{"gene":"KCNG4","run_date":"2026-06-10T01:55:23","timeline":{"discoveries":[{"year":2009,"finding":"Conserved negatively charged aspartates (CDD motif) in the A/B linker of the T1 tetramerization domain are required for efficient assembly of both homotetrameric Kv2.1 and heterotetrameric Kv2.1/Kv6.4 channels; arginine substitution in Kv6.4 at this position prevented its heterotetrameric interaction with Kv2.1, as shown by FRET and co-immunoprecipitation.","method":"Site-directed mutagenesis, co-immunoprecipitation, FRET (confocal microscopy), immunocytochemistry in HEK cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis combined with FRET and co-IP, multiple orthogonal methods in a single focused study","pmids":["19717558"],"is_preprint":false},{"year":2008,"finding":"Histidine 105 in the T1 domain of Kv2.1 is required for heteromerization with Kv6.4; H105V or H105R mutations disrupted T1–T1 interaction with Kv6.4 (and Kv6.3), prevented co-immunoprecipitation of Kv2.1 with Kv6.4, reduced FRET signal, and abolished Kv6.4-mediated shift in voltage dependence of activation.","method":"Yeast two-hybrid, FRET, co-immunoprecipitation, electrophysiology (two-electrode voltage clamp), dominant-negative co-expression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal methods (Y2H, FRET, Co-IP, electrophysiology) in a single rigorous study","pmids":["19074135"],"is_preprint":false},{"year":2015,"finding":"KCNE5 forms a triple complex with Kv2.1 and Kv6.4, modifying Kv2.1/Kv6.4 biophysical properties (accelerated activation, slowed deactivation, steepened inactivation slope, accelerated recovery from closed-state inactivation) without altering current density, as demonstrated by FRET and electrophysiology in HEK293 cells.","method":"Electrophysiology (patch clamp in HEK293 cells), FRET, immunocytochemistry","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal FRET plus electrophysiology, multiple orthogonal methods, single lab","pmids":["26242757"],"is_preprint":false},{"year":2015,"finding":"4-AP potentiates Kv2.1/Kv6.4 currents by suppressing Kv6.4-mediated closed-state inactivation, recovering channels inactivated at resting potential; the lower half of the S6 domain (S6c) of Kv6.4 plays a crucial role in this 4-AP-induced potentiation, as shown by chimeric substitutions between Kv6.4 and Kv9.3.","method":"Electrophysiology (two-electrode voltage clamp in Xenopus oocytes), chimeric subunit substitutions","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — chimeric mutagenesis plus electrophysiological characterization with multiple biophysical measurements","pmids":["26505474"],"is_preprint":false},{"year":2018,"finding":"The S6 activation gate of Kv6.4 contains atypical substitutions that restrict the functional stoichiometry of Kv2.1:Kv6.4 heteromers to 3:1 (Kv2.1:Kv6.4); two amino acid substitutions in the S6 gate of Kv6.4 limit formation and function of 2:2 heteromers, and substituting the self-compatible Kv2.1 T1 domain into Kv6.4 does not alter stoichiometry.","method":"Electrophysiology, concatemer/tandem dimer constructs, mutagenesis, comparative sequence analysis with cnidarian orthologs","journal":"The Journal of general physiology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis combined with electrophysiology and engineered stoichiometry constructs in a single focused study","pmids":["30322883"],"is_preprint":false},{"year":2011,"finding":"Residues T203 in S1 and S347 in S5 of Kv2.1 are energetically coupled and interact with S4 residue R300; double mutant cycle analysis showed these specific S1–S4–S5 interactions are critical for Kv2.1 channel maturation and gating, and are important determinants for the functional Kv2.1/Kv6.4 chimeric channel.","method":"Chimeric Kv2.1/Kv6.4 channels, site-directed mutagenesis, double mutant cycle analysis, electrophysiology","journal":"European biophysics journal","confidence":"Medium","confidence_rationale":"Tier 1–2 / Weak — mutagenesis and double mutant cycle analysis, single lab, focused mechanistic study","pmids":["21455829"],"is_preprint":false},{"year":2020,"finding":"KV6.4 traffics to the plasma membrane to modulate the voltage dependence of KV2.1 inactivation; the rare variant KV6.4-Met419 (rs140124801) fails to traffic to the plasma membrane, exerts a dominant-negative effect, and cannot modulate KV2.1 inactivation, resulting in a more depolarized voltage dependence of inactivation and a higher action potential threshold in neurons overexpressing the mutant.","method":"Functional expression in neurons, electrophysiology (patch clamp), plasma membrane trafficking assay, in vivo retrograde labeling of mouse uterine sensory neurons, human genetic association","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — dominant-negative trafficking assay, neuronal electrophysiology, and in vivo retrograde labeling, multiple orthogonal methods","pmids":["32697988"],"is_preprint":false},{"year":2017,"finding":"Targeted deletion of Kv6.4 (Kcng4-/-) in mice causes male sterility due to disturbed spermiogenesis, manifested by severe reduction in sperm count, absence of motile spermatozoa, and abnormal sperm morphology (smaller head, shorter tail), indicating Kv6.4 is required for normal late-stage spermatogenesis.","method":"Knockout mouse model (targeted gene deletion), semen quality analysis, histology of testicular tissue","journal":"Reproduction, fertility, and development","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KO with defined cellular and morphological phenotype, multiple readouts (sperm count, motility, morphology, histology)","pmids":["27677211"],"is_preprint":false},{"year":2014,"finding":"Dlk1 activates expression of Kv6.4 (Kcng4) in motor neurons to modulate delayed-rectifier K+ currents, promoting a fast biophysical signature; Dlk1 inactivation shifts motor neurons toward slow biophysical properties and abolishes peak force outputs.","method":"Transgenic mouse and chick models, electrophysiology, transcriptome analysis, gain- and loss-of-function experiments","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in multiple species (mouse, chick), electrophysiology, and transcriptome analysis, replicated across models","pmids":["24626931"],"is_preprint":false},{"year":2024,"finding":"The migraine-linked Kv6.4-L360P missense mutation in the S4-S5 linker significantly impairs function of Kv2.1/Kv6.4 