{"gene":"SCN5A","run_date":"2026-06-10T07:46:29","timeline":{"discoveries":[{"year":2000,"finding":"SCN5A encodes the pore-forming α-subunit of the cardiac sodium channel responsible for action potential initiation and propagation; gain-of-function mutations cause persistent (late) sodium current leading to LQT3, while distinct mutations cause loss-of-function associated with Brugada syndrome.","method":"Mutational analysis and patch-clamp electrophysiology in heterologous expression systems","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 1 / Strong — replicated across hundreds of mutations in multiple labs using in vitro electrophysiology; foundational mechanistic framework established by multiple orthogonal studies","pmids":["10973849","19027780","29798782"],"is_preprint":false},{"year":2002,"finding":"The Brugada syndrome double mutant R1232W/T1620M abolishes Nav1.5 functional expression because the channel is retained in the endoplasmic reticulum rather than trafficking to the cell surface; a positively charged residue at position 1232 (R or K) is required for proper channel transport.","method":"Whole-cell patch-clamp, immunofluorescence, confocal microscopy with FLAG-tagged constructs, calnexin co-localization in tsA201 cells","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — direct subcellular localization with functional consequence, mutagenesis rescue experiment, orthogonal imaging and electrophysiology in single focused paper","pmids":["11786529"],"is_preprint":false},{"year":2003,"finding":"The LQT3 mutation L619F in the domain I-II linker of Nav1.5 increases maintained (persistent) Na+ current and causes a +5.8 mV shift in steady-state inactivation, implicating the I-II linker in inactivation gating.","method":"Whole-cell patch-clamp in transiently expressed HEK293 cells","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro electrophysiology with mutagenesis, single lab, single method","pmids":["12673799"],"is_preprint":false},{"year":2003,"finding":"The LQT3 mutation I1768V in domain IVS6 causes faster recovery from inactivation and less slow inactivation without increasing persistent Na+ current, implicating domain IVS6 in inactivation kinetics.","method":"Two-electrode voltage clamp in Xenopus oocytes","journal":"Cardiovascular research","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — reconstituted in Xenopus oocytes, single lab, single method","pmids":["12650885"],"is_preprint":false},{"year":2004,"finding":"Transgenic mice with cardiac-specific expression of the LQT3 mutation N1325S in SCN5A display QT prolongation, spontaneous polymorphic VT/VF, and slow recovery from inactivation of INa; mexiletine (a late Na+ current blocker) suppresses arrhythmias and restores inactivation recovery, establishing that the N1325S-induced persistent Na+ current causes arrhythmogenesis in vivo.","method":"Transgenic mouse model, ECG, action potential recording, voltage-clamp in isolated myocytes, mexiletine pharmacology","journal":"Cardiovascular research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vivo transgenic model with voltage-clamp confirmation and pharmacological rescue, multiple orthogonal methods","pmids":["14736542"],"is_preprint":false},{"year":2005,"finding":"The LQT3 mutant L1825P fails to traffic to the cell surface (only ~9% of wild-type surface expression) and therefore does not prolong QT interval; cisapride rescues misprocessing to ~30% of wild-type surface expression and simultaneously increases late Na+ current, revealing that cell surface trafficking determines LQT3 phenotype expressivity.","method":"Patch-clamp, confocal imaging and cell-counting with epitope-tagged constructs in CHO cells","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — direct surface-expression quantification with pharmacological rescue plus electrophysiology, multiple orthogonal methods in a single focused study","pmids":["16301357"],"is_preprint":false},{"year":2005,"finding":"Two Brugada syndrome mutations (L325R and R535X) cause severe Nav1.5 loss-of-function; the L325R mutant is likely misfolded (rescued partially by mexiletine or curcumin) and exerts a dominant-negative effect on wild-type channels when co-expressed. Fever accelerates Nav1.5 activation and fast inactivation kinetics, providing the mechanism by which reduced INa patients are vulnerable to fever-exacerbated Brugada syndrome.","method":"Whole-cell patch-clamp at various temperatures, Western blot protein expression, co-expression dominant-negative experiments, action potential modelling in HEK293 cells","journal":"Cardiovascular research","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal methods (electrophysiology, biochemistry, temperature manipulation, pharmacological rescue) in single rigorous paper","pmids":["15890323"],"is_preprint":false},{"year":2008,"finding":"Telethonin (encoded by TCAP) is a Nav1.5-interacting protein: it co-localizes with Nav1.5 in gastrointestinal smooth muscle, co-immunoprecipitates with sodium channels, and a disease-associated telethonin mutation doubles the window current of Nav1.5 when co-expressed in HEK293 cells by altering steady-state activation kinetics.","method":"Co-immunoprecipitation, immunofluorescence co-localization, whole-cell patch-clamp in HEK293 cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — reciprocal Co-IP and localization with functional electrophysiology, single lab","pmids":["18408010"],"is_preprint":false},{"year":2010,"finding":"Nav1.5 is mechanosensitive: applying negative patch pressure produces dose-dependent hyperpolarizing shifts in voltage dependence of activation and inactivation (~0.7 mV/mmHg), accelerates voltage-sensor movement but not gate-opening or fast inactivation rate constants, and increases peak current by recruiting more active channels, without changing unitary conductance or maximum open probability.","method":"Cell-attached patch-clamp with controlled pressure in HEK293 cells expressing Nav1.5","journal":"The Journal of physiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — rigorous in vitro single-channel and whole-cell electrophysiology with graded mechanical stimuli, dissecting specific voltage-sensor transitions","pmids":["21041530"],"is_preprint":false},{"year":2011,"finding":"Nav1.5-dependent persistent Na+ influx (late INa) activates CaMKII in cardiomyocytes; Nav1.5 knockdown (but not Nav1.1 or Nav1.2) prevents ATX-II-induced CaMKII phosphorylation, and CaMKII co-immunoprecipitates with Nav1.5 after late INa activation, establishing a feed-forward loop where Nav1.5 late current → CaMKII activation → CaMKII phosphorylation of Nav1.5 → further late current.","method":"siRNA knockdown, CaMKII autophosphorylation assays, co-immunoprecipitation, patch-clamp, transgenic N1325S mouse model, pharmacological inhibition (ranolazine, KN93) in neonatal rat ventricular myocytes","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (KD, Co-IP, in vivo mouse model, pharmacology) replicated in cell and animal systems, single lab","pmids":["21677263"],"is_preprint":false},{"year":2012,"finding":"TBX5 directly drives Scn5a expression in the ventricular conduction system through a TBX5-responsive enhancer downstream of Scn5a that requires canonical T-box binding sites; conditional deletion of Tbx5 from the ventricular conduction system reduces Nav1.5 protein expression and causes severe conduction dysfunction.","method":"Conditional mouse knockout, in vivo enhancer assay, T-box site mutagenesis, immunostaining, optical mapping, ECG","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — enhancer identified by in vivo reporter assay with site-specific mutagenesis plus conditional KO with functional phenotype, multiple orthogonal methods","pmids":["22728936"],"is_preprint":false},{"year":2012,"finding":"αB-crystallin physically interacts with Nav1.5 (validated by yeast two-hybrid, GST pulldown, Co-IP, and immunostaining); overexpression increases peak INa by reducing Nav1.5 internalization and ubiquitination via interaction with Nedd4-2; knockout of αB-crystallin decreases Nav1.5 cell surface expression.","method":"Yeast two-hybrid, GST pulldown, co-immunoprecipitation, immunostaining, whole-cell patch-clamp, mutagenesis of Nav1.5 Nedd4-2 binding site in HEK293 cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — four independent binding assays plus functional mutagenesis and KO, multiple orthogonal methods in single rigorous paper","pmids":["26961874"],"is_preprint":false},{"year":2012,"finding":"The PDZ domain-binding SIV motif (last 3 residues of Nav1.5) is required for Nav1.5 expression and INa specifically at the lateral cardiomyocyte membrane but not at intercalated disks; deletion of the SIV motif in knock-in mice reduces lateral-membrane INa and decreases transverse conduction velocity, increasing anisotropy. The SIV motif regulates Nav1.5 degradation via the proteasome.","method":"Knock-in mouse (ΔSIV), optical mapping, whole-cell patch-clamp, proteasome inhibitor rescue (MG132), HEK293 internalization assay, patient mutation screening","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo knock-in mouse with optical mapping plus mechanistic cell studies and pharmacological rescue, multiple orthogonal methods","pmids":["24895455"],"is_preprint":false},{"year":2012,"finding":"Foxo1 transcription factor negatively regulates Nav1.5 expression by directly binding to insulin-responsive elements (IRE) in the SCN5A promoter; reactive oxygen species (H2O2) promote Foxo1 nuclear localization and thereby suppress Nav1.5 expression and Na+ current.","method":"Chromatin immunoprecipitation, constitutively nuclear Foxo1 mutant expression, RNAi knockdown, whole-cell voltage-clamp in HL-1 cardiomyocytes","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — ChIP demonstrating direct promoter binding, gain- and loss-of-function with electrophysiological readout, multiple orthogonal methods in single paper","pmids":["22400069"],"is_preprint":false},{"year":2014,"finding":"Nav1.5 promotes breast cancer cell invasiveness through activity-dependent mechanisms; shRNA-mediated stable knockdown of Nav1.5 reduces tumor growth, local invasion, and metastasis to liver, lungs and spleen in an orthotopic model, and in vitro Nav1.5 down-regulation reduces CD44 expression, suggesting regulation of invasion via the CD44-src-cortactin axis.","method":"Stable shRNA knockdown, orthotopic mouse xenograft model, in vitro invasion assay, immunohistochemistry, flow cytometry","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo loss-of-function with defined phenotypic readout and in vitro mechanistic follow-up, single lab with multiple methods","pmids":["26452220"],"is_preprint":false},{"year":2015,"finding":"Nav1.5 channels reach the plasma membrane through two distinct N-glycosylation states; only the fully-glycosylated form generates sodium current, while core-glycosylated channels can reach the membrane via an unconventional Golgi-independent route but are non-functional. Brugada syndrome mutants exert dominant-negative effects by trapping the fully-glycosylated form.","method":"Patch-clamp, surface biotinylation assays, enzymatic deglycosylation, brefeldin A treatment, HEK293T cells","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — multiple biochemical and electrophysiological methods in single paper demonstrating glycosylation-function link","pmids":["25721215"],"is_preprint":false},{"year":2016,"finding":"GATA4 and GATA5 transcription factors directly co-occupy SCN5A promoter and intron 1 regions in human cardiac tissue and synergistically activate SCN5A transcription through predicted GATA binding sites; GATA4 mRNA levels are strongly correlated with SCN5A transcript levels in human heart.","method":"Chromatin immunoprecipitation (ChIP) and Re-ChIP in human left ventricle, luciferase reporter with site mutagenesis, ddPCR quantification","journal":"Journal of molecular and cellular cardiology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — direct ChIP in native human cardiac tissue confirmed by reporter assays with site mutagenesis, multiple orthogonal methods","pmids":["27894866"],"is_preprint":false},{"year":2016,"finding":"Wnt/β-catenin signaling transcriptionally suppresses Nav1.5 expression: H2O2 activates β-catenin nuclear localization, β-catenin interacts with TCF4, and the β-catenin/TCF4 complex is recruited to the SCN5A promoter to repress its activity in a dose-dependent