{"gene":"CNGA1","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2021,"finding":"Cryo-EM structures of human CNGA1 in both apo closed and cGMP-bound open states reveal that CNGA1 forms a central channel gate in the middle of the membrane (occluding the central cavity), distinct from most other voltage-gated tetrameric cation channels. Structural analysis of the wild-type channel and the E365Q selectivity filter mutant defined two Ca2+ binding sites inside the selectivity filter, providing a structural basis for Ca2+ blockage and permeation. The E365Q mutant also revealed two alternative side-chain conformations at Q365, explaining voltage-dependent gating acquired upon E365 mutation.","method":"Cryo-EM structure determination of apo and cGMP-bound states; E365Q active-site mutagenesis; ion binding profile analysis","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structures in two functional states combined with mutagenesis and ion binding analysis in a single rigorous study","pmids":["33651975"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM structure of the native heterotetrameric bovine rod CNG channel (3 CNGA1 + 1 CNGB1) in the closed state shows that three CNGA1 subunits are tethered at their C-termini by a coiled-coil region. The CNGB1 subunit has a differently oriented C-helix in its CNBD compared to the three CNGA1 subunits, and CNGB1 residue R994 reaches into the ionic pathway to introduce an additional pore gate distinct from the central hydrophobic gate of homomeric CNGA1 channels.","method":"Cryo-EM structure of native bovine retinal rod CNG channel; subunit-resolved structural analysis","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM of native heterotetrameric channel with subunit-resolved structural detail","pmids":["34969975"],"is_preprint":false},{"year":2004,"finding":"Two salt bridges in the C-linker region of CNGA1 (an intersubunit bridge between neighboring C-linkers, and an intrasubunit bridge between the C-linker and its CNBD) stabilize a closed channel conformation. Breaking these salt bridges by mutation increases the favorability of channel opening; swapping the charged residues restores wild-type behavior. This indicates that in CNGA1, the CNBD can be in a ligand-activated configuration while the C-linker remains in the resting configuration, suggesting a modular gating scheme.","method":"Site-directed mutagenesis of salt bridge residues in CNGA1 (and HCN2) expressed in Xenopus oocytes; electrophysiology","journal":"The Journal of general physiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — charge-swap mutagenesis with functional rescue, two orthogonal channel systems tested, rigorous controls","pmids":["15572346"],"is_preprint":false},{"year":2009,"finding":"Mutations restricted to the region around the ion selectivity filter of CNGA1 render channels essentially fully voltage-gated even in saturating cGMP, with channels remaining mostly closed at physiological voltages. This voltage-dependent gating resembles the selectivity filter-based mechanism of KcsA K+ channels. The mutations loosen the attachment of the selectivity filter to its surrounding structure, shifting gating equilibrium toward closed states. The wild-type structure surrounding the selectivity filter has evolved to suppress inherent voltage-dependent gating, ensuring cGMP binding alone suffices to open the channel.","method":"Site-directed mutagenesis of selectivity filter region; patch-clamp electrophysiology in Xenopus oocytes","journal":"The Journal of general physiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis combined with detailed electrophysiological characterization; mechanistic model supported by multiple mutants","pmids":["19635856"],"is_preprint":false},{"year":2004,"finding":"In homotetrameric CNGA1 channels, at saturating cGMP concentrations, current levels distribute around a single mean with long open times. At low cGMP, current levels become heterogeneous (smaller, equal, or larger than saturating cGMP levels) with short open times. Ions that stay longer in the pore (generating larger single-channel currents) increase heterogeneity of current levels and decrease open probability and open times, suggesting that cGMP and permeating ions jointly modulate the extent and frequency of conformational fluctuations of the channel pore.","method":"Single-channel and macroscopic patch-clamp