id: A0A8I3PI07 gene_symbol: CNNM4 product_type: PROTEIN status: DRAFT taxon: id: NCBITaxon:9615 label: Canis lupus familiaris description: >- CNNM4 (cyclin and CBS domain divalent metal cation transport mediator 4) is a multi-pass plasma-membrane protein of the CNNM/ACDP family. The dog protein (796 aa) has a signal peptide, an N-terminal extracellular immunoglobulin-like domain, the four-helix CNNM transmembrane domain, a cytoplasmic pair of CBS domains that binds MgATP, and a C-terminal cyclic nucleotide-binding homology domain. In mammals CNNM4 sits at the basolateral membrane of intestinal epithelial cells and enamel-forming ameloblasts, where it extrudes Mg2+ from the cell in exchange for extracellular Na+. This basolateral efflux is the exit step of transcellular magnesium absorption, so CNNM4 is needed for systemic magnesium balance and for enamel formation. MgATP binding to the CBS pair organises the full-length protein into a dimer-of-dimers assembly, and CNNM4 also associates with the TRPM7 channel and stimulates divalent-cation entry through it. In humans, biallelic loss-of-function variants cause Jalili syndrome (cone-rod dystrophy with amelogenesis imperfecta). No functional study of the dog protein itself has been reported; its function is inferred from its placement in the CNNM4 subfamily and 93% identity to human CNNM4. existing_annotations: - term: id: GO:0005886 label: plasma membrane evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: is_active_in review: summary: >- PAINT node PTN000248898 assigns plasma-membrane activity across the CNNM family, seeded by experimental annotations of fly uex, mouse Cnnm1/2/4, human CNNM2/CNNM4 and worm homologs. The dog protein is inside this clade (PTHR12064:SF26) and has the signal peptide and four predicted transmembrane helices of the family. action: ACCEPT reason: >- The target is inside the clade that inherited the location, and nothing in its sequence suggests divergence (the transmembrane domain is intact; 93% identity to human CNNM4). Mammalian CNNM4 extrudes Mg2+ across the plasma membrane and localizes to the basolateral surface of epithelia. supported_by: - reference_id: GO_REF:0000033 - reference_id: PMID:24339795 supporting_text: Endogenous CNNM4 is highly expressed in the mature ameloblasts and intestinal epithelia, and localizes at their basolateral membrane. - reference_id: file:CANLF/A0A8I3PI07/A0A8I3PI07-bioinformatics/RESULTS.md supporting_text: 'Dog vs human CNNM4: identity 0.926 over 775 aligned columns (similarity 0.961).' - term: id: GO:0010960 label: magnesium ion homeostasis evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: involved_in review: summary: >- Eukaryote-level PAINT node PTN008532859 (mouse Cnnm4, human CNNM2, worm and zebrafish CNNMs). CNNM4-knockout mice develop hypomagnesemia from defective intestinal Mg2+ absorption. action: ACCEPT reason: >- Magnesium homeostasis is a core, family-wide process and the dog protein is a CNNM4-subfamily member with all functional domains. CNNM4 directly performs the basolateral Mg2+ efflux step, so this is participation and not only necessity. supported_by: - reference_id: GO_REF:0000033 - reference_id: PMID:24339795 supporting_text: In this study, we generated CNNM4-knockout mice; these mice showed defects in amelogenesis and intestinal Mg2+ absorption. - term: id: GO:0015081 label: sodium ion transmembrane transporter activity evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables review: summary: >- PAINT node PTN002635769 (Euteleostomi, seeded by mouse Cnnm4) propagates sodium transporter activity together with magnesium transporter activity. In CNNM4-expressing cells, Mg2+ extrusion requires extracellular Na+ and is accompanied by Na+ entry, and the human CNNM4 structure is described as promoting Mg2+/Na+ exchange. action: MODIFY reason: >- The sodium movement is real but is the counter-ion arm of a coupled Mg2+/Na+ exchange, not independent sodium transport. The more precise coupled term, GO:0061768 magnesium:sodium antiporter activity, captures both IBA MF rows from this node and matches the human CNNM4 review. The node is CNNM4-specific and the target is in the same subfamily (SF26), so transfer of the coupled activity is warranted. proposed_replacement_terms: - id: GO:0061768 