manner.","method":"Immunofluorescence, immunoprecipitation, ChIP, luciferase reporter, siRNA knockdown, whole-cell recording in HL-1 cardiomyocytes","journal":"Free radical biology & medicine","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — ChIP confirming direct promoter occupancy, Co-IP for protein interaction, functional reporter and electrophysiology, multiple orthogonal methods","pmids":["27068063"],"is_preprint":false},{"year":2017,"finding":"Kir2.1 and Nav1.5 channels form complexes in ventricular cardiomyocyte membranes (within <40 nm by proximity ligation assay); CaMKII inhibition decreases INa and IK1 only when channels are co-expressed but not Kir2.1 alone, and Nedd4-2 ubiquitin-ligase promotes Nav1.5 (and Kir2.1-Nav1.5 complex) degradation via the proteasome. The dynein/dynactin motor governs forward trafficking of Nav1.5 and of Kir2.1-Nav1.5 complexes.","method":"Proximity ligation assay, patch-clamp with intracellular antibody dialysis, pharmacological inhibition of CaMKII/14-3-3/dynamin/dynein in heterologous and cardiomyocyte systems","journal":"Frontiers in physiology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — proximity ligation plus functional electrophysiology with pathway inhibitors, single lab","pmids":["29184507"],"is_preprint":false},{"year":2018,"finding":"Brugada syndrome-associated endoplasmic reticulum (ER) trafficking-defective Nav1.5 mutants fail to positively modulate Kir2.1/2.2 channels, thereby reducing IK1 in addition to INa. Golgi trafficking-defective Nav1.5 mutants additionally exert a dominant-negative effect, further reducing IK1. ER-trapped Nav1.5 can be partially rescued by Kir2.1/2.2 through an unconventional secretory route involving GRASPs.","method":"SCN5A haploinsufficiency mouse model, heterologous expression, rat ventricular cardiomyocytes, hiPSC-CMs, patch-clamp","journal":"JCI insight","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple model systems (mouse KO, cardiomyocytes, hiPSC-CMs, heterologous cells) with electrophysiology demonstrating mechanistic interaction","pmids":["30232268"],"is_preprint":false},{"year":2018,"finding":"The RNA-binding protein HuR stabilizes SCN5A mRNA in cardiomyocytes; HuR expression is downregulated in failing hearts. AAV9-mediated overexpression of HuR in a mouse MI model increases SCN5A expression, improves action potential upstroke and conduction velocity in the infarct border zone, and reduces reentrant arrhythmia risk.","method":"Ribonucleoprotein immunoprecipitation, AAV9 overexpression in mice, optical mapping of intact heart, quantitative RT-PCR","journal":"Heart rhythm","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vivo AAV rescue experiment with optical mapping functional readout plus direct RNP-IP binding assay, multiple orthogonal methods","pmids":["29454929"],"is_preprint":false},{"year":2018,"finding":"HuR protects MEF2C mRNA from degradation, thereby maintaining MEF2C protein levels, which in turn binds the SCN5A promoter to enhance its transcription; HuR overexpression increases SCN5A mRNA and this effect is abolished by MEF2C siRNA, placing HuR upstream of MEF2C in an SCN5A regulatory network.","method":"Ribonucleoprotein immunoprecipitation, ChIP-qPCR, EMSA, siRNA knockdown, RT-PCR in cardiomyocytes","journal":"Journal of the American Heart Association","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — ChIP and EMSA for direct promoter binding, RNP-IP for RNA interaction, siRNA epistasis, multiple methods in single lab","pmids":["29678826"],"is_preprint":false},{"year":2018,"finding":"Anti-Nav1.5 autoantibodies are detectable in 90% of Brugada syndrome patients vs. 6% of controls; these autoantibodies reduce sodium current density in cellular models and injection of BrS patient plasma into wild-type mice induces Brugada-like ECG abnormalities, demonstrating a pathogenic immunological mechanism.","method":"Western blot, immunoprecipitation, immunofluorescence screening with HEK293A cells expressing Nav1.5; patch-clamp for current density; plasma injection in vivo mouse ECG","journal":"European heart journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal antibody detection methods plus electrophysiology plus in vivo mouse transfer experiment confirming pathogenicity","pmids":["39078224"],"is_preprint":false},{"year":2019,"finding":"K219T mutation in LMNA causes increased binding of Lamin A/C to the SCN5A promoter and enhanced recruitment of PRC2 protein SUZ12 and deposition of repressive histone mark H3K27me3 at SCN5A, resulting in downregulated Nav1.5 expression, reduced peak sodium current density, and slower conduction velocity in iPSC-derived cardiomyocytes. CRISPR/Cas9 correction of the mutation restores sodium current density and SCN5A expression.","method":"iPSC-derived cardiomyocytes from patient cells, ChIP for Lamin A/C and H3K27me3 at SCN5A promoter, patch-clamp, conduction velocity measurement, CRISPR/Cas9 correction","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — ChIP demonstrating direct epigenetic mechanism, genetic rescue by CRISPR correction, functional electrophysiology and conduction readout in human iPSC-CMs","pmids":["31118417"],"is_preprint":false},{"year":2019,"finding":"An evolutionarily conserved enhancer cluster downstream of SCN5A with super-enhancer characteristics drives cardiac-specific Scn5a expression; genome editing to delete individual enhancer elements or the entire cluster in mice reduces Scn5a expression, impairs cardiac conduction, and affects embryonic development. This cluster also controls chromatin conformation across a >0.5 Mb genomic region.","method":"Genome editing (deletion series) in mice, gene expression analysis, cardiac conduction measurements, chromatin conformation capture (3C/Hi-C)","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple independent genomic deletions in vivo with functional ECG phenotype and chromatin architecture analysis, multiple orthogonal methods","pmids":["31666509"],"is_preprint":false},{"year":2020,"finding":"UBE1 and UBA6 are E1 ubiquitin-activating enzymes that ubiquitinate Nav1.5 at lysine residues K590 and K591; overexpression reduces Nav1.5 expression and sodium current density, knockdown increases them, and the effects require the UBC9 E2 enzyme. Mutation of K590A/K591A abolishes the effects.","method":"Western blot ubiquitination assay, patch-clamp, siRNA knockdown, overexpression, site-directed mutagenesis in HEK293/Nav1.5 cells and neonatal cardiomyocytes","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — direct ubiquitination site identification by mutagenesis, gain- and loss-of-function with electrophysiological readout, multiple orthogonal methods","pmids":["32315024"],"is_preprint":false},{"year":2021,"finding":"FAT10 stabilizes Nav1.5 by binding to lysine residues in the C-terminal fragments of Nav1.5 and competing with Nedd4-2 ubiquitin E3 ligase binding, thereby preventing ubiquitination and proteasomal degradation of Nav1.5. Cardiac-specific Fat10 deletion reduces peak Na+ current and membrane Nav1.5 expression, increases late Na+ current, and increases arrhythmia after MI.","method":"Cardiac-specific conditional knockout mouse (Myh6-Cre × Fat10F/F), patch-clamp, co-immunoprecipitation, immunoblot/immunofluorescence","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vivo conditional KO with electrophysiology plus Co-IP demonstrating direct binding mechanism, multiple orthogonal methods","pmids":["33414395"],"is_preprint":false},{"year":2021,"finding":"Zfhx3 transcription factor directly represses the SCN5A promoter and also suppresses TBX5 expression and increases Nedd4-2 expression, thereby reducing peak INa density in cardiomyocytes through transcriptional and post-translational (proteasomal) mechanisms.","method":"Transcriptional reporter assay, siRNA knockdown, overexpression, patch-clamp in HL-1 cardiomyocytes","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — promoter reporter with functional electrophysiology, gain and loss-of-function, single lab","pmids":["34884836"],"is_preprint":false},{"year":2022,"finding":"TBX5 variant p.F206L fails to transactivate the SCN5A promoter, markedly decreasing peak INa and causing QRS widening in mice; variant p.D111Y increases SCN5A expression but fails to repress CAMK2D and SPTBN4, resulting in increased late INa (INaL) and QT prolongation. This establishes that TBX5 controls both peak INa and INaL in human cardiomyocytes by regulating SCN5A, CaMKIIδ, and βIV-spectrin expression.","method":"hiPSC-CM electrophysiology, HL-1 cell patch-clamp, in vivo ECG of transgenic mice, molecular transcriptional assays","journal":"Cardiovascular research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple model systems (hiPSC-CMs, HL-1, transgenic mice) with electrophysiology, pharmacological rescue (ranolazine), and transcriptional mechanistic follow-up","pmids":["33576403"],"is_preprint":false},{"year":2022,"finding":"Most SCN5A missense loss-of-function variants exert dominant-negative effects on wild-type Nav1.5 when co-expressed in heterozygous conditions; 32 of 35 complete LoF variants reduced peak current to <75% of wild-type alone. Individuals with dominant-negative variants have 2.7-fold higher Brugada syndrome burden than those with haploinsufficient variants.","method":"Automated patch-clamp in HEK293T cells with heterozygous WT + mutant co-expression for 50 variants; clinical cohort comparison with gnomAD","journal":"Genetics in medicine","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic in vitro electrophysiology of 50 variants with heterozygous co-expression design, validated by clinical enrichment data","pmids":["35305865"],"is_preprint":false},{"year":2008,"finding":"SCN5A gain-of-function mutation M1875T causes a +16.4 mV depolarized shift in the voltage-dependence of steady-state inactivation without generating persistent Na+ current, increasing atrial excitability and predisposing to familial atrial fibrillation through a mechanism distinct from LQT3.","method":"Whole-cell patch-clamp in heterologous expression system with M1875T mutant","journal":"Journal of the American College of Cardiology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — single in vitro electrophysiology study in single lab characterizing biophysical mechanism","pmids":["18929244"],"is_preprint":false},{"year":2016,"finding":"Nav1.5 protein is localized to late endosomes and phagolysosomes (Rab7+, LAMP-1+) rather than early endosomes in macrophages within active MS lesions, consistent with a role in endosomal acidification for myelin degradation.","method":"Immunohistochemistry with triple immunolabeling for Nav1.5, endosomal markers, and myelin protein in human MS lesion tissue","journal":"Multiple sclerosis","confidence":"Medium","confidence_rationale":"Tier 2–3 / Weak — direct subcellular localization in native tissue by immunofluorescence, single study, no functional manipulation","pmids":["22951351"],"is_preprint":false},{"year":2014,"finding":"In macrophages, an intracellular SCN5A variant initiates innate immune signaling through ADCY8 and ATF2 transcription factor; cytoplasmic poly(I:C) (viral dsRNA mimic) activates this pathway and markedly augments an inward voltage-sensitive sodium current mediated by the channel, linking pathogen recognition to Nav1.5 channel activation and downstream transcriptional regulation of interferon β.","method":"Electrophysiology (patch-clamp), pharmacological channel stimulation, poly(I:C) treatment, signaling pathway pharmacology in macrophages","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — electrophysiology combined with pathway activation assays, single lab, novel context (macrophages), limited mechanistic detail in abstract","pmids":["25368329"],"is_preprint":false},{"year":2010,"finding":"The common H558R polymorphism has mutation-specific effects on SCN5A-related sick sinus syndrome: it rescues the loss-of-function D1275N mutant by enhancing cell surface targeting and improving steady-state activation, but aggravates defects of E161K, P1298L, and R1632H mutants by reducing steady-state availability.","method":"Whole-cell patch-clamp, cell surface biotinylation in HEK293 cells with all 13 known SSS-related hNav1.5 mutants on H558 vs R558 backgrounds","journal":"Journal of cardiovascular electrophysiology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — systematic in vitro electrophysiology with surface expression quantification across