recordings from CNGA1 channels expressed in Xenopus oocytes; varying cGMP concentrations and permeant ion species","journal":"The Journal of physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct electrophysiological assay with multiple ionic conditions; single lab but orthogonal conditions tested","pmids":["15308684"],"is_preprint":false},{"year":2013,"finding":"Using tail and gating current measurements from homotetrameric CNGA1 channels expressed in Xenopus oocytes, two distinct mechanisms underlie rectification: (1) single-channel current rectification in symmetrical Rb+ and Cs+ dominating macroscopic currents, and (2) voltage-dependent gating becoming prominent with larger organic cations (ethylammonium, dimethylammonium). Isochronal tail current analysis revealed at least two voltage-dependent transitions; only the first is sensitive to S4 helix charge mutations. The number of elementary charges per channel moving across the membrane is less than 2 (vs ~12 in K+ channels), indicating a restricted S4 motion with inefficient coupling to the channel gate that renders CNGA1 poorly sensitive to voltage under physiological conditions.","method":"Tail and gating current recordings from CNGA1 in Xenopus oocytes; S4 charge mutagenesis; multiple permeant ion species","journal":"Physiological reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gating current measurements combined with S4 mutagenesis and multiple ionic conditions; single lab","pmids":["24400150"],"is_preprint":false},{"year":2003,"finding":"Dequalinium (an organic divalent cation) blocks CNGA1 channels from the intracellular side with K1/2 ~190 nM (at 0 mV) in a state-independent, voltage-dependent manner (zdelta ~1), occupying a binding site within the ion-conducting pathway. At the single-channel level, dequalinium acts as a slow blocker without changing unitary conductance. CNGA1+CNGB1 heteromeric channels show reduced sensitivity (K1/2 ~385 nM). Extracellular dequalinium also blocks but with voltage dependence suggesting it crosses the membrane to act from the inside.","method":"Inside-out and outside-out patch clamp from Xenopus oocytes expressing CNGA1 or CNGA1+CNGB1; single-channel recordings; concentration-response analysis","journal":"The Journal of general physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct electrophysiological characterization with single-channel resolution; single lab with multiple approaches","pmids":["12508052"],"is_preprint":false},{"year":2016,"finding":"A missense mutation c.1537G>A (p.G513R) in CNGA1 causes the mutant protein to be largely retained inside the cell rather than being targeted to the plasma membrane, as shown by in vitro expression in HEK293 cells. This suggests that loss of CNGA1 from the plasma membrane underlies the pathogenic mechanism in this form of autosomal recessive retinitis pigmentosa.","method":"In vitro expression of Flag-tagged wild-type and mutant CNGA1 in HEK293 cells; western blot; immunofluorescence localization","journal":"Bioscience reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — two orthogonal methods (western blot + immunofluorescence) in heterologous cells; single lab","pmids":["26802146"],"is_preprint":false},{"year":2019,"finding":"The missense mutation c.622G>A (p.D208N) in CNGA1, predicted to eliminate interactions between transmembrane segments S2 and S4, significantly reduces expression of CNGA1 protein, particularly at the plasma membrane of transfected HEK293T cells.","method":"Expression of Flag-tagged wild-type and G622A mutant CNGA1 in HEK293T cells; western blot; immunofluorescence","journal":"Biochemical genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single set of heterologous cell experiments with two detection methods but no functional electrophysiology","pmids":["30652268"],"is_preprint":false},{"year":2011,"finding":"CNGA1 (but not CNGA2 or CNGA3) is expressed principally in rat alveolar type I cells at the protein level. Activation of CNG channels with 8Br-cGMP stimulates lung liquid absorption ~1.8-fold in a rat in situ lung liquid clearance assay; this stimulation is abolished by the CNGA1-specific inhibitor pseudechetoxin (PsTx) or L-cis-diltiazem, but not by amiloride. Patch-clamp confirmed CNGA1 channels in HEK293 cells are not inhibited by amiloride and ENaC is not inhibited by PsTx. Thus CNGA1 channels in alveolar type I cells underlie the