label: magnesium:sodium antiporter activity supported_by: - reference_id: GO_REF:0000033 - reference_id: PMID:24339795 supporting_text: In the absence of extracellular Na+, Mg2+ depletion did not induce Mg2+ extrusion (Phase 3), but restoration of Na+ instantaneously caused Mg2+ extrusion (Phase 4). - reference_id: PMID:42497866 supporting_text: the outward-facing state, thereby promoting Mg2+/Na+ exchange. propagation_review: root_cause: TERM_SCOPING_PROBLEM failure_modes: - GRANULARITY_MISMATCH source_entities: - source_id: PANTHER:PTN002635769 source_status: SUPPORTS_TRANSFER comment: Euteleostomi CNNM4-subfamily node; the dog target (PTHR12064:SF26) descends from it. The function transfers; only the term granularity is refined. - source_id: MGI:MGI:2151060 source_status: SUPPORTS_TRANSFER comment: Mouse Cnnm4, the sole seed; experimental Mg2+ extrusion with Na+ dependence. - term: id: GO:0015095 label: magnesium ion transmembrane transporter activity evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables review: summary: >- Same CNNM4-specific PAINT node (PTN002635769, seeded by mouse Cnnm4). CNNM4 extrudes Mg2+ from cells in a Na+-dependent manner. action: MODIFY reason: >- Correct but less specific than the evidence supports: CNNM4 moves Mg2+ out of the cell in exchange for Na+. GO:0061768 magnesium:sodium antiporter activity is the specific descendant and is the replacement used for the sodium-transporter row as well. proposed_replacement_terms: - id: GO:0061768 label: magnesium:sodium antiporter activity supported_by: - reference_id: GO_REF:0000033 - reference_id: PMID:24339795 supporting_text: As shown in Figure 4D, Mg2+ depletion from the extracellular medium induced not only the decrease of intracellular Mg2+ but also the increase of intracellular Na+. propagation_review: root_cause: TERM_SCOPING_PROBLEM failure_modes: - GRANULARITY_MISMATCH source_entities: - source_id: PANTHER:PTN002635769 source_status: SUPPORTS_TRANSFER comment: Euteleostomi CNNM4-subfamily node; the dog target (PTHR12064:SF26) descends from it. The function transfers; only the term granularity is refined. - source_id: MGI:MGI:2151060 source_status: SUPPORTS_TRANSFER comment: Mouse Cnnm4, the sole seed; experimental Mg2+ extrusion with Na+ dependence. - term: id: GO:0015693 label: magnesium ion transport evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: involved_in review: summary: >- CNNM4-specific PAINT node PTN002635769. CNNM4 mediates basolateral Mg2+ extrusion in transcellular epithelial Mg2+ transport. action: ACCEPT reason: >- Correct and inherited; the gene also carries GO:1903830 magnesium ion transmembrane transport, which is the more specific process, so this broad term is consistent with it rather than a competing claim. supported_by: - reference_id: GO_REF:0000033 - reference_id: PMID:24339795 supporting_text: Functional analyses at the molecular and organismal levels revealed a common role for CNNM4 in mediating transcellular Mg2+ transport by basolateral Mg2+ extrusion. - term: id: GO:0005886 label: plasma membrane evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: located_in review: summary: >- Combined electronic annotation (human CNNM4 ortholog, UniProt SubCell mapping, PANTHER). Agrees with the IBA row for the same term. action: ACCEPT reason: >- Consistent with the IBA plasma-membrane row and with mammalian CNNM4 localization; the more specific basolateral location is annotated separately for polarized epithelia. supported_by: - reference_id: PMID:24339795 supporting_text: Endogenous CNNM4 is highly expressed in the mature ameloblasts and intestinal epithelia, and localizes at their basolateral membrane. - term: id: GO:0010960 label: magnesium ion homeostasis evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: involved_in review: summary: >- InterPro ACDP family (IPR045095) and PANTHER mapping; same term as the IBA row. action: ACCEPT reason: Same claim as the accepted IBA row; supported by mouse CNNM4 knockout physiology. supported_by: - reference_id: PMID:24339795 supporting_text: In this study, we generated CNNM4-knockout mice; these mice showed defects in amelogenesis and intestinal Mg2+ absorption. - term: id: GO:0015693 label: magnesium ion transport evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: involved_in review: summary: TreeGrafter assignment from the PANTHER CNNM4 subfamily; same term as the IBA row. action: ACCEPT reason: Same claim as the accepted IBA row. supported_by: - reference_id: PMID:24339795 supporting_text: Functional analyses at the molecular and organismal levels revealed a common role for CNNM4 in mediating transcellular Mg2+ transport by basolateral Mg2+ extrusion. - term: id: GO:0016020 label: membrane evidence_type: IEA original_reference_id: GO_REF:0000104 qualifier: located_in review: summary: >- UniRule assignment of the generic membrane term. True (four predicted transmembrane helices) but much less specific than the plasma-membrane location the gene already carries. action: MODIFY reason: >- Correct but general. The specific supported location, GO:0005886 plasma membrane, is already annotated to this gene by IBA and IEA. proposed_replacement_terms: - id: GO:0005886 label: plasma membrane supported_by: - reference_id: PMID:24339795 supporting_text: Endogenous CNNM4 is highly expressed in the mature ameloblasts and intestinal epithelia, and localizes at their basolateral membrane. - term: id: GO:0022857 label: transmembrane transporter activity evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables review: summary: >- PANTHER/UniRule assignment of the generic transporter term (PAINT node PTN000248941 lineage). True but non-specific. action: MODIFY reason: >- Correct but general; the specific supported activity is Mg2+/Na+ exchange (GO:0061768 magnesium:sodium antiporter activity), the same replacement proposed for the IBA transporter rows. proposed_replacement_terms: - id: GO:0061768 label: magnesium:sodium antiporter activity supported_by: - reference_id: PMID:42497866 supporting_text: the outward-facing state, thereby promoting Mg2+/Na+ exchange. - term: id: GO:0035725 label: sodium ion transmembrane transport evidence_type: IEA original_reference_id: GO_REF:0000108 qualifier: involved_in review: summary: >- Logical inference from the sodium transporter activity row. CNNM4-expressing cells take up Na+ while extruding Mg2+. action: ACCEPT reason: >- Na+ entry is the counter-ion arm of CNNM4 Mg2+/Na+ exchange, performed by CNNM4 itself, so the process holds even though the MF is refined to the antiporter term. supported_by: - reference_id: PMID:24339795 supporting_text: As shown in Figure 4A, HEK293 cells transfected with CNNM4-FLAG contained more sodium and less magnesium in comparison to control vector-transfected cells, consistent with the occurrence of Mg2+ extrusion. - term: id: GO:0055085 label: transmembrane transport evidence_type: IEA original_reference_id: GO_REF:0000108 qualifier: involved_in review: summary: Logical inference from the generic transporter activity row. True but non-specific. action: MODIFY reason: >- Correct but general. The specific supported processes, GO:1903830 magnesium ion transmembrane transport and GO:0035725 sodium ion transmembrane transport, are both already annotated to this gene. proposed_replacement_terms: - id: GO:1903830 label: magnesium ion transmembrane transport - id: GO:0035725 label: sodium ion transmembrane transport supported_by: - reference_id: PMID:24339795 supporting_text: As shown in Figure 4D, Mg2+ depletion from the extracellular medium induced not only the decrease of intracellular Mg2+ but also the increase of intracellular Na+. - term: id: GO:1903830 label: magnesium ion transmembrane transport evidence_type: IEA original_reference_id: GO_REF:0000108 qualifier: involved_in review: summary: >- Logical inference from magnesium transporter activity. CNNM4 extrudes Mg2+ across the basolateral plasma membrane. action: ACCEPT reason: The core process carried out by CNNM4; supported by mammalian efflux assays. supported_by: - reference_id: PMID:24339795 supporting_text: Functional analyses at the molecular and organismal levels revealed a common role for CNNM4 in mediating transcellular Mg2+ transport by basolateral Mg2+ extrusion. - term: id: GO:0015081 label: sodium ion transmembrane transporter activity evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables review: summary: >- Combined electronic transfer from mouse Cnnm4 (Q69ZF7) and PANTHER; same claim as the IBA row for this term. action: MODIFY reason: Same