channels; when expressed in fixed 2:2 stoichiometry (monomeric and tandem dimer configurations), L360P alters channel biophysical properties, providing a molecular mechanism for channel dysfunction in migraine.","method":"Electrophysiology with monomeric and tandem dimer (fixed stoichiometry) channel constructs in expression system","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — electrophysiology with engineered stoichiometry, single lab, single study","pmids":["39159549"],"is_preprint":false},{"year":2024,"finding":"Kv6.4-L360P (migraine-linked variant) almost completely abolishes Kv2 currents when co-expressed with Kv2.1, proposed to act through a dominant-negative-like mechanism affecting the trigeminal system.","method":"Electrophysiology (heterologous expression with Kv2.1), molecular characterization of variant","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — electrophysiology in expression system, single lab, single study, mechanistic detail limited in abstract","pmids":["39201645"],"is_preprint":false},{"year":2024,"finding":"Two kcng4b mutations in zebrafish (kcng4b-C1 causing mild loss-of-function with C-terminal truncation, and kcng4b-C2 causing gain-of-function by forming an ectopic 7th transmembrane domain) produce distinct ear developmental defects (failure of kinocilia extension and ectopic otoliths for C1; absent otoliths and reduced kinocilia for C2), demonstrating that different functional states of the Kv6.4-containing channel regulate hollow organ development.","method":"Zebrafish mutant analysis, electrophysiology, developmental biology, in silico structural modelling","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic LOF and GOF alleles with distinct phenotypes, electrophysiology and structural modelling, single lab","pmids":["38492873"],"is_preprint":false},{"year":2025,"finding":"Kv6.4 is specifically expressed in spinal motoneurons (not other spinal cord neuron classes) and co-clusters with Kv2.1 and Kv2.2 at endoplasmic reticulum-plasma membrane (ER-PM) junctions beneath C-bouton synapses; Kv6.4 clustering depends on Kv2.1 (severely reduced in Kv2.1 KO) and moderately on Kv2.2 (moderately reduced in Kv2.2 KO), and requires Kv2.1's ability to bind ER VAP proteins (absent in Kv2.1-S590A mutant).","method":"Immunofluorescence, transgenic/knockout mouse models (Kv2.1 KO, Kv2.2 KO, Kv2.1-S590A knockin), confocal microscopy, subcellular fractionation/localization","journal":"The European journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple KO/knockin models with specific localization readouts, multiple orthogonal approaches, peer-reviewed","pmids":["40919874"],"is_preprint":false},{"year":2025,"finding":"In cortical parvalbumin (PV) neurons, Kv6.4 loss (Kcng4 KO) reduces action potential height and width, hyperpolarizes firing threshold and interspike potential, accelerates AP upstroke during repetitive firing, alters GABA release, and changes paired-pulse depression at PV→pyramidal synapses; effects are amplified at high-frequency firing, consistent with Kv6.4 reducing Kv2-mediated delayed rectifier current.","method":"Conditional/constitutive knockout mice, patch-clamp electrophysiology (intrinsic and synaptic), in vivo imaging of Kcng4 expression","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KO with multiple defined electrophysiological and synaptic phenotypes, multiple orthogonal readouts, peer-reviewed","pmids":["41632839"],"is_preprint":false},{"year":2025,"finding":"Dorsal root ganglion (DRG) neurons in mouse and human show predominantly RY785-resistant but GxTX-sensitive conductances, consistent with Kv2/KvS (including Kv6.4-containing) heteromeric channels, whereas superior cervical ganglion neurons show predominantly Kv2-only (RY785-sensitive) conductances; Kv6.4-containing channels are resistant to pore-blocker RY785 but sensitive to voltage-sensor modulator GxTX.","method":"Pharmacological dissection with two Kv2 inhibitors (RY785, GxTX), patch-clamp electrophysiology in native neurons (mouse SCG, mouse and human DRG)","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Moderate — pharmacological approach with two mechanistically distinct inhibitors validated in native neurons including human DRG, peer-reviewed","pmids":["40423692"],"is_preprint":false},{"year":2025,"finding":"In zebrafish, loss-of-function of kcng4b (modulatory subunit) or kcnb1 (Kv2.1, alpha subunit) reduces locomotor activity and induces seizures with pentylenetetrazole; mutants show increased c-fos and gad2 transcripts and decreased gabra1 transcripts, indicating disrupted inhibitory neurotransmission; local field potential recordings show increased spontaneous electrical activity in the optic tectum.","method":"Zebrafish loss-of-function mutants, behavioral assays, qRT-PCR, local field potential recordings","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic LOF with behavioral and electrophysiological readouts, preprint, single lab","pmids":["40501962"],"is_preprint":true}],"current_model":"KCNG4 (Kv6.4) is an electrically silent voltage-gated K+ channel subunit that cannot form functional homotetramers but assembles with Kv2.1 and Kv2.2 via complementary T1 tetramerization domains (requiring specific residues including Kv2.1-H105 and the CDD motif) into functional heterotetramers with a predominant 3:1 (Kv2.1:Kv6.4) stoichiometry enforced by atypical S6 gate substitutions; within these heteromers, Kv6.4 shifts voltage-dependent activation and inactivation (including imposing closed-state inactivation), modulates pharmacological responses (e.g., 4-AP potentiation via S6c), and can further assemble with KCNE5 in a triple complex; Kv6.4 is specifically localized at ER-PM junctions beneath C-bouton synapses in spinal motoneurons and expressed in cortical PV neurons where its loss alters action potential waveform, high-frequency firing dynamics, and GABAergic synaptic output; physiologically, Kv6.4 is required for normal late-stage spermatogenesis (its knockout causes male sterility), regulates uterine nociceptor excitability (influencing labor pain), contributes to fast motor neuron biophysical properties downstream of Dlk1, and modulates ear development and inhibitory circuit function in zebrafish."