amiloride-insensitive component of lung liquid reabsorption.","method":"RT-PCR and protein expression profiling; in situ rat lung liquid clearance assay with pharmacological inhibitors; patch-clamp of recombinant CNGA1 and ENaC in HEK293 cells","journal":"Pflugers Archiv : European journal of physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in situ functional assay combined with pharmacological specificity controls and patch-clamp validation; single lab, multiple orthogonal methods","pmids":["21559843"],"is_preprint":false},{"year":2012,"finding":"CNG-A1 is localized at the basolateral membrane of proximal and distal tubules of rat renal cortex. Ovariectomy decreases CNG-A1 expression in the renal cortex, and 17β-estradiol benzoate replacement restores CNG-A1 expression to control levels, whereas progesterone replacement does not. This indicates estrogen, but not progesterone, modulates CNGA1 expression in renal cortex.","method":"Immunofluorescence localization in rat kidney; western blot of ovariectomized and hormone-replaced rats","journal":"Cellular physiology and biochemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — localization and expression modulation by hormone shown by immunofluorescence and western blot in a single study; no functional channel assay","pmids":["22759964"],"is_preprint":false},{"year":2021,"finding":"CNGA1 knockout mice (generated by CRISPR/Cas9) show progressive retinal thinning, rod and cone degeneration, and functional deficits consistent with retinitis pigmentosa. RNA-sequencing of retinae revealed down-regulated synaptic transmission and phototransduction pathways as early as postnatal day 9, down-regulation of the PI3K-AKT-mTOR pathway indicating upregulation of autophagic processes, and chaperone-mediated autophagy coinciding with the time course of photoreceptor death.","method":"CRISPR/Cas9 knockout mouse model; ERG; immunofluorescence; TUNEL staining; RNA-sequencing; pathway analysis","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean knockout with multiple orthogonal readouts (functional, histological, transcriptomic); single lab","pmids":["34418172"],"is_preprint":false},{"year":2025,"finding":"AAV8-mediated subretinal delivery of mCnga1 driven by the rhodopsin promoter in Cnga1-/- mice results in correctly localized CNGA1 protein expression, sustained rescue of rod-mediated retinal function, long-term photoreceptor preservation for at least 9 months, improved vision-guided behavior, and upregulation of phototransduction-associated genes by RNA-seq. This demonstrates that CNGA1 restoration is sufficient to rescue rod photoreceptor function and survival.","method":"AAV8 gene augmentation in Cnga1-/- mice; ERG; immunofluorescence; vision-guided behavior assay; RNA-seq","journal":"Communications medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo rescue experiment with multiple functional and structural readouts; single lab, single study","pmids":["40897815"],"is_preprint":false},{"year":2026,"finding":"A medium-throughput aequorin-based functional assay of 86 CNGA1 missense variants showed that 72.1% significantly impaired CNG channel function (classified as functionally abnormal) and 23.3% were functionally normal. Two putative missense variants were shown by in vitro minigene splice assays to induce missplicing rather than amino acid change. AlphaMissense and CPT-1 in silico predictors best correlated with functional outcomes. Functional data enabled reclassification of 62.1% of VUS as likely pathogenic or likely benign.","method":"Aequorin-based medium-throughput cation flux assay for CNGA1 channel function; in vitro minigene splice assays; correlation with in silico predictors","journal":"Molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — medium-throughput functional assay covering 86 variants with two orthogonal methods (flux assay + splice assay); single lab","pmids":["42192302"],"is_preprint":false},{"year":2024,"finding":"Large-scale atomistic MD simulations of homotetrameric CNGA1 under transmembrane voltages reveal that monovalent cation permeation involves hydrated cations in the selectivity filter. Hydration fluctuations in the central gate region, driven by pore flexibility, are proposed as the mechanism for excess single-channel conductance noise and characteristic flickering. An allosteric coupling between the selectivity filter and the central cavity