refinement as the IBA row; Na+ movement is coupled to Mg2+ efflux. proposed_replacement_terms: - id: GO:0061768 label: magnesium:sodium antiporter activity supported_by: - reference_id: PMID:24339795 supporting_text: In the absence of extracellular Na+, Mg2+ depletion did not induce Mg2+ extrusion (Phase 3), but restoration of Na+ instantaneously caused Mg2+ extrusion (Phase 4). - term: id: GO:0015095 label: magnesium ion transmembrane transporter activity evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables review: summary: >- Combined electronic transfer from mouse Cnnm4 (Q69ZF7) and PANTHER; same claim as the IBA row for this term. action: MODIFY reason: Same refinement as the IBA row. proposed_replacement_terms: - id: GO:0061768 label: magnesium:sodium antiporter activity supported_by: - reference_id: PMID:42497866 supporting_text: the outward-facing state, thereby promoting Mg2+/Na+ exchange. - term: id: GO:0016323 label: basolateral plasma membrane evidence_type: IEA original_reference_id: GO_REF:0000107 qualifier: located_in review: summary: >- Ensembl Compara transfer from mouse Cnnm4 (Q69ZF7). Endogenous mouse CNNM4 is at the basolateral membrane of intestinal epithelia and ameloblasts, and human CNNM4 expressed in polarized MDCK (dog kidney) cells is also basolateral. action: ACCEPT reason: >- Ortholog transfer within the CNNM4 subfamily of a location that underlies the vectorial transport function. This is the location in polarized epithelia; it does not exclude plasma-membrane localization in non-polarized cells. supported_by: - reference_id: PMID:24339795 supporting_text: Endogenous CNNM4 is highly expressed in the mature ameloblasts and intestinal epithelia, and localizes at their basolateral membrane. - term: id: GO:0030003 label: intracellular monoatomic cation homeostasis evidence_type: IEA original_reference_id: GO_REF:0000107 qualifier: involved_in review: summary: >- Ensembl Compara transfer from human CNNM4. The human source annotation rests on metal-toxicity assays; the cation CNNM4 is known to handle is Mg2+, and CNNM4 efflux directly lowers intracellular Mg2+. action: MODIFY reason: >- Correct but general. CNNM4-dependent efflux lowers intracellular Mg2+, so the specific descendant GO:0010961 intracellular magnesium ion homeostasis is supported (it is_a GO:0030003 and GO:0010960, the latter already annotated). Other cations are not established as CNNM4 substrates. proposed_replacement_terms: - id: GO:0010961 label: intracellular magnesium ion homeostasis supported_by: - reference_id: PMID:24339795 supporting_text: As shown in Figure 4D, Mg2+ depletion from the extracellular medium induced not only the decrease of intracellular Mg2+ but also the increase of intracellular Na+. - term: id: GO:0032991 label: protein-containing complex evidence_type: IEA original_reference_id: GO_REF:0000107 qualifier: part_of review: summary: >- Ensembl Compara transfer from human CNNM4. Full-length human CNNM4 forms an MgATP-dependent asymmetric tetramer, and CNNM4 associates with TRPM7. action: KEEP_AS_NON_CORE reason: >- True (homo-oligomer; TRPM7 association) but no specific GO complex term exists for either assembly, so the broad term is as precise as available. Kept as non-core. supported_by: - reference_id: PMID:42497866 supporting_text: These structures reveal that CNNM4 does not assemble as a simple symmetric dimer but forms an asymmetric tetramer organized as a dimer-of-dimers. references: - id: GO_REF:0000033 title: Annotation inferences using phylogenetic trees findings: [] - id: GO_REF:0000104 title: Electronic Gene Ontology annotations created by transferring manual GO annotations between related proteins based on shared sequence features findings: [] - id: GO_REF:0000107 title: Automatic transfer of experimentally verified manual GO annotation data to orthologs using Ensembl Compara findings: [] - id: GO_REF:0000108 title: Automatic assignment of GO terms using logical inference, based on on inter-ontology links findings: [] - id: GO_REF:0000118 title: TreeGrafter-generated GO annotations findings: [] - id: GO_REF:0000120 title: Combined Automated Annotation using Multiple IEA Methods findings: [] - id: PMID:24339795 title: 'Basolateral Mg2+ extrusion via CNNM4 mediates transcellular Mg2+ transport