},"narrative":{"mechanistic_narrative":"KCNG4 encodes Kv6.4, an electrically silent voltage-gated K+ channel subunit that does not form functional homotetramers but assembles into functional heterotetramers with the Kv2.1 pore-forming subunit, where it tunes the voltage dependence and kinetics of channel gating [PMID:19074135, PMID:32697988]. Heteromerization is mediated by complementary T1 tetramerization domains, requiring conserved CDD-motif aspartates in the Kv6.4 A/B linker and Kv2.1 residue His105; disrupting either interface abolishes co-assembly and Kv6.4-imposed gating shifts [PMID:19717558, PMID:19074135]. Atypical substitutions in the Kv6.4 S6 activation gate restrict functional assembly to a predominant 3:1 Kv2.1:Kv6.4 stoichiometry [PMID:30322883]. Within these heteromers Kv6.4 shifts voltage-dependent activation and inactivation, imposes closed-state inactivation that can be relieved pharmacologically by 4-AP through its S6c segment, and the complex can further recruit KCNE5 into a triple complex that further modifies gating kinetics [PMID:26242757, PMID:26505474]. Kv6.4-containing channels are distinguished from Kv2-only channels pharmacologically by resistance to the pore blocker RY785 with retained sensitivity to the voltage-sensor modulator GxTX [PMID:40423692]. In native tissue Kv6.4 co-clusters with Kv2.1 and Kv2.2 at ER-PM junctions beneath C-bouton synapses in spinal motoneurons in a manner dependent on Kv2.1 and its VAP-binding capacity, and in cortical parvalbumin interneurons it shapes action potential waveform, high-frequency firing, and GABAergic synaptic output by reducing Kv2-mediated delayed-rectifier current [PMID:40919874, PMID:41632839]. Physiologically, Kv6.4 confers fast biophysical properties on motor neurons downstream of Dlk1 and is required for normal late-stage spermatogenesis, as its knockout causes male sterility [PMID:27677211, PMID:24626931]. Human Kv6.4 variants link the channel to disease: a non-trafficking dominant-negative variant elevates uterine nociceptor action-potential threshold and influences labor pain, and an S4-S5 linker missense mutation associated with migraine impairs Kv2.1/Kv6.4 function [PMID:32697988, PMID:39159549].","teleology":[{"year":2008,"claim":"Established the molecular interface for Kv6.4 heteromerization, showing that a specific T1-domain residue in the Kv2.1 partner is required for assembly and for Kv6.4's functional gating effect.","evidence":"Yeast two-hybrid, FRET, co-IP and electrophysiology with Kv2.1 H105 mutants","pmids":["19074135"],"confidence":"High","gaps":["Did not define the reciprocal Kv6.4 residues","Stoichiometry of the heteromer not resolved"]},{"year":2009,"claim":"Identified the Kv6.4 side of the assembly interface, showing conserved CDD-motif aspartates in the T1 A/B linker are required for heterotetramerization with Kv2.1.","evidence":"Site-directed mutagenesis, co-IP and FRET in HEK cells","pmids":["19717558"],"confidence":"High","gaps":["Did not establish assembly stoichiometry","Structural basis of the linker contact not resolved"]},{"year":2011,"claim":"Mapped intramembrane S1-S4-S5 residue couplings critical for maturation and gating of functional Kv2.1/Kv6.4 chimeric channels.","evidence":"Chimeric channels and double mutant cycle analysis with electrophysiology","pmids":["21455829"],"confidence":"Medium","gaps":["Used chimeras rather than native heteromers","Single-lab mechanistic study"]},{"year":2014,"claim":"Placed Kv6.4 in a developmental gene regulatory program, showing Dlk1 activates Kcng4 to confer fast biophysical properties on motor neurons.","evidence":"Transgenic mouse/chick gain- and loss-of-function, electrophysiology, transcriptomics","pmids":["24626931"],"confidence":"High","gaps":["Mechanism by which Dlk1 induces Kcng4 transcription not defined","Direct contribution of Kv6.4 currents versus other Dlk1 targets not isolated"]},{"year":2015,"claim":"Defined the pharmacological and accessory-subunit modulation of the heteromer: KCNE5 forms a triple complex altering kinetics, and 4-AP potentiates currents by relieving Kv6.4-imposed closed-state inactivation via S6c.","evidence":"FRET and electrophysiology in HEK293, chimeric Kv6.4/Kv9.3 substitutions in oocytes","pmids":["26242757","26505474"],"confidence":"High","gaps":["Physiological relevance of the KCNE5 triple complex in native tissue not shown","4-AP mechanism mapped to S6c but exact binding determinants unresolved"]},{"year":2017,"claim":"Revealed an unexpected reproductive requirement, showing Kcng4 knockout causes male sterility through disturbed spermiogenesis.","evidence":"Targeted knockout mouse, semen analysis, testicular histology","pmids":["27677211"],"confidence":"High","gaps":["Channel partner and current responsible in germ cells not identified","Cellular mechanism linking K+ conductance to sperm morphogenesis unknown"]},{"year":2018,"claim":"Explained why heteromers adopt a fixed stoichiometry, showing atypical Kv6.4 S6 gate substitutions restrict functional assembly to 3:1 Kv2.1:Kv6.4.","evidence":"Concatemer/tandem-dimer constructs, mutagenesis, electrophysiology","pmids":["30322883"],"confidence":"High","gaps":["Whether 2:2 species assemble but are non-functional not fully resolved","Structural conformation of the mixed gate not determined"]},{"year":2020,"claim":"Connected Kv6.4 to human physiology and disease, showing it traffics to the membrane to modulate Kv2.1 inactivation and that a non-trafficking dominant-negative variant raises action-potential threshold in uterine nociceptors.","evidence":"Neuronal expression, trafficking assays, patch clamp, in vivo retrograde labeling, human genetic association","pmids":["32697988"],"confidence":"High","gaps":["Causal link from nociceptor threshold to labor pain phenotype indirect","Trafficking determinants of the variant not mapped"]},{"year":2024,"claim":"Provided a molecular mechanism for migraine association, showing the S4-S5 linker L360P mutation impairs or abolishes Kv2.1/Kv6.4 currents.","evidence":"Electrophysiology with monomeric and fixed-stoichiometry tandem dimer constructs co-expressed with Kv2.1","pmids":["39159549","39201645"],"confidence":"Medium","gaps":["Dominant-negative mechanism inferred from heterologous expression only","Direct demonstration in trigeminal neurons lacking"]},{"year":2024,"claim":"Demonstrated that distinct functional states of Kv6.4-containing channels control hollow organ morphogenesis, using zebrafish loss- and gain-of-function ear alleles.","evidence":"Zebrafish