multiple mutations, single lab","pmids":["20384651"],"is_preprint":false},{"year":2016,"finding":"Pan-HDAC inhibitors (TSA, vorinostat, romidepsin) reduce peak INa density and increase Nav1.5 acetylation in cardiomyocytes; Nav1.5 protein levels are significantly reduced after HDAC inhibition without changes in Scn5a mRNA, and co-immunoprecipitation with anti-acetyl-lysine antibody confirms Nav1.5 acetylation is increased in vivo after HDAC inhibition.","method":"Whole-cell patch-clamp, Western blot, immunoprecipitation with anti-acetyl-lysine antibody in neonatal mouse ventricular cardiomyocytes and hiPSC-CMs","journal":"American journal of physiology. Heart and circulatory physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct demonstration of Nav1.5 acetylation by Co-IP/WB with functional electrophysiological consequence, single lab","pmids":["27638876"],"is_preprint":false},{"year":2018,"finding":"A synonymous SNP (rs1805126) in the SCN5A coding sequence adjacent to a miR-24 binding site modulates miR-24-mediated suppression of SCN5A expression; miR-24 potently suppresses SCN5A expression and the minor allele of rs1805126 is associated with decreased cardiac SCN5A expression. In mice, decreased Scn5a expression leads to accumulation of myocardial reactive oxygen species.","method":"miRNA binding site mapping, luciferase reporter assays, human heart tissue expression correlation, mouse Scn5a reduction model with ROS measurement","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — reporter assay for miR-24/SCN5A interaction, in vivo mouse ROS phenotype, human tissue expression data, single lab","pmids":["29457789"],"is_preprint":false}],"current_model":"SCN5A encodes the pore-forming α-subunit Nav1.5 of the cardiac voltage-gated Na+ channel, which generates the rapid upstroke of the cardiac action potential; gain-of-function mutations (e.g., persistent late INa, shifted inactivation) cause LQT3/AF while loss-of-function mutations cause Brugada syndrome/conduction disease; Nav1.5 function and membrane expression are tightly regulated by a network of post-translational modifications (phosphorylation by CaMKII, ubiquitination by Nedd4-2/UBE1/UBA6, acetylation, glycosylation), interacting proteins (αB-crystallin, telethonin, Kir2.1, FAT10, syntrophins via PDZ-SIV motif), transcription factors (TBX5, GATA4/5, MEF2C, Foxo1, Zfhx3, β-catenin/TCF4), and enhancer elements; Nav1.5 is distributed in subcellular microdomains (lateral membrane vs. intercalated disk) with distinct regulation, and also functions in non-cardiac contexts including macrophage endosomal acidification, cancer cell invasiveness via NHE-1, olfactory neuron spontaneous firing, and innate immune signaling."},"narrative":{"mechanistic_narrative":"SCN5A encodes Nav1.5, the pore-forming α-subunit of the cardiac voltage-gated Na+ channel that initiates and propagates the action potential, with gain-of-function mutations producing persistent (late) Na+ current to cause LQT3 and loss-of-function mutations causing Brugada syndrome and conduction disease [PMID:10973849, PMID:19027780, PMID:29798782]. The channel's biophysical phenotype is gated by discrete structural determinants: the domain I-II linker and domain IVS6 govern inactivation gating and recovery [PMID:12673799, PMID:12650885], a depolarizing shift in steady-state inactivation can predispose to atrial fibrillation independent of late current [PMID:18929244], and an in vivo transgenic LQT3 model confirms that persistent Na+ current is directly arrhythmogenic and reversible by late-current blockade [PMID:14736542]. A recurring theme is that disease severity tracks with how much functional channel reaches the surface: trafficking-defective mutants are retained in the ER or fail to mature, with surface expression and dominant-negative interference determining penetrance [PMID:11786529, PMID:16301357, PMID:15890323, PMID:35305865]. Nav1.5 abundance and surface density are set by a multi-layered regulatory network — transcriptional activators (TBX5, GATA4/5, MEF2C) and repressors (Foxo1, β-catenin/TCF4, Zfhx3), a cardiac super-enhancer cluster, mRNA stabilization by HuR, and post-translational control by ubiquitination (Nedd4-2, UBE1/UBA6), acetylation, glycosylation, and stabilizing partners (αB-crystallin, FAT10) [PMID:22728936, PMID:26961874, PMID:22400069, PMID:27894866, PMID:27068063, PMID:29454929, PMID:31666509, PMID:32315024, PMID:33414395, PMID:27638876]. A feed-forward loop in which late INa activates CaMKII, which in turn phosphorylates Nav1.5 to enhance late current, couples channel activity to its own regulation [PMID:21677263]. Nav1.5 occupies distinct cardiomyocyte microdomains — the SIV PDZ-binding motif targets it to the lateral membrane and controls proteasomal turnover there — and it functionally interacts with Kir2.1/2.2 to co-regulate IK1 [PMID:24895455, PMID:29184507, PMID:30232268]. Beyond the heart, Nav1.5 has documented roles in breast cancer invasiveness, macrophage endosomal localization and innate immune signaling, and it is the target of pathogenic autoantibodies in Brugada syndrome [PMID:26452220, PMID:39078224, PMID:22951351, PMID:25368329].","teleology":[{"year":2000,"claim":"Established the core disease paradigm that opposite biophysical defects in a single channel gene produce distinct arrhythmia syndromes, defining the field's mechanistic framework.","evidence":"Mutational analysis and patch-clamp in heterologous expression systems","pmids":["10973849","19027780","29798782"],"confidence":"High","gaps":["Does not resolve which structural elements control late current vs. peak current loss","In vitro biophysics not yet linked to in vivo arrhythmia"]},{"year":2003,"claim":"Mapped specific channel domains to specific gating defects, showing the I-II linker and domain IVS6 control inactivation gating and recovery kinetics.","evidence":"Whole-cell patch-clamp in HEK293 and two-electrode voltage clamp in Xenopus oocytes for LQT3 mutants L619F and I1768V","pmids":["12673799","12650885"],"confidence":"Medium","gaps":["Single-lab, single-method biophysics","No structural model of how these residues couple to the inactivation gate"]},{"year":2008,"claim":"Distinguished an AF-causing gain-of-function mechanism (depolarizing shift in inactivation) from the LQT3 late-current mechanism, broadening the genotype-phenotype map.","evidence":"Whole-cell patch-clamp of M1875T mutant in heterologous cells","pmids":["18929244"],"confidence":"Medium","gaps":["Single in vitro study","Atrial-specific consequences not tested in vivo"]},{"year":2004,"claim":"Demonstrated in vivo that LQT3 persistent Na+ current is causally arrhythmogenic and pharmacologically reversible, validating the in vitro mechanism in a whole animal.","evidence":"Cardiac-specific N1325S transgenic mouse with ECG, action potential recording, voltage-clamp, and mexiletine rescue","pmids":["14736542"],"confidence":"High","gaps":["Mouse repolarization differs from human","Does not address mutation-specific differences in surface expression"]},{"year":2005,"claim":"Showed that channel surface trafficking, not just gating, determines disease expressivity, with misfolded mutants rescuable pharmacologically and capable of dominant-negative effects.","evidence":"Patch-clamp, surface quantification, temperature manipulation, and pharmacological rescue (cisapride, mexiletine, curcumin) of trafficking-defective mutants in CHO/HEK293","pmids":["16301357","15890323","11786529"],"confidence":"High","gaps":["Identity of the trafficking machinery and chaperones not defined","Fever-modulation mechanism at the molecular level not resolved"]},{"year":2008,"claim":"Identified telethonin as a Nav1.5-interacting partner that alters channel window current, extending Nav1.5 regulation to cytoskeletal/sarcomeric proteins.","evidence":"Reciprocal Co-IP, immunofluorescence co-localization, and patch-clamp in HEK293 and GI smooth muscle","pmids":["18408010"],"confidence":"Medium","gaps":["Single lab","Binding interface on Nav1.5 not mapped"]},{"year":2010,"claim":"Revealed Nav1.5 mechanosensitivity, showing that mechanical force shifts voltage dependence by accelerating voltage-sensor movement, a property relevant to mechano-electric feedback.","evidence":"Cell-attached patch-clamp with controlled pressure in HEK293","pmids":["21041530"],"confidence":"High","gaps":["Physiological relevance in intact myocardium not established","Structural basis of force-sensing unknown"]},{"year":2010,"claim":"Showed that a common polymorphism (H558R) modifies mutant channel phenotype in a mutation-specific manner, providing a mechanism for variable penetrance.","evidence":"Patch-clamp and surface biotinylation across 13 SSS mutants on H558 vs R558 backgrounds in HEK293","pmids":["20384651"],"confidence":"Medium","gaps":["Mechanism of allele-specific rescue/aggravation not resolved structurally","Single lab heterologous data"]},{"year":2011,"claim":"Defined a feed-forward loop linking Nav1.5 late current to CaMKII activation and subsequent CaMKII phosphorylation of the channel, explaining self-amplifying late INa.","evidence":"siRNA knockdown, CaMKII autophosphorylation assays, Co-IP, patch-clamp, and N1325S mouse with pharmacology in cardiomyocytes","pmids":["21677263"],"confidence":"High","gaps":["Phosphorylation sites mediating the loop not fully mapped here","In vivo contribution to arrhythmia quantification incomplete"]},{"year":2012,"claim":"Established transcriptional control of SCN5A by both activators and repressors, identifying TBX5 as an enhancer-driven activator in the conduction system and Foxo1 as a redox-responsive repressor.","evidence":"Conditional KO, in vivo enhancer reporter with T-box mutagenesis, optical mapping (TBX5); ChIP, nuclear-Foxo1 mutant, RNAi, voltage-clamp (Foxo1)","pmids":["22728936","22400069"],"confidence":"High","gaps":["Combinatorial logic among transcription factors not resolved","Link between Foxo1 redox sensing and physiological INa modulation incomplete"]},{"year":2012,"claim":"Showed microdomain-specific regulation: the C-terminal SIV PDZ-binding motif targets Nav1.5 to the lateral membrane and controls its proteasomal degradation, distinct from intercalated-disk pools.","evidence":"ΔSIV knock-in mouse, optical mapping, patch-clamp, MG132 rescue, HEK293 internalization assay","pmids":["24895455"],"confidence":"High","gaps":["Identity of all PDZ partners at the lateral membrane not fully defined","Disk-specific scaffolding mechanism not addressed here"]},{"year":2012,"claim":"Identified αB-crystallin as a chaperone-like partner that stabilizes surface Nav1.5 by limiting Nedd4-2-dependent ubiquitination and internalization.","evidence":"Yeast two-hybrid, GST pulldown, Co-IP, immunostaining, patch-clamp, and Nedd4-2 site mutagenesis in HEK293","pmids":["26961874"],"confidence":"High","gaps":["In vivo cardiac contribution not tested","Stoichiometry and binding interface not defined"]},{"year":2016,"claim":"Expanded the transcriptional network with GATA4/5 as direct co-occupying activators and β-catenin/TCF4 as a redox-driven repressor of SCN5A.","evidence":"ChIP/Re-ChIP in human ventricle and luciferase assays (GATA); IF, Co-IP, ChIP, reporter, and recording in HL-1 (β-catenin/TCF4)","pmids":["27894866","27068063"],"confidence":"High","gaps":["Hierarchy among activators/repressors at the locus unresolved","Physiological triggers in vivo not defined"]},{"year":2015,"claim":"Linked N-glycosylation state to channel functionality, showing only fully-glycosylated Nav1.5 conducts and that Brugada mutants act dominant-negatively by trapping this form.","evidence":"Patch-clamp, surface biotinylation, deglycosylation, and brefeldin A treatment in HEK293T","pmids":["25721215"],"confidence":"Medium","gaps":["Glycosyltransferases responsible not identified","Unconventional Golgi-independent route mechanism incompletely defined"]},{"year":2016,"claim":"Demonstrated acetylation as a post-translational regulator of Nav1.5 abundance, with HDAC inhibition reducing channel protein and current without affecting mRNA.","evidence":"Patch-clamp, Western blot, and anti-acetyl-lysine Co-IP in cardiomyocytes and hiPSC-CMs","pmids":["27638876"],"confidence":"Medium","gaps":["Specific acetylated residues not mapped","Responsible acetyltransferase/deacetylase not identified"]},{"year":2017,"claim":"Showed physical and functional coupling of Nav1.5 with Kir2.1 in cardiomyocyte membranes, with shared trafficking and