized by AP-1A/AP-1B adaptors.\",\n      \"evidence\": \"Co-IP of PRL–CNNM4, cellular Mg²⁺ flux assays, Cnnm4-KO mice on Apc-mutant background with rapamycin rescue; dileucine mutagenesis with μ1A/μ1B siRNA in MDCK cells\",\n      \"pmids\": [\"25347473\", \"25449265\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct structural basis of PRL–CNNM4 inhibition was not determined\", \"Whether AP-1A and AP-1B are fully redundant or context-dependent remained unresolved\", \"Whether Mg²⁺ transport is electrogenic or coupled to a counter-ion was not established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"CNNM4 was shown to be required for Ca²⁺ influx during sperm capacitation, revealing that its Mg²⁺ efflux activity indirectly regulates Ca²⁺ signaling; Cnnm4-deficient sperm phenocopied CatSper-null sperm, and forced Ca²⁺ entry rescued tyrosine phosphorylation.\",\n      \"evidence\": \"Germline and germ-cell-specific Cnnm4 KO mice with Ca²⁺ imaging, motility analysis, and forced Ca²⁺ entry rescue\",\n      \"pmids\": [\"27006114\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism linking Mg²⁺ efflux to CatSper-dependent Ca²⁺ influx was not identified\", \"Whether CNNM4 loss contributes to human male infertility was not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"A physical interaction between CNNM4 and IQCB1 (a Leber congenital amaurosis gene product) was identified, and a Jalili-associated truncation (p.R605X) enhanced this interaction and increased apoptosis, suggesting a functional bridge between the two retinal-disease pathways.\",\n      \"evidence\": \"Co-IP of CNNM4 and IQCB1, apoptosis assay with truncated construct\",\n      \"pmids\": [\"29322253\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP without reciprocal validation\", \"Physiological relevance of enhanced interaction to retinal degeneration not demonstrated in vivo\", \"IQCB1 interaction not confirmed by an independent group\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"In colonic epithelium, CNNM4 deficiency was shown to suppress TRPV1-mediated Ca²⁺ influx and constitutively activate EGFR signaling, directly linking CNNM4-dependent Mg²⁺ homeostasis to proliferative control; gefitinib rescue proved EGFR as the downstream effector.\",\n      \"evidence\": \"Cnnm4-KO mice, organoid Ca²⁺ imaging, EGFR phosphorylation assay, gefitinib pharmacological rescue\",\n      \"pmids\": [\"30670776\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How altered intracellular Mg²⁺ suppresses TRPV1 activity at the channel level is unknown\", \"Whether this EGFR axis operates in non-colonic epithelia was not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Multiple studies expanded CNNM4's physiological scope: pathogenic CBS-domain missense variants were shown to reduce protein/mRNA stability (not localization) and impair Mg²⁺ extrusion, cold-induced CNNM4 expression in adipocytes established a paracrine Mg²⁺ signal that activates mTORC2 in macrophages, and hepatic CNNM4 silencing restored PCMT1 activity by improving mitochondrial SAM metabolism.\",\n      \"evidence\": \"Mg²⁺ efflux assay and stability measurements for CBS-domain variants; ADRB3-PKA-CREB pathway dissection with CNNM4 gain/loss-of-function in adipocytes and mTORC2 activity in macrophages; GalNAc-siRNA Cnnm4 silencing in alcohol-fed mice with PCMT1/SAM assays\",\n      \"pmids\": [\"39580587\", \"39517124\", \"39641635\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CNNM4 is the sole Mg²⁺ efflux pathway in adipocytes is unclear\", \"The DFG-motif Mg²⁺ sensor model on mTOR lacks structural validation\", \"Hepatic CNNM4 silencing effects on Mg²⁺-dependent enzymes beyond PCMT1 are unexplored\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Post-transcriptional regulation of CNNM4 by miRNAs was demonstrated through direct binding to its 3′UTR in hepatocytes, showing that cellular Mg²⁺ levels can be tuned at the mRNA level.\",\n      \"evidence\": \"High-throughput miRNA–3′UTR binding assay with cellular Mg²⁺ concentration measurement in hepatocytes\",\n      \"pmids\": [\"40862638\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific miRNAs that are physiologically dominant in regulating CNNM4 in vivo are not identified\", \"Whether miRNA regulation is tissue-specific was not addressed\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"CNNM4 silencing in cholangiocarcinoma was shown to attenuate ferroptosis via a NUPR1-dependent pathway; iron chelation reversed the effect while HO-1 inhibition did not, establishing a Mg²⁺-linked ferroptosis axis distinct from canonical heme oxygenase pathways.\",\n      \"evidence\": \"siRNA/shRNA KD in CCA cell lines, GalNAc-siRNA in transposon-based mouse CCA model, proteomics, deferiprone rescue\",\n      \"pmids\": [\"40764063\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting Mg²⁺ efflux to NUPR1 expression is not resolved\", \"Single-lab finding awaiting independent replication\", \"Whether this ferroptosis link generalizes beyond cholangiocarcinoma is unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include the transport mechanism (electrogenic vs. coupled), a high-resolution full-length structure, the molecular basis of PRL-mediated inhibition, and whether CNNM4-dependent Mg²⁺ efflux directly or indirectly regulates CatSper and TRPV1 channel gating.