mutant analysis, electrophysiology, structural modelling","pmids":["38492873"],"confidence":"Medium","gaps":["Channel partner in developing ear not defined","Mechanism linking conductance state to kinocilia/otolith defects unknown"]},{"year":2025,"claim":"Localized native Kv6.4 to ER-PM junctions beneath C-bouton synapses in spinal motoneurons and showed clustering depends on Kv2.1 and its VAP-binding ability.","evidence":"Immunofluorescence and confocal microscopy in Kv2.1 KO, Kv2.2 KO and Kv2.1-S590A knockin mice","pmids":["40919874"],"confidence":"High","gaps":["Functional consequence of ER-PM clustering for motoneuron firing not directly tested","Role of the C-bouton localization in motor output undefined"]},{"year":2025,"claim":"Defined Kv6.4's role in cortical inhibitory circuits and its native pharmacological signature, showing loss alters PV-neuron action potential waveform, high-frequency firing and GABA release, and that Kv6.4-containing channels are RY785-resistant/GxTX-sensitive in native neurons.","evidence":"Knockout mouse patch-clamp (intrinsic and synaptic) plus pharmacological dissection with RY785/GxTX in mouse and human DRG and SCG neurons","pmids":["41632839","40423692"],"confidence":"High","gaps":["Network-level behavioral consequences in mammals not established","Relative contribution of Kv2.1 versus Kv2.2 partners in PV neurons not resolved"]},{"year":2025,"claim":"Linked Kv6.4/Kv2.1 loss to disrupted inhibitory neurotransmission and seizure susceptibility in zebrafish.","evidence":"Zebrafish loss-of-function mutants, behavioral assays, qRT-PCR, local field potential recordings (preprint)","pmids":["40501962"],"confidence":"Medium","gaps":["Preprint, not peer-reviewed","Mechanism connecting channel loss to altered GABAergic transcripts not established"]},{"year":null,"claim":"How Kv6.4 K+ conductance is mechanistically coupled to its diverse organ-level roles—spermiogenesis, ear morphogenesis, and labor/migraine nociception—remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the assembled Kv2.1/Kv6.4 heteromer","Tissue-specific partners outside neurons not identified","Causal chain from channel biophysics to morphogenetic phenotypes unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[1,4,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,6,3]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[6,12]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[12]}],"pathway":[{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[6,13]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[8,11]}],"complexes":["Kv2.1/Kv6.4 heterotetramer","Kv2.1/Kv6.4/KCNE5 triple complex"],"partners":["KCNB1","KCNB2","KCNE5"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8TDN1","full_name":"Voltage-gated potassium channel regulatory subunit KCNG4","aliases":["Potassium voltage-gated channel subfamily G member 4","Voltage-gated potassium channel subunit Kv6.3","Voltage-gated potassium channel subunit Kv6.4"],"length_aa":519,"mass_kda":59.0,"function":"Regulatory subunit of the voltage-gated potassium (Kv) channel which, when coassembled with KCNB1, modulates the kinetics parameters of the heterotetrameric channel namely the time course of activation, deactivation and inactivation and on the voltage-dependence of activation (PubMed:12060745, PubMed:19074135). Potassium channel subunit that does not form functional channels by itself (Probable) (PubMed:12060745). Reduces the deactivation rate (PubMed:12060745, PubMed:19074135). Modulates the threshold for activation by shifting by approximately 20 mV in hyperpolarizing direction (PubMed:12060745). Markedly changes the inactivation by shifting the voltage dependence of inactivation by approximately 40 mV in hyperpolarizing direction (PubMed:12060745). Acceleratee activation and enhances the time course of activation (PubMed:12060745)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q8TDN1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/KCNG4","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/KCNG4","total_profiled":1310},"omim":[{"mim_id":"607603","title":"POTASSIUM CHANNEL, VOLTAGE-GATED, SUBFAMILY G, MEMBER 4; KCNG4","url":"https://www.omim.org/entry/607603"},{"mim_id":"606767","title":"POTASSIUM CHANNEL, VOLTAGE-GATED, SUBFAMILY G, MEMBER 3; KCNG3","url":"https://www.omim.org/entry/606767"},{"mim_id":"600397","title":"POTASSIUM CHANNEL, VOLTAGE-GATED, SHAB-RELATED SUBFAMILY, MEMBER 1; KCNB1","url":"https://www.omim.org/entry/600397"},{"mim_id":"176290","title":"DELTA-LIKE NONCANONICAL NOTCH LIGAND 1; DLK1","url":"https://www.omim.org/entry/176290"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in single","driving_tissues":[{"tissue":"retina","ntpm":3.6}],"url":"https://www.proteinatlas.org/search/KCNG4"},"hgnc":{"alias_symbol":["Kv6.4"],"prev_symbol":[]},"alphafold":{"accession":"Q8TDN1","domains":[{"cath_id":"3.30.710.10","chopping":"39-156","consensus_level":"high","plddt":83.7448,"start":39,"end":156},{"cath_id":"1.20.120.350","chopping":"213-315_329-352","consensus_level":"high","plddt":85.8254,"start":213,"end":352},{"cath_id":"1.10.287.70","chopping":"358-476","consensus_level":"high","plddt":91.1139,"start":358,"end":476}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TDN1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TDN1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TDN1-F1-predicted_aligned_error_v6.png","plddt_mean":77.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=KCNG4","jax_strain_url":"https://www.jax.org/strain/search?query=KCNG4"},"sequence":{"accession":"Q8TDN1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TDN1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TDN1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TDN1"}},"corpus_meta":[{"pmid":"24626931","id":"PMC_24626931","title":"Dlk1 promotes a fast motor neuron biophysical signature required for peak force execution.","date":"2014","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/24626931","citation_count":59,"is_preprint":false},{"pmid":"33731450","id":"PMC_33731450","title":"Dissociable Roles of Pallidal Neuron Subtypes in Regulating Motor Patterns.","date":"2021","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/33731450","citation_count":49,"is_preprint":false},{"pmid":"31170158","id":"PMC_31170158","title":"Exome sequencing in multiple sclerosis families identifies 12 candidate