degradation machinery (dynein/dynactin, Nedd4-2).","evidence":"Proximity ligation assay, patch-clamp with antibody dialysis, and pathway inhibitors in heterologous and cardiomyocyte systems","pmids":["29184507"],"confidence":"Medium","gaps":["Stoichiometry of the channelosome unresolved","Single-lab functional inference"]},{"year":2018,"claim":"Established post-transcriptional and additional transcriptional control via HuR, which stabilizes SCN5A mRNA directly and indirectly through MEF2C, with therapeutic rescue of conduction post-MI.","evidence":"RNP-IP, AAV9 overexpression with optical mapping (HuR); RNP-IP, ChIP-qPCR, EMSA, siRNA epistasis (HuR→MEF2C)","pmids":["29454929","29678826"],"confidence":"High","gaps":["Signals controlling HuR downregulation in failing hearts not defined","Relative contribution of direct vs. MEF2C-mediated pathway not quantified"]},{"year":2018,"claim":"Revealed an autoimmune mechanism in Brugada syndrome, with anti-Nav1.5 autoantibodies reducing current and transferring the ECG phenotype to mice.","evidence":"Antibody screening, patch-clamp, and BrS plasma injection in wild-type mice","pmids":["39078224"],"confidence":"High","gaps":["Epitopes on Nav1.5 not mapped","Trigger for autoantibody generation unknown"]},{"year":2018,"claim":"Refined the trafficking-defect model by showing Brugada mutants disrupt Kir2.1/2.2 modulation (reducing IK1) and that ER-trapped Nav1.5 can be partially rescued via a GRASP-dependent unconventional secretory route.","evidence":"SCN5A haploinsufficiency mouse, rat cardiomyocytes, hiPSC-CMs, and heterologous patch-clamp","pmids":["30232268"],"confidence":"High","gaps":["Mechanistic detail of GRASP-mediated rescue incomplete","Clinical relevance of dual INa/IK1 loss not quantified"]},{"year":2018,"claim":"Identified a coding synonymous SNP that modulates miR-24-mediated suppression of SCN5A, linking microRNA regulation to channel expression and myocardial ROS.","evidence":"miRNA binding site mapping, luciferase reporters, human tissue correlation, and mouse Scn5a-reduction ROS model","pmids":["29457789"],"confidence":"Medium","gaps":["Causal arrhythmia link not directly tested","Mechanism connecting reduced Nav1.5 to ROS accumulation undefined"]},{"year":2019,"claim":"Defined epigenetic and enhancer-architecture control of SCN5A, with LMNA mutation recruiting PRC2/H3K27me3 to repress the locus and a downstream super-enhancer cluster organizing chromatin to drive cardiac expression.","evidence":"Patient iPSC-CMs with ChIP and CRISPR correction (LMNA); enhancer deletion series in mice with conduction phenotype and 3C/Hi-C","pmids":["31118417","31666509"],"confidence":"High","gaps":["How LMNA mutation recruits PRC2 mechanistically not resolved","Individual enhancer element functions within the cluster not fully dissected"]},{"year":2020,"claim":"Pinpointed ubiquitination as a direct degradation signal, identifying K590/K591 as E1-dependent (UBE1/UBA6, UBC9) ubiquitination sites controlling channel abundance and current.","evidence":"Ubiquitination assay, patch-clamp, knockdown/overexpression, and K590A/K591A mutagenesis in HEK293 and neonatal cardiomyocytes","pmids":["32315024"],"confidence":"High","gaps":["Relationship to Nedd4-2-mediated ubiquitination not fully integrated","In vivo significance not established"]},{"year":2021,"claim":"Identified FAT10 as a stabilizing modifier that competes with Nedd4-2 to protect Nav1.5 from degradation, with cardiac deletion reducing peak INa and increasing arrhythmia.","evidence":"Cardiac-specific Fat10 knockout mouse, patch-clamp, Co-IP, and immunoblot","pmids":["33414395"],"confidence":"High","gaps":["FAT10 lysine binding sites on Nav1.5 not precisely mapped","Regulation of FAT10 expression in disease not defined"]},{"year":2021,"claim":"Added Zfhx3 as a transcriptional repressor acting both directly on the SCN5A promoter and indirectly by suppressing TBX5 and inducing Nedd4-2, integrating transcriptional and proteasomal control.","evidence":"Reporter assay, siRNA, overexpression, and patch-clamp in HL-1 cardiomyocytes","pmids":["34884836"],"confidence":"Medium","gaps":["In vivo validation lacking","Direct vs. indirect contributions not separately quantified"]},{"year":2022,"claim":"Showed TBX5 simultaneously controls peak INa and late INa by regulating SCN5A, CAMK2D, and SPTBN4, with variant-specific failures producing distinct conduction vs. repolarization phenotypes.","evidence":"hiPSC-CM and HL-1 electrophysiology, transgenic mouse ECG, ranolazine rescue, and transcriptional assays for TBX5 variants","pmids":["33576403"],"confidence":"High","gaps":["Full TBX5 target network in human cardiomyocytes not mapped","Mechanism distinguishing activation vs. repression by the same factor unresolved"]},{"year":2022,"claim":"Systematically established that most SCN5A loss-of-function missense variants act dominant-negatively, with dominant-negative variants conferring markedly higher Brugada burden than haploinsufficient ones.","evidence":"Automated heterozygous co-expression patch-clamp of 50 variants in HEK293T with clinical cohort comparison","pmids":["35305865"],"confidence":"High","gaps":["Molecular basis of dominant-negative interference per variant not resolved","Modifier effects on penetrance not addressed"]},{"year":2016,"claim":"Documented non-cardiac Nav1.5 localization to late endosomes/phagolysosomes in macrophages, consistent with a role in endosomal acidification for myelin degradation.","evidence":"Triple immunolabeling of Nav1.5 and endosomal markers in human MS lesion tissue","pmids":["22951351"],"confidence":"Medium","gaps":["No functional manipulation","Endosomal Na+ flux contribution to acidification not directly measured"]},{"year":2014,"claim":"Extended Nav1.5 function to innate immunity and cancer, linking an intracellular channel variant to ADCY8/ATF2 interferon signaling in macrophages and channel activity to breast cancer invasiveness.","evidence":"Patch-clamp with poly(I:C) and pathway pharmacology in macrophages; shRNA knockdown with orthotopic xenograft and invasion assays for breast cancer","pmids":["25368329","26452220"],"confidence":"Medium","gaps":["Mechanistic detail of intracellular variant activation limited","CD44-src-cortactin axis link inferential, not directly demonstrated"]},{"year":null,"claim":"How the multilayered regulatory network (transcription factors, enhancers, miRNAs, ubiquitin/acetyl/glyco modifications, scaffolding partners, and microdomain targeting) is integrated to set Nav1.5 surface density and microdomain distribution in vivo remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unified model coupling transcriptional and post-translational control","Crosstalk between lateral-membrane and intercalated-disk channel pools mechanistically undefined","Translation of variant-specific biophysics to individual clinical phenotype incomplete"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[0,2,3,4,8]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,8,9]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,5,12,15]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[1,15,19]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[31]}],"pathway":[{"term_id":"R-HSA-397014","term_label":"Muscle contraction","supporting_discovery_ids":[0,4]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[0,6,22,29]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[10,13,16,17,24,28]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[11,25,26,34]}],"complexes":["Kir2.1-Nav1.5 channel complex"],"partners":["NEDD4L","CRYAB","TCAP","KCNJ2","CAMK2D","FAT10","TBX5","HUR"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q14524","full_name":"Sodium channel protein type 5 subunit alpha","aliases":["Sodium channel protein cardiac muscle subunit alpha","Sodium channel protein type V subunit alpha","Voltage-gated sodium channel subunit alpha Nav1.5","hH1"],"length_aa":2016,"mass_kda":226.9,"function":"Pore-forming subunit of Nav1.5, a voltage-gated sodium (Nav) channel that directly mediates the depolarizing phase of action potentials in excitable membranes. Navs, also called VGSCs (voltage-gated sodium channels) or VDSCs (voltage-dependent sodium channels), operate by switching between closed and open conformations depending on the voltage difference across the membrane. In the open conformation they allow Na(+) ions to selectively pass through the pore, along their electrochemical gradient. The influx of Na(+) ions provokes membrane depolarization, initiating the propagation of electrical signals throughout cells and tissues (PubMed:1309946, PubMed:21447824, PubMed:23085483, PubMed:23420830, PubMed:25370050, PubMed:26279430, PubMed:26392562, PubMed:26776555). Nav1.5 is the predominant sodium channel expressed in myocardial cells and it is responsible for the initial upstroke of the action potential in cardiac myocytes, thereby initiating the heartbeat (PubMed:11234013, PubMed:11804990, PubMed:12569159, PubMed:1309946). Required for normal electrical conduction including formation of the infranodal ventricular conduction system and normal action potential configuration, as a result of its interaction with XIRP2 (By similarity)","subcellular_location":"Cell membrane; Cytoplasm, perinuclear region; Cell membrane, sarcolemma, T-tubule; Cell junction","url":"https://www.uniprot.org/uniprotkb/Q14524/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SCN5A","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SCN5A","total_profiled":1310},"omim":[{"mim_id":"621529","title":"TRANSMEMBRANE PROTEIN 168; TMEM168","url":"https://www.omim.org/entry/621529"},{"mim_id":"616249","title":"LONG QT SYNDROME 15; LQT15","url":"https://www.omim.org/entry/616249"},{"mim_id":"616247","title":"LONG QT SYNDROME 14; LQT14","url":"https://www.omim.org/entry/616247"},{"mim_id":"615396","title":"LEFT VENTRICULAR NONCOMPACTION 10; LVNC10","url":"https://www.omim.org/entry/615396"},{"mim_id":"615378","title":"ATRIAL FIBRILLATION, FAMILIAL, 14; ATFB14","url":"https://www.omim.org/entry/615378"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Nucleoli","reliability":"Additional"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"heart muscle","ntpm":49.7}],"url":"https://www.proteinatlas.org/search/SCN5A"},"hgnc":{"alias_symbol":["Nav1.5","LQT3","HB1","HBBD","PFHB1","IVF","HB2","HH1","SSS1","CDCD2","CMPD2","ICCD"],"prev_symbol":["CMD1E"]},"alphafold":{"accession":"Q14524","domains":[{"cath_id":"1.20.120.350","chopping":"19-36_56-240","consensus_level":"medium","plddt":78.4854,"start":19,"end":240},{"cath_id":"1.20.120.350","chopping":"1171-1309","consensus_level":"medium","plddt":82.1878,"start":1171,"end":1309},{"cath_id":"-","chopping":"1362-1398","consensus_level":"medium","plddt":79.8895,"start":1362,"end":1398},{"cath_id":"1.20.120.350","chopping":"1510-1549_1556-1641","consensus_level":"medium","plddt":81.3883,"start":1510,"end":1641},{"cath_id":"1.10.287,1.20.1440","chopping":"1642-1781","consensus_level":"medium","plddt":82.8552,"start":1642,"end":1781},{"cath_id":"1.10.238.10","chopping":"1785-1903","consensus_level":"medium","plddt":74.3493,"start":1785,"end":1903}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14524","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q14524-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q14524-F1-predicted_aligned_error_v6.png","plddt_mean":67.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SCN5A","jax_strain_url":"https://www.jax.org/strain/search?query=SCN5A"},"sequence":{"accession":"Q14524","fasta_url":"https://rest.uniprot.org/uniprotkb/Q14524.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q14524/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14524"}},"corpus_meta":[{"pmid":"10973849","id":"PMC_10973849","title":"Spectrum of mutations in long-QT syndrome genes. 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vaisseaux","url":"https://pubmed.ncbi.nlm.nih.gov/18033008","citation_count":16,"is_preprint":false},{"pmid":"26907222","id":"PMC_26907222","title":"Negative-dominance phenomenon with genetic variants of the cardiac sodium channel Nav1.5.","date":"2016","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/26907222","citation_count":16,"is_preprint":false},{"pmid":"25923670","id":"PMC_25923670","title":"Arrhythmogenic Biophysical Phenotype for SCN5A Mutation S1787N Depends upon Splice Variant Background and Intracellular Acidosis.