gate is proposed as the mechanism for intrinsic voltage sensitivity of the pore.","method":"Atomistic molecular dynamics simulations under transmembrane voltage; analysis of cation hydration and pore dynamics","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational simulation only, no experimental validation reported; preprint","pmids":["bio_10.1101_2024.12.19.629380"],"is_preprint":true}],"current_model":"CNGA1 is the principal alpha subunit of the rod photoreceptor cyclic nucleotide-gated (CNG) channel: it forms a homotetramer (or 3:1 heterotetrameric complex with CNGB1 in native rods) whose pore gate is located in the middle of the membrane, opens upon cGMP binding to its C-terminal cyclic nucleotide-binding domain via rearrangement of the C-linker region (stabilized in the closed state by intersubunit and intrasubunit salt bridges), has a selectivity filter that defines Ca2+ binding sites and suppresses inherent voltage-dependent gating so that cGMP binding alone suffices to open the channel at physiological voltages, and whose loss causes progressive rod photoreceptor degeneration that can be rescued by AAV-mediated gene augmentation."},"narrative":{"mechanistic_narrative":"CNGA1 is the principal pore-forming alpha subunit of the rod photoreceptor cyclic nucleotide-gated (CNG) cation channel, the effector that transduces light-driven changes in cGMP into membrane current [PMID:33651975, PMID:34418172]. It assembles as a homotetramer in heterologous systems and as a 3:1 heterotetramer with CNGB1 in native rods, where the three CNGA1 subunits are tethered at their C-termini through a coiled-coil region while the incorporated CNGB1 subunit contributes an additional pore gate (via residue R994) distinct from the central hydrophobic gate of homomeric channels [PMID:34969975]. Unlike most voltage-gated tetrameric cation channels, CNGA1 positions its activation gate in the middle of the membrane, occluding the central cavity, and opens upon cGMP binding to its C-terminal cyclic nucleotide-binding domain [PMID:33651975]. Gating is modular: intersubunit and intrasubunit salt bridges in the C-linker stabilize the closed state, so that the CNBD can adopt a ligand-bound configuration while the C-linker remains at rest, and breaking these bridges favors opening [PMID:15572346]. The selectivity filter defines two intrapore Ca2+ binding sites underlying Ca2+ block and permeation, and the wild-type filter architecture has evolved to suppress an inherent voltage-dependent gating mode so that cGMP binding alone suffices to open the channel at physiological voltages [PMID:33651975, PMID:19635856]. Consistent with this, CNGA1 moves fewer than two elementary gating charges across the membrane and is poorly voltage-sensitive under physiological conditions [PMID:24400150]. Loss of CNGA1 causes progressive rod and cone degeneration: knockout mice develop a retinitis pigmentosa-like phenotype with down-regulated phototransduction and synaptic transmission and engagement of autophagic pathways [PMID:34418172], and pathogenic missense variants act by retaining the protein intracellularly and depleting it from the plasma membrane [PMID:26802146]. AAV-mediated CNGA1 gene augmentation restores correctly localized protein, rod function, and photoreceptor survival, establishing that CNGA1 restoration is sufficient for rescue [PMID:40897815]. A distinct pool of CNGA1 functions outside the retina, mediating the amiloride-insensitive component of cGMP-stimulated lung liquid reabsorption in alveolar type I cells [PMID:21559843].","teleology":[{"year":2003,"claim":"Establishing where blockers bind within the conduction pathway clarified the architecture of the open pore and provided a pharmacological tool to distinguish homomeric from heteromeric channels.","evidence":"Inside-out/outside-out patch clamp with dequalinium block of CNGA1 and CNGA1+CNGB1 in Xenopus oocytes","pmids":["12508052"],"confidence":"Medium","gaps":["Blocker binding site not resolved at atomic resolution","Functional consequence of differential CNGB1 sensitivity in native rods not addressed"]},{"year":2004,"claim":"Identifying C-linker salt bridges that stabilize the closed state revealed that ligand binding and gate opening are mechanically separable, defining a modular allosteric gating scheme.","evidence":"Charge-swap mutagenesis with functional rescue in CNGA1 and HCN2 expressed in