genes and nominates biological pathways for the genesis of disease.","date":"2019","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/31170158","citation_count":46,"is_preprint":false},{"pmid":"32697988","id":"PMC_32697988","title":"Human Labor Pain Is Influenced by the Voltage-Gated Potassium Channel KV6.4 Subunit.","date":"2020","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/32697988","citation_count":31,"is_preprint":false},{"pmid":"23030542","id":"PMC_23030542","title":"Identification of novel genes involved in migraine.","date":"2012","source":"Headache","url":"https://pubmed.ncbi.nlm.nih.gov/23030542","citation_count":30,"is_preprint":false},{"pmid":"26242757","id":"PMC_26242757","title":"Auxiliary KCNE subunits modulate both homotetrameric Kv2.1 and heterotetrameric Kv2.1/Kv6.4 channels.","date":"2015","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/26242757","citation_count":26,"is_preprint":false},{"pmid":"36292658","id":"PMC_36292658","title":"Identification of Genomic Regions and Candidate Genes Associated with Body Weight and Body Conformation Traits in Karachai Goats.","date":"2022","source":"Genes","url":"https://pubmed.ncbi.nlm.nih.gov/36292658","citation_count":26,"is_preprint":false},{"pmid":"19717558","id":"PMC_19717558","title":"Conserved negative charges in the N-terminal tetramerization domain mediate efficient assembly of Kv2.1 and Kv2.1/Kv6.4 channels.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19717558","citation_count":23,"is_preprint":false},{"pmid":"26505474","id":"PMC_26505474","title":"Modulation of Closed-State Inactivation in Kv2.1/Kv6.4 Heterotetramers as Mechanism for 4-AP Induced Potentiation.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26505474","citation_count":21,"is_preprint":false},{"pmid":"19074135","id":"PMC_19074135","title":"Mutation of histidine 105 in the T1 domain of the potassium channel Kv2.1 disrupts heteromerization with Kv6.3 and Kv6.4.","date":"2008","source":"The Journal of biological 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development","date":"2024-12-20","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.20.629661","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":20129,"output_tokens":4432,"usd":0.063434,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12226,"output_tokens":4288,"usd":0.084165,"stage2_stop_reason":"end_turn"},"total_usd":0.147599,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"Conserved negatively charged aspartates (CDD motif) in the A/B linker of the T1 tetramerization domain are required for efficient assembly of both homotetrameric Kv2.1 and heterotetrameric Kv2.1/Kv6.4 channels; arginine substitution in Kv6.4 at this position prevented its heterotetrameric interaction with Kv2.1, as shown by FRET and co-immunoprecipitation.\",\n      \"method\": \"Site-directed mutagenesis, co-immunoprecipitation, FRET (confocal microscopy), immunocytochemistry in HEK cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis combined with FRET and co-IP, multiple orthogonal methods in a single focused study\",\n      \"pmids\": [\"19717558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Histidine 105 in the T1 domain of Kv2.1 is required for heteromerization with Kv6.4; H105V or H105R mutations disrupted T1–T1 interaction with Kv6.4 (and Kv6.3), prevented co-immunoprecipitation of Kv2.1 with Kv6.4, reduced FRET signal, and abolished Kv6.4-mediated shift in voltage dependence of activation.\",\n      \"method\": \"Yeast two-hybrid, FRET, co-immunoprecipitation, electrophysiology (two-electrode voltage clamp), dominant-negative co-expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal methods (Y2H, FRET, Co-IP, electrophysiology) in a single rigorous study\",\n      \"pmids\": [\"19074135\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"KCNE5 forms a triple complex with Kv2.1 and Kv6.4, modifying Kv2.1/Kv6.4 biophysical properties (accelerated activation, slowed deactivation, steepened inactivation slope, accelerated recovery from closed-state inactivation) without altering current density, as demonstrated by FRET and electrophysiology in HEK293 cells.\",\n      \"method\": \"Electrophysiology (patch clamp in HEK293 cells), FRET, immunocytochemistry\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal FRET plus electrophysiology, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"26242757\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"4-AP potentiates Kv2.1/Kv6.4 currents by suppressing Kv6.4-mediated closed-state inactivation, recovering channels inactivated at resting potential; the lower half of the S6 domain (S6c) of Kv6.4 plays a crucial role in this 4-AP-induced potentiation, as shown by chimeric substitutions between Kv6.4 and Kv9.3.\",\n      \"method\": \"Electrophysiology (two-electrode voltage clamp in Xenopus oocytes), chimeric subunit substitutions\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — chimeric mutagenesis plus electrophysiological characterization with multiple biophysical measurements\",\n      \"pmids\": [\"26505474\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The S6 activation gate of Kv6.4 contains atypical substitutions that restrict the functional stoichiometry of Kv2.1:Kv6.4 heteromers to 3:1 (Kv2.1:Kv6.4); two amino acid substitutions in the S6 gate of Kv6.4 limit formation and function of 2:2 heteromers, and substituting the self-compatible Kv2.1 T1 domain into Kv6.4 does not alter stoichiometry.\",\n      \"method\": \"Electrophysiology, concatemer/tandem dimer constructs, mutagenesis, comparative sequence analysis with cnidarian orthologs\",\n      \"journal\": \"The Journal of general physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis combined with electrophysiology and engineered stoichiometry constructs in a single focused study\",\n      \"pmids\": [\"30322883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Residues T203 in S1 and S347 in S5 of Kv2.1 are energetically coupled and interact with S4 residue R300; double mutant cycle analysis showed these specific S1–S4–S5 interactions are critical for Kv2.1 channel maturation and gating, and are important determinants for the functional Kv2.1/Kv6.4 chimeric channel.