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25923670","citation_count":16,"is_preprint":false},{"pmid":"37369559","id":"PMC_37369559","title":"Beneficial effects of chronic mexiletine treatment in a human model of SCN5A overlap syndrome.","date":"2023","source":"Europace : European pacing, arrhythmias, and cardiac electrophysiology : journal of the working groups on cardiac pacing, arrhythmias, and cardiac cellular electrophysiology of the European Society of Cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/37369559","citation_count":16,"is_preprint":false},{"pmid":"18662696","id":"PMC_18662696","title":"In calcineurin-induced cardiac hypertrophy expression of Nav1.5, Cx40 and Cx43 is reduced by different mechanisms.","date":"2008","source":"Journal of molecular and cellular cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/18662696","citation_count":16,"is_preprint":false},{"pmid":"34884836","id":"PMC_34884836","title":"Zfhx3 Transcription Factor Represses the Expression of SCN5A Gene and Decreases Sodium Current Density (INa).","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/34884836","citation_count":15,"is_preprint":false},{"pmid":"32315024","id":"PMC_32315024","title":"Ubiquitination-activating enzymes UBE1 and UBA6 regulate ubiquitination and expression of cardiac sodium channel Nav1.5.","date":"2020","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/32315024","citation_count":15,"is_preprint":false},{"pmid":"30828412","id":"PMC_30828412","title":"Protein structure aids predicting functional perturbation of missense variants in SCN5A and KCNQ1.","date":"2019","source":"Computational and structural biotechnology journal","url":"https://pubmed.ncbi.nlm.nih.gov/30828412","citation_count":15,"is_preprint":false},{"pmid":"35351625","id":"PMC_35351625","title":"SCN5A overlap syndromes: An open-minded approach.","date":"2022","source":"Heart rhythm","url":"https://pubmed.ncbi.nlm.nih.gov/35351625","citation_count":14,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":53311,"output_tokens":9221,"usd":0.149124,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":19471,"output_tokens":7919,"usd":0.147665,"stage2_stop_reason":"end_turn"},"total_usd":0.296789,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"SCN5A encodes the pore-forming α-subunit of the cardiac sodium channel responsible for action potential initiation and propagation; gain-of-function mutations cause persistent (late) sodium current leading to LQT3, while distinct mutations cause loss-of-function associated with Brugada syndrome.\",\n      \"method\": \"Mutational analysis and patch-clamp electrophysiology in heterologous expression systems\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — replicated across hundreds of mutations in multiple labs using in vitro electrophysiology; foundational mechanistic framework established by multiple orthogonal studies\",\n      \"pmids\": [\"10973849\", \"19027780\", \"29798782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The Brugada syndrome double mutant R1232W/T1620M abolishes Nav1.5 functional expression because the channel is retained in the endoplasmic reticulum rather than trafficking to the cell surface; a positively charged residue at position 1232 (R or K) is required for proper channel transport.\",\n      \"method\": \"Whole-cell patch-clamp, immunofluorescence, confocal microscopy with FLAG-tagged constructs, calnexin co-localization in tsA201 cells\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct subcellular localization with functional consequence, mutagenesis rescue experiment, orthogonal imaging and electrophysiology in single focused paper\",\n      \"pmids\": [\"11786529\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The LQT3 mutation L619F in the domain I-II linker of Nav1.5 increases maintained (persistent) Na+ current and causes a +5.8 mV shift in steady-state inactivation, implicating the I-II linker in inactivation gating.\",\n      \"method\": \"Whole-cell patch-clamp in transiently expressed HEK293 cells\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro electrophysiology with mutagenesis, single lab, single method\",\n      \"pmids\": [\"12673799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The LQT3 mutation I1768V in domain IVS6 causes faster recovery from inactivation and less slow inactivation without increasing persistent Na+ current, implicating domain IVS6 in inactivation kinetics.\",\n      \"method\": \"Two-electrode voltage clamp in Xenopus oocytes\",\n      \"journal\": \"Cardiovascular research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — reconstituted in Xenopus oocytes, single lab, single method\",\n      \"pmids\": [\"12650885\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Transgenic mice with cardiac-specific expression of the LQT3 mutation N1325S in SCN5A display QT prolongation, spontaneous polymorphic VT/VF, and slow recovery from inactivation of INa; mexiletine (a late Na+ current blocker) suppresses arrhythmias and restores inactivation recovery, establishing that the N1325S-induced persistent Na+ current causes arrhythmogenesis in vivo.\",\n      \"method\": \"Transgenic mouse model, ECG, action potential recording, voltage-clamp in isolated myocytes, mexiletine pharmacology\",\n      \"journal\": \"Cardiovascular research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo transgenic model with voltage-clamp confirmation and pharmacological rescue, multiple orthogonal methods\",\n      \"pmids\": [\"14736542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The LQT3 mutant L1825P fails to traffic to the cell surface (only ~9% of wild-type surface expression) and therefore does not prolong QT interval; cisapride rescues misprocessing to ~30% of wild-type surface expression and simultaneously increases late Na+ current, revealing that cell surface trafficking determines LQT3 phenotype expressivity.\",\n      \"method\": \"Patch-clamp, confocal imaging and cell-counting with epitope-tagged constructs in CHO cells\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct surface-expression quantification with pharmacological rescue plus electrophysiology, multiple orthogonal methods in a single focused study\",\n      \"pmids\": [\"16301357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Two Brugada syndrome mutations (L325R and R535X) cause severe Nav1.5 loss-of-function; the L325R mutant is likely misfolded (rescued partially by mexiletine or curcumin) and exerts a dominant-negative effect on wild-type channels when co-expressed. Fever accelerates Nav1.5 activation and fast inactivation kinetics, providing the mechanism by which reduced INa patients are vulnerable to fever-exacerbated Brugada syndrome.\",\n      \"method\": \"Whole-cell patch-clamp at various temperatures, Western blot protein expression, co-expression dominant-negative experiments, action potential modelling in HEK293 cells\",\n      \"journal\": \"Cardiovascular research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal methods (electrophysiology, biochemistry, temperature manipulation, pharmacological rescue) in single rigorous paper\",\n      \"pmids\": [\"15890323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Telethonin (encoded by TCAP) is a Nav1.5-interacting protein: it co-localizes with Nav1.5 in gastrointestinal smooth muscle, co-immunoprecipitates with sodium channels, and a disease-associated telethonin mutation doubles the window current of Nav1.5 when co-expressed in HEK293 cells by altering steady-state activation kinetics.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence co-localization, whole-cell patch-clamp in HEK293 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — reciprocal Co-IP and localization with functional electrophysiology, single lab\",\n      \"pmids\": [\"18408010\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Nav1.5 is mechanosensitive: applying negative patch pressure produces dose-dependent hyperpolarizing shifts in voltage dependence of activation and inactivation (~0.7 mV/mmHg), accelerates voltage-sensor movement but not gate-opening or fast inactivation rate constants, and increases peak current by recruiting more active channels, without changing unitary conductance or maximum open probability.\",\n      \"method\": \"Cell-attached patch-clamp with controlled pressure in HEK293 cells expressing Nav1.5\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — rigorous in vitro single-channel and whole-cell electrophysiology with graded mechanical stimuli, dissecting specific voltage-sensor transitions\",\n      \"pmids\": [\"21041530\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Nav1.5-dependent persistent Na+ influx (late INa) activates CaMKII in cardiomyocytes; Nav1.5 knockdown (but not Nav1.1 or Nav1.2) prevents ATX-II-induced CaMKII phosphorylation, and CaMKII co-immunoprecipitates with Nav1.5 after late INa activation, establishing a feed-forward loop where Nav1.5 late current → CaMKII activation → CaMKII phosphorylation of Nav1.5 → further late current.\",\n      \"method\": \"siRNA knockdown, CaMKII autophosphorylation assays, co-immunoprecipitation, patch-clamp, transgenic N1325S mouse model, pharmacological inhibition (ranolazine, KN93) in neonatal rat ventricular myocytes\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (KD, Co-IP, in vivo mouse model, pharmacology) replicated in cell and animal systems, single lab\",\n      \"pmids\": [\"21677263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TBX5 directly drives Scn5a expression in the ventricular conduction system through a TBX5-responsive enhancer downstream of Scn5a that requires canonical T-box binding sites; conditional deletion of Tbx5 from the ventricular conduction system reduces Nav1.5 protein expression and causes severe conduction dysfunction.\",\n      \"method\": \"Conditional mouse knockout, in vivo enhancer assay, T-box site mutagenesis, immunostaining, optical mapping, ECG\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — enhancer identified by in vivo reporter assay with site-specific mutagenesis plus conditional KO with functional phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"22728936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"αB-crystallin physically interacts with Nav1.5 (validated by yeast two-hybrid, GST pulldown, Co-IP, and immunostaining); overexpression increases peak INa by reducing Nav1.5 internalization and ubiquitination via interaction with Nedd4-2; knockout of αB-crystallin decreases Nav1.5 cell surface expression.\",\n      \"method\": \"Yeast two-hybrid, GST pulldown, co-immunoprecipitation, immunostaining, whole-cell patch-clamp, mutagenesis of Nav1.5 Nedd4-2 binding site in HEK293 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — four independent binding assays plus functional mutagenesis and KO, multiple orthogonal methods in single rigorous paper\",\n      \"pmids\": [\"26961874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The PDZ domain-binding SIV motif (last 3 residues of Nav1.5) is required for Nav1.5 expression and INa specifically at the lateral cardiomyocyte membrane but not at intercalated disks; deletion of the SIV motif in knock-in mice reduces lateral-membrane INa and decreases transverse conduction velocity, increasing anisotropy. The SIV motif regulates Nav1.5 degradation via the proteasome.\",\n      \"method\": \"Knock-in mouse (ΔSIV), optical mapping, whole-cell patch-clamp, proteasome inhibitor rescue (MG132), HEK293 internalization assay, patient mutation screening\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo knock-in mouse with optical mapping plus mechanistic cell studies and pharmacological rescue, multiple orthogonal methods\",\n      \"pmids\": [\"24895455\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Foxo1 transcription factor negatively regulates Nav1.5 expression by directly binding to insulin-responsive elements (IRE) in the SCN5A promoter; reactive oxygen species (H2O2) promote Foxo1 nuclear localization and thereby suppress Nav1.5 expression and Na+ current.\",\n      \"method\": \"Chromatin immunoprecipitation, constitutively nuclear Foxo1 mutant expression, RNAi knockdown, whole-cell voltage-clamp in HL-1 cardiomyocytes\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — ChIP demonstrating direct promoter binding, gain- and loss-of-function with electrophysiological readout, multiple orthogonal methods in single paper\",\n      \"pmids\": [\"22400069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Nav1.5 promotes breast cancer cell invasiveness through activity-dependent mechanisms; shRNA-mediated stable knockdown of Nav1.5 reduces tumor growth, local invasion, and metastasis to liver, lungs and spleen in an orthotopic model, and in vitro Nav1.5 down-regulation reduces CD44 expression, suggesting regulation of invasion via the CD44-src-cortactin axis.