Xenopus oocytes; electrophysiology","pmids":["15572346"],"confidence":"High","gaps":["Did not resolve the structural transition coupling CNBD to the membrane gate","Quantitative energetic contribution of each bridge not fully partitioned"]},{"year":2004,"claim":"Single-channel analysis showed cGMP and permeant ions jointly shape pore conformational fluctuations, linking ligand occupancy and ion occupancy to gating behavior.","evidence":"Single-channel and macroscopic patch-clamp from CNGA1 in Xenopus oocytes varying cGMP and permeant ion species","pmids":["15308684"],"confidence":"Medium","gaps":["Structural basis of ion-dependent fluctuations not defined","Single lab"]},{"year":2009,"claim":"Mutations around the selectivity filter that rendered the channel fully voltage-gated demonstrated that the wild-type filter architecture actively suppresses intrinsic voltage sensitivity, explaining why cGMP alone opens the channel physiologically.","evidence":"Selectivity filter region mutagenesis with patch-clamp in Xenopus oocytes","pmids":["19635856"],"confidence":"High","gaps":["Did not provide the atomic structure of the filter in open and closed states","Coupling between filter and central gate not directly observed"]},{"year":2011,"claim":"Identifying CNGA1 in alveolar type I cells and its pharmacological control of fluid clearance extended its role beyond phototransduction to epithelial liquid transport.","evidence":"Protein expression profiling, in situ rat lung liquid clearance with PsTx/L-cis-diltiazem/amiloride, and patch-clamp validation in HEK293 cells","pmids":["21559843"],"confidence":"Medium","gaps":["cGMP source and upstream signaling in alveolar cells not defined","Physiological relevance to human lung not tested"]},{"year":2013,"claim":"Gating current measurements quantified that CNGA1 moves fewer than two charges with weak S4-to-gate coupling, mechanistically explaining its poor physiological voltage sensitivity.","evidence":"Tail and gating current recordings with S4 charge mutagenesis and multiple permeant ions in Xenopus oocytes","pmids":["24400150"],"confidence":"Medium","gaps":["Structural motion of S4 not visualized","Two voltage-dependent transitions not fully assigned to structural elements"]},{"year":2016,"claim":"Demonstrating that a pathogenic missense variant traps CNGA1 intracellularly established loss of plasma membrane targeting as a disease mechanism in autosomal recessive retinitis pigmentosa.","evidence":"Heterologous expression of WT and p.G513R CNGA1 in HEK293 cells; western blot and immunofluorescence","pmids":["26802146"],"confidence":"Medium","gaps":["No electrophysiology to confirm channel function loss","Trafficking defect studied in heterologous cells, not rods"]},{"year":2019,"claim":"A second trafficking-defective missense variant reinforced reduced plasma-membrane expression as a recurring pathogenic theme.","evidence":"Expression of WT and p.D208N CNGA1 in HEK293T cells; western blot and immunofluorescence","pmids":["30652268"],"confidence":"Low","gaps":["No functional electrophysiology","Single lab, single heterologous system","S2-S4 interaction loss inferred, not structurally confirmed"]},{"year":2021,"claim":"Cryo-EM of human CNGA1 in apo-closed and cGMP-bound open states defined a mid-membrane central gate and two intrapore Ca2+ sites, providing the structural framework for ligand gating and ion permeation.","evidence":"Cryo-EM of apo and cGMP-bound CNGA1 with E365Q selectivity filter mutagenesis and ion binding analysis","pmids":["33651975"],"confidence":"High","gaps":["Dynamics of the gating transition not captured","Structures of homomeric channel may differ from native heteromer"]},{"year":2021,"claim":"Cryo-EM of the native bovine 3:1 CNGA1-CNGB1 channel revealed C-terminal coiled-coil tethering and a CNGB1-specific second pore gate, defining how the native rod channel differs from the homomer.","evidence":"Cryo-EM of native bovine retinal rod CNG channel with subunit-resolved analysis","pmids":["34969975"],"confidence":"High","gaps":["Open-state structure of the heteromer not resolved","Functional role of the CNGB1 R994 gate not directly tested"]},{"year":2021,"claim":"A CRISPR knockout mouse causally linked CNGA1 loss to progressive photoreceptor degeneration and identified early down-regulated phototransduction and autophagy engagement as molecular correlates of cell