\",\n      \"method\": \"Chimeric Kv2.1/Kv6.4 channels, site-directed mutagenesis, double mutant cycle analysis, electrophysiology\",\n      \"journal\": \"European biophysics journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Weak — mutagenesis and double mutant cycle analysis, single lab, focused mechanistic study\",\n      \"pmids\": [\"21455829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"KV6.4 traffics to the plasma membrane to modulate the voltage dependence of KV2.1 inactivation; the rare variant KV6.4-Met419 (rs140124801) fails to traffic to the plasma membrane, exerts a dominant-negative effect, and cannot modulate KV2.1 inactivation, resulting in a more depolarized voltage dependence of inactivation and a higher action potential threshold in neurons overexpressing the mutant.\",\n      \"method\": \"Functional expression in neurons, electrophysiology (patch clamp), plasma membrane trafficking assay, in vivo retrograde labeling of mouse uterine sensory neurons, human genetic association\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — dominant-negative trafficking assay, neuronal electrophysiology, and in vivo retrograde labeling, multiple orthogonal methods\",\n      \"pmids\": [\"32697988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Targeted deletion of Kv6.4 (Kcng4-/-) in mice causes male sterility due to disturbed spermiogenesis, manifested by severe reduction in sperm count, absence of motile spermatozoa, and abnormal sperm morphology (smaller head, shorter tail), indicating Kv6.4 is required for normal late-stage spermatogenesis.\",\n      \"method\": \"Knockout mouse model (targeted gene deletion), semen quality analysis, histology of testicular tissue\",\n      \"journal\": \"Reproduction, fertility, and development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined cellular and morphological phenotype, multiple readouts (sperm count, motility, morphology, histology)\",\n      \"pmids\": [\"27677211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Dlk1 activates expression of Kv6.4 (Kcng4) in motor neurons to modulate delayed-rectifier K+ currents, promoting a fast biophysical signature; Dlk1 inactivation shifts motor neurons toward slow biophysical properties and abolishes peak force outputs.\",\n      \"method\": \"Transgenic mouse and chick models, electrophysiology, transcriptome analysis, gain- and loss-of-function experiments\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in multiple species (mouse, chick), electrophysiology, and transcriptome analysis, replicated across models\",\n      \"pmids\": [\"24626931\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The migraine-linked Kv6.4-L360P missense mutation in the S4-S5 linker significantly impairs function of Kv2.1/Kv6.4 channels; when expressed in fixed 2:2 stoichiometry (monomeric and tandem dimer configurations), L360P alters channel biophysical properties, providing a molecular mechanism for channel dysfunction in migraine.\",\n      \"method\": \"Electrophysiology with monomeric and tandem dimer (fixed stoichiometry) channel constructs in expression system\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — electrophysiology with engineered stoichiometry, single lab, single study\",\n      \"pmids\": [\"39159549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Kv6.4-L360P (migraine-linked variant) almost completely abolishes Kv2 currents when co-expressed with Kv2.1, proposed to act through a dominant-negative-like mechanism affecting the trigeminal system.\",\n      \"method\": \"Electrophysiology (heterologous expression with Kv2.1), molecular characterization of variant\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — electrophysiology in expression system, single lab, single study, mechanistic detail limited in abstract\",\n      \"pmids\": [\"39201645\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Two kcng4b mutations in zebrafish (kcng4b-C1 causing mild loss-of-function with C-terminal truncation, and kcng4b-C2 causing gain-of-function by forming an ectopic 7th transmembrane domain) produce distinct ear developmental defects (failure of kinocilia extension and ectopic otoliths for C1; absent otoliths and reduced kinocilia for C2), demonstrating that different functional states of the Kv6.4-containing channel regulate hollow organ development.\",\n      \"method\": \"Zebrafish mutant analysis, electrophysiology, developmental biology, in silico structural modelling\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic LOF and GOF alleles with distinct phenotypes, electrophysiology and structural modelling, single lab\",\n      \"pmids\": [\"38492873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Kv6.4 is specifically expressed in spinal motoneurons (not other spinal cord neuron classes) and co-clusters with Kv2.1 and Kv2.2 at endoplasmic reticulum-plasma membrane (ER-PM) junctions beneath C-bouton synapses; Kv6.4 clustering depends on Kv2.1 (severely reduced in Kv2.1 KO) and moderately on Kv2.2 (moderately reduced in Kv2.2 KO), and requires Kv2.1's ability to bind ER VAP proteins (absent in Kv2.1-S590A mutant).\",\n      \"method\": \"Immunofluorescence, transgenic/knockout mouse models (Kv2.1 KO, Kv2.2 KO, Kv2.1-S590A knockin), confocal microscopy, subcellular fractionation/localization\",\n      \"journal\": \"The European journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple KO/knockin models with specific localization readouts, multiple orthogonal approaches, peer-reviewed\",\n      \"pmids\": [\"40919874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In cortical parvalbumin (PV) neurons, Kv6.4 loss (Kcng4 KO) reduces action potential height and width, hyperpolarizes firing threshold and interspike potential, accelerates AP upstroke during repetitive firing, alters GABA release, and changes paired-pulse depression at PV→pyramidal synapses; effects are amplified at high-frequency firing, consistent with Kv6.4 reducing Kv2-mediated delayed rectifier current.\",\n      \"method\": \"Conditional/constitutive knockout mice, patch-clamp electrophysiology (intrinsic and synaptic), in vivo imaging of Kcng4 expression\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with multiple defined electrophysiological and synaptic phenotypes, multiple orthogonal readouts, peer-reviewed\",\n      \"pmids\": [\"41632839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Dorsal root ganglion (DRG) neurons in mouse and human show predominantly RY785-resistant but GxTX-sensitive conductances, consistent with Kv2/KvS (including Kv6.4-containing) heteromeric channels, whereas superior cervical ganglion neurons show predominantly Kv2-only (RY785-sensitive) conductances; Kv6.4-containing channels are resistant to pore-blocker RY785 but sensitive to voltage-sensor modulator GxTX.