\",\n      \"method\": \"Stable shRNA knockdown, orthotopic mouse xenograft model, in vitro invasion assay, immunohistochemistry, flow cytometry\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo loss-of-function with defined phenotypic readout and in vitro mechanistic follow-up, single lab with multiple methods\",\n      \"pmids\": [\"26452220\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Nav1.5 channels reach the plasma membrane through two distinct N-glycosylation states; only the fully-glycosylated form generates sodium current, while core-glycosylated channels can reach the membrane via an unconventional Golgi-independent route but are non-functional. Brugada syndrome mutants exert dominant-negative effects by trapping the fully-glycosylated form.\",\n      \"method\": \"Patch-clamp, surface biotinylation assays, enzymatic deglycosylation, brefeldin A treatment, HEK293T cells\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple biochemical and electrophysiological methods in single paper demonstrating glycosylation-function link\",\n      \"pmids\": [\"25721215\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"GATA4 and GATA5 transcription factors directly co-occupy SCN5A promoter and intron 1 regions in human cardiac tissue and synergistically activate SCN5A transcription through predicted GATA binding sites; GATA4 mRNA levels are strongly correlated with SCN5A transcript levels in human heart.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP) and Re-ChIP in human left ventricle, luciferase reporter with site mutagenesis, ddPCR quantification\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct ChIP in native human cardiac tissue confirmed by reporter assays with site mutagenesis, multiple orthogonal methods\",\n      \"pmids\": [\"27894866\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Wnt/β-catenin signaling transcriptionally suppresses Nav1.5 expression: H2O2 activates β-catenin nuclear localization, β-catenin interacts with TCF4, and the β-catenin/TCF4 complex is recruited to the SCN5A promoter to repress its activity in a dose-dependent manner.\",\n      \"method\": \"Immunofluorescence, immunoprecipitation, ChIP, luciferase reporter, siRNA knockdown, whole-cell recording in HL-1 cardiomyocytes\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — ChIP confirming direct promoter occupancy, Co-IP for protein interaction, functional reporter and electrophysiology, multiple orthogonal methods\",\n      \"pmids\": [\"27068063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Kir2.1 and Nav1.5 channels form complexes in ventricular cardiomyocyte membranes (within <40 nm by proximity ligation assay); CaMKII inhibition decreases INa and IK1 only when channels are co-expressed but not Kir2.1 alone, and Nedd4-2 ubiquitin-ligase promotes Nav1.5 (and Kir2.1-Nav1.5 complex) degradation via the proteasome. The dynein/dynactin motor governs forward trafficking of Nav1.5 and of Kir2.1-Nav1.5 complexes.\",\n      \"method\": \"Proximity ligation assay, patch-clamp with intracellular antibody dialysis, pharmacological inhibition of CaMKII/14-3-3/dynamin/dynein in heterologous and cardiomyocyte systems\",\n      \"journal\": \"Frontiers in physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — proximity ligation plus functional electrophysiology with pathway inhibitors, single lab\",\n      \"pmids\": [\"29184507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Brugada syndrome-associated endoplasmic reticulum (ER) trafficking-defective Nav1.5 mutants fail to positively modulate Kir2.1/2.2 channels, thereby reducing IK1 in addition to INa. Golgi trafficking-defective Nav1.5 mutants additionally exert a dominant-negative effect, further reducing IK1. ER-trapped Nav1.5 can be partially rescued by Kir2.1/2.2 through an unconventional secretory route involving GRASPs.\",\n      \"method\": \"SCN5A haploinsufficiency mouse model, heterologous expression, rat ventricular cardiomyocytes, hiPSC-CMs, patch-clamp\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple model systems (mouse KO, cardiomyocytes, hiPSC-CMs, heterologous cells) with electrophysiology demonstrating mechanistic interaction\",\n      \"pmids\": [\"30232268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The RNA-binding protein HuR stabilizes SCN5A mRNA in cardiomyocytes; HuR expression is downregulated in failing hearts. AAV9-mediated overexpression of HuR in a mouse MI model increases SCN5A expression, improves action potential upstroke and conduction velocity in the infarct border zone, and reduces reentrant arrhythmia risk.\",\n      \"method\": \"Ribonucleoprotein immunoprecipitation, AAV9 overexpression in mice, optical mapping of intact heart, quantitative RT-PCR\",\n      \"journal\": \"Heart rhythm\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo AAV rescue experiment with optical mapping functional readout plus direct RNP-IP binding assay, multiple orthogonal methods\",\n      \"pmids\": [\"29454929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HuR protects MEF2C mRNA from degradation, thereby maintaining MEF2C protein levels, which in turn binds the SCN5A promoter to enhance its transcription; HuR overexpression increases SCN5A mRNA and this effect is abolished by MEF2C siRNA, placing HuR upstream of MEF2C in an SCN5A regulatory network.\",\n      \"method\": \"Ribonucleoprotein immunoprecipitation, ChIP-qPCR, EMSA, siRNA knockdown, RT-PCR in cardiomyocytes\",\n      \"journal\": \"Journal of the American Heart Association\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — ChIP and EMSA for direct promoter binding, RNP-IP for RNA interaction, siRNA epistasis, multiple methods in single lab\",\n      \"pmids\": [\"29678826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Anti-Nav1.5 autoantibodies are detectable in 90% of Brugada syndrome patients vs. 6% of controls; these autoantibodies reduce sodium current density in cellular models and injection of BrS patient plasma into wild-type mice induces Brugada-like ECG abnormalities, demonstrating a pathogenic immunological mechanism.\",\n      \"method\": \"Western blot, immunoprecipitation, immunofluorescence screening with HEK293A cells expressing Nav1.5; patch-clamp for current density; plasma injection in vivo mouse ECG\",\n      \"journal\": \"European heart journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal antibody detection methods plus electrophysiology plus in vivo mouse transfer experiment confirming pathogenicity\",\n      \"pmids\": [\"39078224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"K219T mutation in LMNA causes increased binding of Lamin A/C to the SCN5A promoter and enhanced recruitment of PRC2 protein SUZ12 and deposition of repressive histone mark H3K27me3 at SCN5A, resulting in downregulated Nav1.5 expression, reduced peak sodium current density, and slower conduction velocity in iPSC-derived cardiomyocytes. CRISPR/Cas9 correction of the mutation restores sodium current density and SCN5A expression.\",\n      \"method\": \"iPSC-derived cardiomyocytes from patient cells, ChIP for Lamin A/C and H3K27me3 at SCN5A promoter, patch-clamp, conduction velocity measurement, CRISPR/Cas9 correction\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — ChIP demonstrating direct epigenetic mechanism, genetic rescue by CRISPR correction, functional electrophysiology and conduction readout in human iPSC-CMs\",\n      \"pmids\": [\"31118417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"An evolutionarily conserved enhancer cluster downstream of SCN5A with super-enhancer characteristics drives cardiac-specific Scn5a expression; genome editing to delete individual enhancer elements or the entire cluster in mice reduces Scn5a expression, impairs cardiac conduction, and affects embryonic development. This cluster also controls chromatin conformation across a >0.5 Mb genomic region.\",\n      \"method\": \"Genome editing (deletion series) in mice, gene expression analysis, cardiac conduction measurements, chromatin conformation capture (3C/Hi-C)\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple independent genomic deletions in vivo with functional ECG phenotype and chromatin architecture analysis, multiple orthogonal methods\",\n      \"pmids\": [\"31666509\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"UBE1 and UBA6 are E1 ubiquitin-activating enzymes that ubiquitinate Nav1.5 at lysine residues K590 and K591; overexpression reduces Nav1.5 expression and sodium current density, knockdown increases them, and the effects require the UBC9 E2 enzyme. Mutation of K590A/K591A abolishes the effects.\",\n      \"method\": \"Western blot ubiquitination assay, patch-clamp, siRNA knockdown, overexpression, site-directed mutagenesis in HEK293/Nav1.5 cells and neonatal cardiomyocytes\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct ubiquitination site identification by mutagenesis, gain- and loss-of-function with electrophysiological readout, multiple orthogonal methods\",\n      \"pmids\": [\"32315024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"FAT10 stabilizes Nav1.5 by binding to lysine residues in the C-terminal fragments of Nav1.5 and competing with Nedd4-2 ubiquitin E3 ligase binding, thereby preventing ubiquitination and proteasomal degradation of Nav1.5. Cardiac-specific Fat10 deletion reduces peak Na+ current and membrane Nav1.5 expression, increases late Na+ current, and increases arrhythmia after MI.\",\n      \"method\": \"Cardiac-specific conditional knockout mouse (Myh6-Cre × Fat10F/F), patch-clamp, co-immunoprecipitation, immunoblot/immunofluorescence\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo conditional KO with electrophysiology plus Co-IP demonstrating direct binding mechanism, multiple orthogonal methods\",\n      \"pmids\": [\"33414395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Zfhx3 transcription factor directly represses the SCN5A promoter and also suppresses TBX5 expression and increases Nedd4-2 expression, thereby reducing peak INa density in cardiomyocytes through transcriptional and post-translational (proteasomal) mechanisms.\",\n      \"method\": \"Transcriptional reporter assay, siRNA knockdown, overexpression, patch-clamp in HL-1 cardiomyocytes\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — promoter reporter with functional electrophysiology, gain and loss-of-function, single lab\",\n      \"pmids\": [\"34884836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TBX5 variant p.F206L fails to transactivate the SCN5A promoter, markedly decreasing peak INa and causing QRS widening in mice; variant p.D111Y increases SCN5A expression but fails to repress CAMK2D and SPTBN4, resulting in increased late INa (INaL) and QT prolongation. This establishes that TBX5 controls both peak INa and INaL in human cardiomyocytes by regulating SCN5A, CaMKIIδ, and βIV-spectrin expression.\",\n      \"method\": \"hiPSC-CM electrophysiology, HL-1 cell patch-clamp, in vivo ECG of transgenic mice, molecular transcriptional assays\",\n      \"journal\": \"Cardiovascular research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple model systems (hiPSC-CMs, HL-1, transgenic mice) with electrophysiology, pharmacological rescue (ranolazine), and transcriptional mechanistic follow-up\",\n      \"pmids\": [\"33576403\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Most SCN5A missense loss-of-function variants exert dominant-negative effects on wild-type Nav1.5 when co-expressed in heterozygous conditions; 32 of 35 complete LoF variants reduced peak current to <75% of wild-type alone. Individuals with dominant-negative variants have 2.7-fold higher Brugada syndrome burden than those with haploinsufficient variants.