across epithelia: a mouse model.' findings: - statement: Mouse CNNM4 localizes to the basolateral membrane of ameloblasts and intestinal epithelia and extrudes Mg2+ in a Na+-dependent manner; knockout mice have defective intestinal Mg2+ absorption and amelogenesis. supporting_text: Functional analyses at the molecular and organismal levels revealed a common role for CNNM4 in mediating transcellular Mg2+ transport by basolateral Mg2+ extrusion. reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text cached; primary experimental source for CNNM4 Mg2+/Na+ exchange and basolateral localization (mouse and human CNNM4 constructs). - id: PMID:42497866 title: Dynamic dimer-of-dimers architecture defines Mg(2+) transport in human CNNM4. findings: - statement: Full-length human CNNM4 forms an MgATP-dependent asymmetric tetramer and its conformational cycle promotes Mg2+/Na+ exchange. supporting_text: Tetramerization is MgATP-dependent, with the intracellular modules remaining highly dynamic otherwise. reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text cached; human CNNM4 cryo-EM structures. - id: PMID:34928937 title: CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells. findings: - statement: CNNM4 coexpression stimulates TRPM7-dependent divalent cation uptake. supporting_text: Coexpression of TRPM7 with CNNM2 and CNNM4 produced the greatest stimulation of TRPM7-dependent Zn2+ uptake. reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Full text cached; supports the TRPM7-regulatory role mentioned in the description. Not used to add a NEW annotation for the dog protein. - id: PMID:19200525 title: Mutations in CNNM4 cause Jalili syndrome, consisting of autosomal-recessive cone-rod dystrophy and amelogenesis imperfecta. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Human disease association cited in the description; phenotype, not a process annotation. - id: file:CANLF/A0A8I3PI07/A0A8I3PI07-bioinformatics/RESULTS.md title: Dog CNNM4 sequence comparison with human CNNM4 and CNNM2 findings: - statement: A0A8I3PI07 is a full-length CNNM4 ortholog (93% identity to human CNNM4, 66% to CNNM2) with a ~21-aa C-terminal insertion outside the transport and CBS regions. supporting_text: The benchmark accession is a full-length CNNM4 gene model, not a fragment. core_functions: - description: >- CNNM4 exchanges intracellular Mg2+ for extracellular Na+ across the basolateral plasma membrane of polarized epithelia (intestine, ameloblasts), providing the exit step of transcellular magnesium absorption and supporting systemic and intracellular Mg2+ balance. MgATP binding to the CBS pair organises the full-length protein into an asymmetric dimer-of-dimers. For the dog protein this is inferred from CNNM4-subfamily placement (PTHR12064:SF26, PAINT node PTN002635769) and 93% identity to human CNNM4. molecular_function: id: GO:0061768 label: magnesium:sodium antiporter activity directly_involved_in: - id: GO:1903830 label: magnesium ion transmembrane transport - id: GO:0035725 label: sodium ion transmembrane transport - id: GO:0010960 label: magnesium ion homeostasis locations: - id: GO:0016323 label: basolateral plasma membrane supported_by: - reference_id: PMID:24339795 supporting_text: In the absence of extracellular Na+, Mg2+ depletion did not induce Mg2+ extrusion (Phase 3), but restoration of Na+ instantaneously caused Mg2+ extrusion (Phase 4). - reference_id: PMID:42497866 supporting_text: the outward-facing state, thereby promoting Mg2+/Na+ exchange. - reference_id: file:CANLF/A0A8I3PI07/A0A8I3PI07-bioinformatics/RESULTS.md supporting_text: 'Dog vs human CNNM4: identity 0.926 over 775 aligned columns (similarity 0.961).' suggested_questions: - question: >- GO:0061768 magnesium:sodium antiporter activity is written as Na+(in) + Mg2+(out) = Na+(out) + Mg2+(in), whereas CNNM4 extrudes Mg2+ and takes up Na+. Should the definition be direction-neutral, or is a separate export-direction term needed? - question: >- Does the ~21-residue C-terminal insertion in the dog CNNM4 gene model correspond to an alternative exon present in other carnivores, and does it affect the CNBH domain? - question: >- Should the TRPM7-stimulating (ion channel regulator) activity shown for human CNNM4 be propagated phylogenetically to CNNM4 orthologs?