\",\n      \"method\": \"Pharmacological dissection with two Kv2 inhibitors (RY785, GxTX), patch-clamp electrophysiology in native neurons (mouse SCG, mouse and human DRG)\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological approach with two mechanistically distinct inhibitors validated in native neurons including human DRG, peer-reviewed\",\n      \"pmids\": [\"40423692\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In zebrafish, loss-of-function of kcng4b (modulatory subunit) or kcnb1 (Kv2.1, alpha subunit) reduces locomotor activity and induces seizures with pentylenetetrazole; mutants show increased c-fos and gad2 transcripts and decreased gabra1 transcripts, indicating disrupted inhibitory neurotransmission; local field potential recordings show increased spontaneous electrical activity in the optic tectum.\",\n      \"method\": \"Zebrafish loss-of-function mutants, behavioral assays, qRT-PCR, local field potential recordings\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic LOF with behavioral and electrophysiological readouts, preprint, single lab\",\n      \"pmids\": [\"40501962\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"KCNG4 (Kv6.4) is an electrically silent voltage-gated K+ channel subunit that cannot form functional homotetramers but assembles with Kv2.1 and Kv2.2 via complementary T1 tetramerization domains (requiring specific residues including Kv2.1-H105 and the CDD motif) into functional heterotetramers with a predominant 3:1 (Kv2.1:Kv6.4) stoichiometry enforced by atypical S6 gate substitutions; within these heteromers, Kv6.4 shifts voltage-dependent activation and inactivation (including imposing closed-state inactivation), modulates pharmacological responses (e.g., 4-AP potentiation via S6c), and can further assemble with KCNE5 in a triple complex; Kv6.4 is specifically localized at ER-PM junctions beneath C-bouton synapses in spinal motoneurons and expressed in cortical PV neurons where its loss alters action potential waveform, high-frequency firing dynamics, and GABAergic synaptic output; physiologically, Kv6.4 is required for normal late-stage spermatogenesis (its knockout causes male sterility), regulates uterine nociceptor excitability (influencing labor pain), contributes to fast motor neuron biophysical properties downstream of Dlk1, and modulates ear development and inhibitory circuit function in zebrafish.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"KCNG4 encodes Kv6.4, an electrically silent voltage-gated K+ channel subunit that does not form functional homotetramers but assembles into functional heterotetramers with the Kv2.1 pore-forming subunit, where it tunes the voltage dependence and kinetics of channel gating [#1, #6]. Heteromerization is mediated by complementary T1 tetramerization domains, requiring conserved CDD-motif aspartates in the Kv6.4 A/B linker and Kv2.1 residue His105; disrupting either interface abolishes co-assembly and Kv6.4-imposed gating shifts [#0, #1]. Atypical substitutions in the Kv6.4 S6 activation gate restrict functional assembly to a predominant 3:1 Kv2.1:Kv6.4 stoichiometry [#4]. Within these heteromers Kv6.4 shifts voltage-dependent activation and inactivation, imposes closed-state inactivation that can be relieved pharmacologically by 4-AP through its S6c segment, and the complex can further recruit KCNE5 into a triple complex that further modifies gating kinetics [#2, #3]. Kv6.4-containing channels are distinguished from Kv2-only channels pharmacologically by resistance to the pore blocker RY785 with retained sensitivity to the voltage-sensor modulator GxTX [#14]. In native tissue Kv6.4 co-clusters with Kv2.1 and Kv2.2 at ER-PM junctions beneath C-bouton synapses in spinal motoneurons in a manner dependent on Kv2.1 and its VAP-binding capacity, and in cortical parvalbumin interneurons it shapes action potential waveform, high-frequency firing, and GABAergic synaptic output by reducing Kv2-mediated delayed-rectifier current [#12, #13]. Physiologically, Kv6.4 confers fast biophysical properties on motor neurons downstream of Dlk1 and is required for normal late-stage spermatogenesis, as its knockout causes male sterility [#7, #8]. Human Kv6.4 variants link the channel to disease: a non-trafficking dominant-negative variant elevates uterine nociceptor action-potential threshold and influences labor pain, and an S4-S5 linker missense mutation associated with migraine impairs Kv2.1/Kv6.4 function [#6, #9].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Established the molecular interface for Kv6.4 heteromerization, showing that a specific T1-domain residue in the Kv2.1 partner is required for assembly and for Kv6.4's functional gating effect.\",\n      \"evidence\": \"Yeast two-hybrid, FRET, co-IP and electrophysiology with Kv2.1 H105 mutants\",\n      \"pmids\": [\"19074135\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the reciprocal Kv6.4 residues\", \"Stoichiometry of the heteromer not resolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified the Kv6.4 side of the assembly interface, showing conserved CDD-motif aspartates in the T1 A/B linker are required for heterotetramerization with Kv2.1.\",\n      \"evidence\": \"Site-directed mutagenesis, co-IP and FRET in HEK cells\",\n      \"pmids\": [\"19717558\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish assembly stoichiometry\", \"Structural basis of the linker contact not resolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Mapped intramembrane S1-S4-S5 residue couplings critical for maturation and gating of functional Kv2.1/Kv6.4 chimeric channels.\",\n      \"evidence\": \"Chimeric channels and double mutant cycle analysis with electrophysiology\",\n      \"pmids\": [\"21455829\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Used chimeras rather than native heteromers\", \"Single-lab mechanistic study\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Placed Kv6.4 in a developmental gene regulatory program, showing Dlk1 activates Kcng4 to confer fast biophysical properties on motor neurons.