\",\n      \"method\": \"Automated patch-clamp in HEK293T cells with heterozygous WT + mutant co-expression for 50 variants; clinical cohort comparison with gnomAD\",\n      \"journal\": \"Genetics in medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic in vitro electrophysiology of 50 variants with heterozygous co-expression design, validated by clinical enrichment data\",\n      \"pmids\": [\"35305865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"SCN5A gain-of-function mutation M1875T causes a +16.4 mV depolarized shift in the voltage-dependence of steady-state inactivation without generating persistent Na+ current, increasing atrial excitability and predisposing to familial atrial fibrillation through a mechanism distinct from LQT3.\",\n      \"method\": \"Whole-cell patch-clamp in heterologous expression system with M1875T mutant\",\n      \"journal\": \"Journal of the American College of Cardiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — single in vitro electrophysiology study in single lab characterizing biophysical mechanism\",\n      \"pmids\": [\"18929244\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Nav1.5 protein is localized to late endosomes and phagolysosomes (Rab7+, LAMP-1+) rather than early endosomes in macrophages within active MS lesions, consistent with a role in endosomal acidification for myelin degradation.\",\n      \"method\": \"Immunohistochemistry with triple immunolabeling for Nav1.5, endosomal markers, and myelin protein in human MS lesion tissue\",\n      \"journal\": \"Multiple sclerosis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Weak — direct subcellular localization in native tissue by immunofluorescence, single study, no functional manipulation\",\n      \"pmids\": [\"22951351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In macrophages, an intracellular SCN5A variant initiates innate immune signaling through ADCY8 and ATF2 transcription factor; cytoplasmic poly(I:C) (viral dsRNA mimic) activates this pathway and markedly augments an inward voltage-sensitive sodium current mediated by the channel, linking pathogen recognition to Nav1.5 channel activation and downstream transcriptional regulation of interferon β.\",\n      \"method\": \"Electrophysiology (patch-clamp), pharmacological channel stimulation, poly(I:C) treatment, signaling pathway pharmacology in macrophages\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — electrophysiology combined with pathway activation assays, single lab, novel context (macrophages), limited mechanistic detail in abstract\",\n      \"pmids\": [\"25368329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The common H558R polymorphism has mutation-specific effects on SCN5A-related sick sinus syndrome: it rescues the loss-of-function D1275N mutant by enhancing cell surface targeting and improving steady-state activation, but aggravates defects of E161K, P1298L, and R1632H mutants by reducing steady-state availability.\",\n      \"method\": \"Whole-cell patch-clamp, cell surface biotinylation in HEK293 cells with all 13 known SSS-related hNav1.5 mutants on H558 vs R558 backgrounds\",\n      \"journal\": \"Journal of cardiovascular electrophysiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic in vitro electrophysiology with surface expression quantification across multiple mutations, single lab\",\n      \"pmids\": [\"20384651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Pan-HDAC inhibitors (TSA, vorinostat, romidepsin) reduce peak INa density and increase Nav1.5 acetylation in cardiomyocytes; Nav1.5 protein levels are significantly reduced after HDAC inhibition without changes in Scn5a mRNA, and co-immunoprecipitation with anti-acetyl-lysine antibody confirms Nav1.5 acetylation is increased in vivo after HDAC inhibition.\",\n      \"method\": \"Whole-cell patch-clamp, Western blot, immunoprecipitation with anti-acetyl-lysine antibody in neonatal mouse ventricular cardiomyocytes and hiPSC-CMs\",\n      \"journal\": \"American journal of physiology. Heart and circulatory physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct demonstration of Nav1.5 acetylation by Co-IP/WB with functional electrophysiological consequence, single lab\",\n      \"pmids\": [\"27638876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A synonymous SNP (rs1805126) in the SCN5A coding sequence adjacent to a miR-24 binding site modulates miR-24-mediated suppression of SCN5A expression; miR-24 potently suppresses SCN5A expression and the minor allele of rs1805126 is associated with decreased cardiac SCN5A expression. In mice, decreased Scn5a expression leads to accumulation of myocardial reactive oxygen species.\",\n      \"method\": \"miRNA binding site mapping, luciferase reporter assays, human heart tissue expression correlation, mouse Scn5a reduction model with ROS measurement\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — reporter assay for miR-24/SCN5A interaction, in vivo mouse ROS phenotype, human tissue expression data, single lab\",\n      \"pmids\": [\"29457789\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SCN5A encodes the pore-forming α-subunit Nav1.5 of the cardiac voltage-gated Na+ channel, which generates the rapid upstroke of the cardiac action potential; gain-of-function mutations (e.g., persistent late INa, shifted inactivation) cause LQT3/AF while loss-of-function mutations cause Brugada syndrome/conduction disease; Nav1.5 function and membrane expression are tightly regulated by a network of post-translational modifications (phosphorylation by CaMKII, ubiquitination by Nedd4-2/UBE1/UBA6, acetylation, glycosylation), interacting proteins (αB-crystallin, telethonin, Kir2.1, FAT10, syntrophins via PDZ-SIV motif), transcription factors (TBX5, GATA4/5, MEF2C, Foxo1, Zfhx3, β-catenin/TCF4), and enhancer elements; Nav1.5 is distributed in subcellular microdomains (lateral membrane vs. intercalated disk) with distinct regulation, and also functions in non-cardiac contexts including macrophage endosomal acidification, cancer cell invasiveness via NHE-1, olfactory neuron spontaneous firing, and innate immune signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SCN5A encodes Nav1.5, the pore-forming \\u03b1-subunit of the cardiac voltage-gated Na+ channel that initiates and propagates the action potential, with gain-of-function mutations producing persistent (late) Na+ current to cause LQT3 and loss-of-function mutations causing Brugada syndrome and conduction disease [#0]. The channel's biophysical phenotype is gated by discrete structural determinants: the domain I-II linker and domain IVS6 govern inactivation gating and recovery [#2, #3], a depolarizing shift in steady-state inactivation can predispose to atrial fibrillation independent of late current [#30], and an in vivo transgenic LQT3 model confirms that persistent Na+ current is directly arrhythmogenic and reversible by late-current blockade [#4]. A recurring theme is that disease severity tracks with how much functional channel reaches the surface: trafficking-defective mutants are retained in the ER or fail to mature, with surface expression and dominant-negative interference determining penetrance [#1, #5, #6, #29]. Nav1.5 abundance and surface density are set by a multi-layered regulatory network \\u2014 transcriptional activators (TBX5, GATA4/5, MEF2C) and repressors (Foxo1, \\u03b2-catenin/TCF4, Zfhx3), a cardiac super-enhancer cluster, mRNA stabilization by HuR, and post-translational control by ubiquitination (Nedd4-2, UBE1/UBA6), acetylation, glycosylation, and stabilizing partners (\\u03b1B-crystallin, FAT10) [#10, #11, #13, #16, #17, #20, #24, #25, #26, #34]. A feed-forward loop in which late INa activates CaMKII, which in turn phosphorylates Nav1.5 to enhance late current, couples channel activity to its own regulation [#9]. Nav1.5 occupies distinct cardiomyocyte microdomains \\u2014 the SIV PDZ-binding motif targets it to the lateral membrane and controls proteasomal turnover there \\u2014 and it functionally interacts with Kir2.1/2.2 to co-regulate IK1 [#12, #18, #19]. Beyond the heart, Nav1.5 has documented roles in breast cancer invasiveness, macrophage endosomal localization and innate immune signaling, and it is the target of pathogenic autoantibodies in Brugada syndrome [#14, #22, #31, #32].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established the core disease paradigm that opposite biophysical defects in a single channel gene produce distinct arrhythmia syndromes, defining the field's mechanistic framework.\",\n      \"evidence\": \"Mutational analysis and patch-clamp in heterologous expression systems\",\n      \"pmids\": [\"10973849\", \"19027780\", \"29798782\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not resolve which structural elements control late current vs. peak current loss\", \"In vitro biophysics not yet linked to in vivo arrhythmia\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Mapped specific channel domains to specific gating defects, showing the I-II linker and domain IVS6 control inactivation gating and recovery kinetics.\",\n      \"evidence\": \"Whole-cell patch-clamp in HEK293 and two-electrode voltage clamp in Xenopus oocytes for LQT3 mutants L619F and I1768V\",\n      \"pmids\": [\"12673799\", \"12650885\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab, single-method biophysics\", \"No structural model of how these residues couple to the inactivation gate\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Distinguished an AF-causing gain-of-function mechanism (depolarizing shift in inactivation) from the LQT3 late-current mechanism, broadening the genotype-phenotype map.\",\n      \"evidence\": \"Whole-cell patch-clamp of M1875T mutant in heterologous cells\",\n      \"pmids\": [\"18929244\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single in vitro study\", \"Atrial-specific consequences not tested in vivo\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrated in vivo that LQT3 persistent Na+ current is causally arrhythmogenic and pharmacologically reversible, validating the in vitro mechanism in a whole animal.\",\n      \"evidence\": \"Cardiac-specific N1325S transgenic mouse with ECG, action potential recording, voltage-clamp, and mexiletine rescue\",\n      \"pmids\": [\"14736542\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mouse repolarization differs from human\", \"Does not address mutation-specific differences in surface expression\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed that channel surface trafficking, not just gating, determines disease expressivity, with misfolded mutants rescuable pharmacologically and capable of dominant-negative effects.\",\n      \"evidence\": \"Patch-clamp, surface quantification, temperature manipulation, and pharmacological rescue (cisapride, mexiletine, curcumin) of trafficking-defective mutants in CHO/HEK293\",\n      \"pmids\": [\"16301357\", \"15890323\", \"11786529\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the trafficking machinery and chaperones not defined\", \"Fever-modulation mechanism at the molecular level not resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified telethonin as a Nav1.5-interacting partner that alters channel window current, extending Nav1.5 regulation to cytoskeletal/sarcomeric proteins.\",\n      \"evidence\": \"Reciprocal Co-IP, immunofluorescence co-localization, and patch-clamp in HEK293 and GI smooth muscle\",\n      \"pmids\": [\"18408010\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Binding interface on Nav1.5 not mapped\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Revealed Nav1.5 mechanosensitivity, showing that mechanical force shifts voltage dependence by accelerating voltage-sensor movement, a property relevant to mechano-electric feedback.\",\n      \"evidence\": \"Cell-attached patch-clamp with controlled pressure in HEK293\",\n      \"pmids\": [\"21041530\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance in intact myocardium not established\", \"Structural basis of force-sensing unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed that a common polymorphism (H558R) modifies mutant channel phenotype in a mutation-specific manner, providing a mechanism for variable penetrance.\",\n      \"evidence\": \"Patch-clamp and surface biotinylation across 13 SSS mutants on H558 vs R558 backgrounds in HEK293\",\n      \"pmids\": [\"20384651\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of allele-specific rescue/aggravation not resolved structurally\", \"Single lab heterologous data\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined a feed-forward loop linking Nav1.5 late current to CaMKII activation and subsequent CaMKII phosphorylation of the channel, explaining self-amplifying late INa.