death.","evidence":"CRISPR/Cas9 Cnga1 knockout mice with ERG, immunofluorescence, TUNEL, and RNA-seq","pmids":["34418172"],"confidence":"Medium","gaps":["Causal role of autophagy/PI3K-AKT-mTOR changes in death not tested","Cone degeneration mechanism downstream of rod loss not defined"]},{"year":2025,"claim":"AAV8 gene augmentation rescued rod function and photoreceptor survival in knockout mice, establishing that restoring CNGA1 alone is sufficient and providing therapeutic proof of concept.","evidence":"AAV8 subretinal delivery of mCnga1 in Cnga1-/- mice; ERG, immunofluorescence, vision-guided behavior, RNA-seq","pmids":["40897815"],"confidence":"Medium","gaps":["Durability beyond 9 months and large-animal/human efficacy not shown","Optimal therapeutic window relative to degeneration stage not defined"]},{"year":2026,"claim":"A medium-throughput functional assay across 86 missense variants enabled large-scale variant classification and revealed that some apparent missense changes act through missplicing.","evidence":"Aequorin-based cation flux assay, minigene splice assays, and in silico predictor correlation","pmids":["42192302"],"confidence":"Medium","gaps":["Assay measures function in heterologous cells, not native rods","Trafficking versus gating defects not distinguished for each variant"]},{"year":null,"claim":"How allosteric coupling between the selectivity filter and the central cavity gate generates the channel's characteristic flicker and residual voltage sensitivity awaits direct experimental confirmation.","evidence":"","pmids":[],"confidence":"Low","gaps":["MD-predicted hydration fluctuations at the gate not experimentally validated","No time-resolved structural data on the gating transition","Coupling mechanism inferred computationally"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[0,4,9]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,2,3]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[7,9,10]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[11,12]}],"pathway":[{"term_id":"R-HSA-9709957","term_label":"Sensory Perception","supporting_discovery_ids":[11,12]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,2]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[9]}],"complexes":["Rod CNG channel (CNGA1/CNGB1 heterotetramer)"],"partners":["CNGB1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P29973","full_name":"Cyclic nucleotide-gated channel alpha-1","aliases":["Cyclic nucleotide-gated channel, photoreceptor","Rod photoreceptor cGMP-gated cation channel subunit alpha","cGMP-gated cation channel alpha-1"],"length_aa":686,"mass_kda":79.1,"function":"Pore-forming subunit of the rod cyclic nucleotide-gated channel. Mediates rod photoresponses at dim light converting transient changes in intracellular cGMP levels into electrical signals. In the dark, cGMP levels are high and keep the channel open enabling a steady inward current carried by Na(+) and Ca(2+) ions that leads to membrane depolarization and neurotransmitter release from synaptic terminals. Upon photon absorption cGMP levels decline leading to channel closure and membrane hyperpolarization that ultimately slows neurotransmitter release and signals the presence of light, the end point of the phototransduction cascade. Conducts cGMP- and cAMP-gated ion currents, with permeability for monovalent and divalent cations. The selectivity for Ca(2+) over Na(+) increases with cGMP concentrations, whereas the selectivity among monovalent ions is independent of the cGMP levels","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/P29973/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CNGA1","classification":"Not Classified","n_dependent_lines":12,"n_total_lines":1208,"dependency_fraction":0.009933774834437087},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CNGA1","total_profiled":1310},"omim":[{"mim_id":"613756","title":"RETINITIS PIGMENTOSA 49; RP49","url":"https://www.omim.org/entry/613756"},{"mim_id":"612095","title":"RETINITIS PIGMENTOSA 41; RP41","url":"https://www.omim.org/entry/612095"},{"mim_id":"603617","title":"SOLUTE CARRIER FAMILY 24 (SODIUM/POTASSIUM/CALCIUM EXCHANGER), MEMBER 1; SLC24A1","url":"https://www.omim.org/entry/603617"},{"mim_id":"600724","title":"CYCLIC NUCLEOTIDE-GATED CHANNEL, BETA-1; CNGB1","url":"https://www.omim.org/entry/600724"},{"mim_id":"268000","title":"RETINITIS