\",\n      \"evidence\": \"Transgenic mouse/chick gain- and loss-of-function, electrophysiology, transcriptomics\",\n      \"pmids\": [\"24626931\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which Dlk1 induces Kcng4 transcription not defined\", \"Direct contribution of Kv6.4 currents versus other Dlk1 targets not isolated\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined the pharmacological and accessory-subunit modulation of the heteromer: KCNE5 forms a triple complex altering kinetics, and 4-AP potentiates currents by relieving Kv6.4-imposed closed-state inactivation via S6c.\",\n      \"evidence\": \"FRET and electrophysiology in HEK293, chimeric Kv6.4/Kv9.3 substitutions in oocytes\",\n      \"pmids\": [\"26242757\", \"26505474\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance of the KCNE5 triple complex in native tissue not shown\", \"4-AP mechanism mapped to S6c but exact binding determinants unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Revealed an unexpected reproductive requirement, showing Kcng4 knockout causes male sterility through disturbed spermiogenesis.\",\n      \"evidence\": \"Targeted knockout mouse, semen analysis, testicular histology\",\n      \"pmids\": [\"27677211\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Channel partner and current responsible in germ cells not identified\", \"Cellular mechanism linking K+ conductance to sperm morphogenesis unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Explained why heteromers adopt a fixed stoichiometry, showing atypical Kv6.4 S6 gate substitutions restrict functional assembly to 3:1 Kv2.1:Kv6.4.\",\n      \"evidence\": \"Concatemer/tandem-dimer constructs, mutagenesis, electrophysiology\",\n      \"pmids\": [\"30322883\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether 2:2 species assemble but are non-functional not fully resolved\", \"Structural conformation of the mixed gate not determined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Connected Kv6.4 to human physiology and disease, showing it traffics to the membrane to modulate Kv2.1 inactivation and that a non-trafficking dominant-negative variant raises action-potential threshold in uterine nociceptors.\",\n      \"evidence\": \"Neuronal expression, trafficking assays, patch clamp, in vivo retrograde labeling, human genetic association\",\n      \"pmids\": [\"32697988\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Causal link from nociceptor threshold to labor pain phenotype indirect\", \"Trafficking determinants of the variant not mapped\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided a molecular mechanism for migraine association, showing the S4-S5 linker L360P mutation impairs or abolishes Kv2.1/Kv6.4 currents.\",\n      \"evidence\": \"Electrophysiology with monomeric and fixed-stoichiometry tandem dimer constructs co-expressed with Kv2.1\",\n      \"pmids\": [\"39159549\", \"39201645\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Dominant-negative mechanism inferred from heterologous expression only\", \"Direct demonstration in trigeminal neurons lacking\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated that distinct functional states of Kv6.4-containing channels control hollow organ morphogenesis, using zebrafish loss- and gain-of-function ear alleles.\",\n      \"evidence\": \"Zebrafish mutant analysis, electrophysiology, structural modelling\",\n      \"pmids\": [\"38492873\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Channel partner in developing ear not defined\", \"Mechanism linking conductance state to kinocilia/otolith defects unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Localized native Kv6.4 to ER-PM junctions beneath C-bouton synapses in spinal motoneurons and showed clustering depends on Kv2.1 and its VAP-binding ability.\",\n      \"evidence\": \"Immunofluorescence and confocal microscopy in Kv2.1 KO, Kv2.2 KO and Kv2.1-S590A knockin mice\",\n      \"pmids\": [\"40919874\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of ER-PM clustering for motoneuron firing not directly tested\", \"Role of the C-bouton localization in motor output undefined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined Kv6.4's role in cortical inhibitory circuits and its native pharmacological signature, showing loss alters PV-neuron action potential waveform, high-frequency firing and GABA release, and that Kv6.4-containing channels are RY785-resistant/GxTX-sensitive in native neurons.\",\n      \"evidence\": \"Knockout mouse patch-clamp (intrinsic and synaptic) plus pharmacological dissection with RY785/GxTX in mouse and human DRG and SCG neurons\",\n      \"pmids\": [\"41632839\", \"40423692\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Network-level behavioral consequences in mammals not established\", \"Relative contribution of Kv2.1 versus Kv2.2 partners in PV neurons not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked Kv6.4/Kv2.1 loss to disrupted inhibitory neurotransmission and seizure susceptibility in zebrafish.\",\n      \"evidence\": \"Zebrafish loss-of-function mutants, behavioral assays, qRT-PCR, local field potential recordings (preprint)\",\n      \"pmids\": [\"40501962\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not peer-reviewed\", \"Mechanism connecting channel loss to altered GABAergic transcripts not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How Kv6.4 K+ conductance is mechanistically coupled to its diverse organ-level roles—spermiogenesis, ear morphogenesis, and labor/migraine nociception—remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the assembled Kv2.1/Kv6.4 heteromer\", \"Tissue-specific partners outside neurons not identified\", \"Causal chain from channel biophysics to morphogenetic phenotypes unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [1, 4, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 6, 3]},\n      {\"term_id\": \"GO:0005216\", \"supporting_discovery_ids\": [1, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [6, 12]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [6, 13]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [8, 11]}\n    ],\n    \"complexes\": [\n      \"Kv2.1/Kv6.4 heterotetramer\",\n      \"Kv2.1/Kv6.4/KCNE5 triple complex\"\n    ],\n    \"partners\": [\n      \"KCNB1\",\n      \"KCNB2\",\n      \"KCNE5\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}