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length CNNM4 structure exists\", \"Ion coupling stoichiometry and electrogenicity unresolved\", \"PRL inhibition mechanism at atomic detail unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [1, 3, 6, 9, 10, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 2, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PRL\", \"COX11\", \"IQCB1\", \"AP1M1\", \"AP1M2\"],\n    \"other_free_text\": []\n  }\n}\n```\n\nWait, I need to fix the pathway entries. The pathway should use Reactome IDs. Let me reconsider.\n\n```json\n{\n  \"mechanistic_narrative\": \"CNNM4 is a plasma-membrane Mg²⁺ efflux transporter that couples intracellular magnesium homeostasis to diverse signaling outputs including AMPK/mTOR energy metabolism, Ca²⁺ influx, and EGFR activation in epithelia, sperm, adipocytes, and hepatocytes [PMID:25347473, PMID:27006114, PMID:30670776, PMID:39517124]. Its CBS-pair domain binds ATP and is the site of pathogenic missense variants that reduce protein and mRNA stability and impair Mg²⁺ extrusion without mislocalizing the transporter [PMID:39580587]. Basolateral sorting in polarized epithelia depends on three dileucine motifs recognized by AP-1A and AP-1B clathrin adaptor complexes, and the oncogenic phosphatase PRL directly binds CNNM4 to inhibit Mg²⁺ efflux, thereby promoting tumor-associated metabolic reprogramming [PMID:25449265, PMID:25347473]. Loss-of-function mutations in CNNM4 cause Jalili syndrome, a recessive disorder of cone-rod dystrophy and amelogenesis imperfecta, reflecting the transporter's essential roles in retinal photoreceptors and ameloblasts [PMID:19200525, PMID:19200527].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Before CNNM4's transport function was known, a yeast two-hybrid screen identified the metal chaperone COX11 as a physical interactor and showed that co-expression sensitized cells to copper, manganese, and cobalt, providing the first evidence that CNNM4 participates in metal ion homeostasis.\",\n      \"evidence\": \"Yeast two-hybrid screen plus metal toxicity assay in HEK293 cells\",\n      \"pmids\": [\"15840172\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct metal transport assay was performed\", \"COX11 interaction not validated by reciprocal Co-IP\", \"Relevance to Mg²⁺ versus other divalent cations unclear\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Positional cloning in multiple consanguineous families established CNNM4 as the causative gene for Jalili syndrome (cone-rod dystrophy with amelogenesis imperfecta), linking a putative metal transporter to both retinal and dental mineralization biology.\",\n      \"evidence\": \"Homozygosity mapping and candidate-gene sequencing in seven independent families by two groups, with expression confirmed in neural retina and ameloblasts\",\n      \"pmids\": [\"19200525\", \"19200527\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The mechanism by which CNNM4 loss causes photoreceptor degeneration was not resolved\", \"Which metal ion CNNM4 transports was still unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Crystallization of the CBS-pair regulatory domain provided the first structural information on CNNM4, establishing the feasibility of atomic-resolution studies of its regulatory module.\",\n      \"evidence\": \"Protein purification, crystallization, and preliminary X-ray diffraction at 3.6 Å\",\n      \"pmids\": [\"21393841\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Resolution too low for detailed atomic model\", \"No ligand-bound structure obtained\", \"Functional consequences of CBS domain conformation not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Two concurrent studies resolved the core molecular function and trafficking mechanism of CNNM4: it mediates Mg²⁺ efflux at the plasma membrane, the PRL phosphatase binds and inhibits this efflux to activate AMPK/mTOR signaling in colorectal tumors, and basolateral sorting in polarized epithelia requires three dileucine motifs recognized by AP-1A/AP-1B adaptors.\",\n      \"evidence\": \"Co-IP of PRL–CNNM4, cellular Mg²⁺ flux assays, Cnnm4-KO mice on Apc-mutant background with rapamycin rescue; dileucine mutagenesis with μ1A/μ1B siRNA in MDCK cells\",\n      \"pmids\": [\"25347473\", \"25449265\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct structural basis of PRL–CNNM4 inhibition was not determined\", \"Whether AP-1A and AP-1B are fully redundant or context-dependent remained unresolved\", \"Whether Mg²⁺ transport is electrogenic or coupled to a counter-ion was not established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"CNNM4 was shown to be required for Ca²⁺ influx during sperm capacitation, revealing that its Mg²⁺ efflux activity indirectly regulates Ca²⁺ signaling; Cnnm4-deficient sperm phenocopied CatSper-null sperm, and forced Ca²⁺ entry rescued tyrosine phosphorylation.