\",\n      \"evidence\": \"siRNA knockdown, CaMKII autophosphorylation assays, Co-IP, patch-clamp, and N1325S mouse with pharmacology in cardiomyocytes\",\n      \"pmids\": [\"21677263\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphorylation sites mediating the loop not fully mapped here\", \"In vivo contribution to arrhythmia quantification incomplete\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Established transcriptional control of SCN5A by both activators and repressors, identifying TBX5 as an enhancer-driven activator in the conduction system and Foxo1 as a redox-responsive repressor.\",\n      \"evidence\": \"Conditional KO, in vivo enhancer reporter with T-box mutagenesis, optical mapping (TBX5); ChIP, nuclear-Foxo1 mutant, RNAi, voltage-clamp (Foxo1)\",\n      \"pmids\": [\"22728936\", \"22400069\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Combinatorial logic among transcription factors not resolved\", \"Link between Foxo1 redox sensing and physiological INa modulation incomplete\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed microdomain-specific regulation: the C-terminal SIV PDZ-binding motif targets Nav1.5 to the lateral membrane and controls its proteasomal degradation, distinct from intercalated-disk pools.\",\n      \"evidence\": \"\\u0394SIV knock-in mouse, optical mapping, patch-clamp, MG132 rescue, HEK293 internalization assay\",\n      \"pmids\": [\"24895455\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of all PDZ partners at the lateral membrane not fully defined\", \"Disk-specific scaffolding mechanism not addressed here\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified \\u03b1B-crystallin as a chaperone-like partner that stabilizes surface Nav1.5 by limiting Nedd4-2-dependent ubiquitination and internalization.\",\n      \"evidence\": \"Yeast two-hybrid, GST pulldown, Co-IP, immunostaining, patch-clamp, and Nedd4-2 site mutagenesis in HEK293\",\n      \"pmids\": [\"26961874\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo cardiac contribution not tested\", \"Stoichiometry and binding interface not defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Expanded the transcriptional network with GATA4/5 as direct co-occupying activators and \\u03b2-catenin/TCF4 as a redox-driven repressor of SCN5A.\",\n      \"evidence\": \"ChIP/Re-ChIP in human ventricle and luciferase assays (GATA); IF, Co-IP, ChIP, reporter, and recording in HL-1 (\\u03b2-catenin/TCF4)\",\n      \"pmids\": [\"27894866\", \"27068063\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Hierarchy among activators/repressors at the locus unresolved\", \"Physiological triggers in vivo not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Linked N-glycosylation state to channel functionality, showing only fully-glycosylated Nav1.5 conducts and that Brugada mutants act dominant-negatively by trapping this form.\",\n      \"evidence\": \"Patch-clamp, surface biotinylation, deglycosylation, and brefeldin A treatment in HEK293T\",\n      \"pmids\": [\"25721215\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Glycosyltransferases responsible not identified\", \"Unconventional Golgi-independent route mechanism incompletely defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated acetylation as a post-translational regulator of Nav1.5 abundance, with HDAC inhibition reducing channel protein and current without affecting mRNA.\",\n      \"evidence\": \"Patch-clamp, Western blot, and anti-acetyl-lysine Co-IP in cardiomyocytes and hiPSC-CMs\",\n      \"pmids\": [\"27638876\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific acetylated residues not mapped\", \"Responsible acetyltransferase/deacetylase not identified\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed physical and functional coupling of Nav1.5 with Kir2.1 in cardiomyocyte membranes, with shared trafficking and degradation machinery (dynein/dynactin, Nedd4-2).\",\n      \"evidence\": \"Proximity ligation assay, patch-clamp with antibody dialysis, and pathway inhibitors in heterologous and cardiomyocyte systems\",\n      \"pmids\": [\"29184507\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry of the channelosome unresolved\", \"Single-lab functional inference\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established post-transcriptional and additional transcriptional control via HuR, which stabilizes SCN5A mRNA directly and indirectly through MEF2C, with therapeutic rescue of conduction post-MI.\",\n      \"evidence\": \"RNP-IP, AAV9 overexpression with optical mapping (HuR); RNP-IP, ChIP-qPCR, EMSA, siRNA epistasis (HuR\\u2192MEF2C)\",\n      \"pmids\": [\"29454929\", \"29678826\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signals controlling HuR downregulation in failing hearts not defined\", \"Relative contribution of direct vs. MEF2C-mediated pathway not quantified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Revealed an autoimmune mechanism in Brugada syndrome, with anti-Nav1.5 autoantibodies reducing current and transferring the ECG phenotype to mice.\",\n      \"evidence\": \"Antibody screening, patch-clamp, and BrS plasma injection in wild-type mice\",\n      \"pmids\": [\"39078224\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Epitopes on Nav1.5 not mapped\", \"Trigger for autoantibody generation unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Refined the trafficking-defect model by showing Brugada mutants disrupt Kir2.1/2.2 modulation (reducing IK1) and that ER-trapped Nav1.5 can be partially rescued via a GRASP-dependent unconventional secretory route.\",\n      \"evidence\": \"SCN5A haploinsufficiency mouse, rat cardiomyocytes, hiPSC-CMs, and heterologous patch-clamp\",\n      \"pmids\": [\"30232268\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic detail of GRASP-mediated rescue incomplete\", \"Clinical relevance of dual INa/IK1 loss not quantified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified a coding synonymous SNP that modulates miR-24-mediated suppression of SCN5A, linking microRNA regulation to channel expression and myocardial ROS.\",\n      \"evidence\": \"miRNA binding site mapping, luciferase reporters, human tissue correlation, and mouse Scn5a-reduction ROS model\",\n      \"pmids\": [\"29457789\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal arrhythmia link not directly tested\", \"Mechanism connecting reduced Nav1.5 to ROS accumulation undefined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined epigenetic and enhancer-architecture control of SCN5A, with LMNA mutation recruiting PRC2/H3K27me3 to repress the locus and a downstream super-enhancer cluster organizing chromatin to drive cardiac expression.\",\n      \"evidence\": \"Patient iPSC-CMs with ChIP and CRISPR correction (LMNA); enhancer deletion series in mice with conduction phenotype and 3C/Hi-C\",\n      \"pmids\": [\"31118417\", \"31666509\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How LMNA mutation recruits PRC2 mechanistically not resolved\", \"Individual enhancer element functions within the cluster not fully dissected\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Pinpointed ubiquitination as a direct degradation signal, identifying K590/K591 as E1-dependent (UBE1/UBA6, UBC9) ubiquitination sites controlling channel abundance and current.\",\n      \"evidence\": \"Ubiquitination assay, patch-clamp, knockdown/overexpression, and K590A/K591A mutagenesis in HEK293 and neonatal cardiomyocytes\",\n      \"pmids\": [\"32315024\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relationship to Nedd4-2-mediated ubiquitination not fully integrated\", \"In vivo significance not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified FAT10 as a stabilizing modifier that competes with Nedd4-2 to protect Nav1.5 from degradation, with cardiac deletion reducing peak INa and increasing arrhythmia.\",\n      \"evidence\": \"Cardiac-specific Fat10 knockout mouse, patch-clamp, Co-IP, and immunoblot\",\n      \"pmids\": [\"33414395\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"FAT10 lysine binding sites on Nav1.5 not precisely mapped\", \"Regulation of FAT10 expression in disease not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Added Zfhx3 as a transcriptional repressor acting both directly on the SCN5A promoter and indirectly by suppressing TBX5 and inducing Nedd4-2, integrating transcriptional and proteasomal control.\",\n      \"evidence\": \"Reporter assay, siRNA, overexpression, and patch-clamp in HL-1 cardiomyocytes\",\n      \"pmids\": [\"34884836\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo validation lacking\", \"Direct vs. indirect contributions not separately quantified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed TBX5 simultaneously controls peak INa and late INa by regulating SCN5A, CAMK2D, and SPTBN4, with variant-specific failures producing distinct conduction vs. repolarization phenotypes.\",\n      \"evidence\": \"hiPSC-CM and HL-1 electrophysiology, transgenic mouse ECG, ranolazine rescue, and transcriptional assays for TBX5 variants\",\n      \"pmids\": [\"33576403\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full TBX5 target network in human cardiomyocytes not mapped\", \"Mechanism distinguishing activation vs. repression by the same factor unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Systematically established that most SCN5A loss-of-function missense variants act dominant-negatively, with dominant-negative variants conferring markedly higher Brugada burden than haploinsufficient ones.\",\n      \"evidence\": \"Automated heterozygous co-expression patch-clamp of 50 variants in HEK293T with clinical cohort comparison\",\n      \"pmids\": [\"35305865\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of dominant-negative interference per variant not resolved\", \"Modifier effects on penetrance not addressed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Documented non-cardiac Nav1.5 localization to late endosomes/phagolysosomes in macrophages, consistent with a role in endosomal acidification for myelin degradation.\",\n      \"evidence\": \"Triple immunolabeling of Nav1.5 and endosomal markers in human MS lesion tissue\",\n      \"pmids\": [\"22951351\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional manipulation\", \"Endosomal Na+ flux contribution to acidification not directly measured\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extended Nav1.5 function to innate immunity and cancer, linking an intracellular channel variant to ADCY8/ATF2 interferon signaling in macrophages and channel activity to breast cancer invasiveness.\",\n      \"evidence\": \"Patch-clamp with poly(I:C) and pathway pharmacology in macrophages; shRNA knockdown with orthotopic xenograft and invasion assays for breast cancer\",\n      \"pmids\": [\"25368329\", \"26452220\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic detail of intracellular variant activation limited\", \"CD44-src-cortactin axis link inferential, not directly demonstrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multilayered regulatory network (transcription factors, enhancers, miRNAs, ubiquitin/acetyl/glyco modifications, scaffolding partners, and microdomain targeting) is integrated to set Nav1.5 surface density and microdomain distribution in vivo remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified model coupling transcriptional and post-translational control\", \"Crosstalk between lateral-membrane and intercalated-disk channel pools mechanistically undefined\", \"Translation of variant-specific biophysics to individual clinical phenotype incomplete\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [0, 2, 3, 4, 8]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 8, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 5, 12, 15]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [1, 15, 19]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [31]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-397014\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 6, 22, 29]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [10, 13, 16, 17, 24, 28]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [11, 25, 26, 34]}\n    ],\n    \"complexes\": [\"Kir2.1-Nav1.5 channel complex\"],\n    \"partners\": [\"NEDD4L\", \"CRYAB\", \"TCAP\", \"KCNJ2\", \"CAMK2D\", \"FAT10\", \"TBX5\", \"HuR\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}