\",\n      \"evidence\": \"Germline and germ-cell-specific Cnnm4 KO mice with Ca²⁺ imaging, motility analysis, and forced Ca²⁺ entry rescue\",\n      \"pmids\": [\"27006114\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism linking Mg²⁺ efflux to CatSper-dependent Ca²⁺ influx was not identified\", \"Whether CNNM4 loss contributes to human male infertility was not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"A physical interaction between CNNM4 and IQCB1 (a Leber congenital amaurosis gene product) was identified, and a Jalili-associated truncation (p.R605X) enhanced this interaction and increased apoptosis, suggesting a functional bridge between the two retinal-disease pathways.\",\n      \"evidence\": \"Co-IP of CNNM4 and IQCB1, apoptosis assay with truncated construct\",\n      \"pmids\": [\"29322253\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP without reciprocal validation\", \"Physiological relevance of enhanced interaction to retinal degeneration not demonstrated in vivo\", \"IQCB1 interaction not confirmed by an independent group\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"In colonic epithelium, CNNM4 deficiency was shown to suppress TRPV1-mediated Ca²⁺ influx and constitutively activate EGFR signaling, directly linking CNNM4-dependent Mg²⁺ homeostasis to proliferative control; gefitinib rescue proved EGFR as the downstream effector.\",\n      \"evidence\": \"Cnnm4-KO mice, organoid Ca²⁺ imaging, EGFR phosphorylation assay, gefitinib pharmacological rescue\",\n      \"pmids\": [\"30670776\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How altered intracellular Mg²⁺ suppresses TRPV1 activity at the channel level is unknown\", \"Whether this EGFR axis operates in non-colonic epithelia was not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Multiple studies expanded CNNM4's physiological scope: pathogenic CBS-domain missense variants were shown to reduce protein/mRNA stability (not localization) and impair Mg²⁺ extrusion, cold-induced CNNM4 expression in adipocytes established a paracrine Mg²⁺ signal activating mTORC2 in macrophages, and hepatic CNNM4 silencing restored PCMT1 activity by improving mitochondrial SAM metabolism.\",\n      \"evidence\": \"Mg²⁺ efflux assay and stability measurements for CBS-domain variants; ADRB3-PKA-CREB pathway dissection with CNNM4 gain/loss-of-function in adipocytes and mTORC2 activity in macrophages; GalNAc-siRNA Cnnm4 silencing in alcohol-fed mice with PCMT1/SAM assays\",\n      \"pmids\": [\"39580587\", \"39517124\", \"39641635\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CNNM4 is the sole Mg²⁺ efflux pathway in adipocytes is unclear\", \"The DFG-motif Mg²⁺ sensor model on mTOR lacks structural validation\", \"Hepatic CNNM4 silencing effects on Mg²⁺-dependent enzymes beyond PCMT1 are unexplored\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Post-transcriptional regulation of CNNM4 by miRNAs was demonstrated through direct binding to its 3′UTR in hepatocytes, showing that cellular Mg²⁺ levels can be tuned at the mRNA level.\",\n      \"evidence\": \"High-throughput miRNA–3′UTR binding assay with cellular Mg²⁺ concentration measurement in hepatocytes\",\n      \"pmids\": [\"40862638\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific miRNAs that are physiologically dominant in regulating CNNM4 in vivo are not identified\", \"Whether miRNA regulation is tissue-specific was not addressed\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"CNNM4 silencing in cholangiocarcinoma was shown to attenuate ferroptosis via a NUPR1-dependent pathway; iron chelation reversed the effect while HO-1 inhibition did not, establishing a Mg²⁺-linked ferroptosis axis distinct from canonical heme oxygenase pathways.\",\n      \"evidence\": \"siRNA/shRNA KD in CCA cell lines, GalNAc-siRNA in transposon-based mouse CCA model, proteomics, deferiprone rescue\",\n      \"pmids\": [\"40764063\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting Mg²⁺ efflux to NUPR1 expression is not resolved\", \"Single-lab finding awaiting independent replication\", \"Whether this ferroptosis link generalizes beyond cholangiocarcinoma is unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include the transport mechanism (electrogenic vs. coupled), a high-resolution full-length structure, the molecular basis of PRL-mediated inhibition, and whether CNNM4-dependent Mg²⁺ efflux directly or indirectly regulates CatSper and TRPV1 channel gating.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length CNNM4 structure exists\", \"Ion coupling stoichiometry and electrogenicity unresolved\", \"PRL inhibition mechanism at atomic detail unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [1, 3, 6, 9, 10, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 2, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [1, 3, 9, 10, 11]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 6, 10]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 7, 9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PRL\", \"COX11\", \"IQCB1\", \"AP1M1\", \"AP1M2\"],\n    \"other_free_text\": []\n  }\n}\n```"}