PIGMENTOSA; RP","url":"https://www.omim.org/entry/268000"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Nucleoplasm","reliability":"Uncertain"},{"location":"Vesicles","reliability":"Uncertain"},{"location":"Mid piece","reliability":"Uncertain"},{"location":"Principal piece","reliability":"Uncertain"},{"location":"Plasma membrane","reliability":"Additional"},{"location":"Mitotic spindle","reliability":"Additional"},{"location":"Primary cilium","reliability":"Additional"},{"location":"Primary cilium tip","reliability":"Additional"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"retina","ntpm":244.8}],"url":"https://www.proteinatlas.org/search/CNGA1"},"hgnc":{"alias_symbol":["RCNC1","RCNCa","CNG1","RP49"],"prev_symbol":["CNCG1","CNCG"]},"alphafold":{"accession":"P29973","domains":[{"cath_id":"-","chopping":"154-291","consensus_level":"medium","plddt":92.6153,"start":154,"end":291},{"cath_id":"1.10.287","chopping":"293-447","consensus_level":"medium","plddt":91.9349,"start":293,"end":447},{"cath_id":"2.60.120.10","chopping":"449-608","consensus_level":"high","plddt":85.4701,"start":449,"end":608}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P29973","model_url":"https://alphafold.ebi.ac.uk/files/AF-P29973-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P29973-F1-predicted_aligned_error_v6.png","plddt_mean":76.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CNGA1","jax_strain_url":"https://www.jax.org/strain/search?query=CNGA1"},"sequence":{"accession":"P29973","fasta_url":"https://rest.uniprot.org/uniprotkb/P29973.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P29973/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P29973"}},"corpus_meta":[{"pmid":"15572346","id":"PMC_15572346","title":"Salt bridges and gating in the COOH-terminal region of HCN2 and CNGA1 channels.","date":"2004","source":"The Journal of general physiology","url":"https://pubmed.ncbi.nlm.nih.gov/15572346","citation_count":121,"is_preprint":false},{"pmid":"11454763","id":"PMC_11454763","title":"DNA variation at the rp49 gene region of Drosophila simulans: evolutionary inferences from an unusual haplotype structure.","date":"2001","source":"Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/11454763","citation_count":87,"is_preprint":false},{"pmid":"25268133","id":"PMC_25268133","title":"Whole exome analysis identifies frequent CNGA1 mutations in Japanese population with autosomal recessive retinitis pigmentosa.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25268133","citation_count":58,"is_preprint":false},{"pmid":"33651975","id":"PMC_33651975","title":"Structural mechanisms of gating and selectivity of human rod CNGA1 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absorption.","date":"2011","source":"Pflugers Archiv : European journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/21559843","citation_count":14,"is_preprint":false},{"pmid":"15308684","id":"PMC_15308684","title":"Effects of permeating ions and cGMP on gating and conductance of rod-type cyclic nucleotide-gated (CNGA1) channels.","date":"2004","source":"The Journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/15308684","citation_count":14,"is_preprint":false},{"pmid":"36115851","id":"PMC_36115851","title":"Mutations within the cGMP-binding domain of CNGA1 causing autosomal recessive retinitis pigmentosa in human and animal model.","date":"2022","source":"Cell death discovery","url":"https://pubmed.ncbi.nlm.nih.gov/36115851","citation_count":13,"is_preprint":false},{"pmid":"34418172","id":"PMC_34418172","title":"Retinal degeneration in mice lacking the cyclic nucleotide-gated channel subunit CNGA1.","date":"2021","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/34418172","citation_count":12,"is_preprint":false},{"pmid":"24400150","id":"PMC_24400150","title":"Multiple mechanisms underlying rectification in retinal cyclic nucleotide-gated (CNGA1) channels.","date":"2013","source":"Physiological reports","url":"https://pubmed.ncbi.nlm.nih.gov/24400150","citation_count":11,"is_preprint":false},{"pmid":"33332786","id":"PMC_33332786","title":"Genome-wide Association Analysis Across 16,956 Patients Identifies a Novel Genetic Association Between BMP6, NIPAL1, CNGA1 and Spondylosis.","date":"2021","source":"Spine","url":"https://pubmed.ncbi.nlm.nih.gov/33332786","citation_count":11,"is_preprint":false},{"pmid":"30652268","id":"PMC_30652268","title":"A Novel CNGA1 Gene Mutation (c.G622A) of Autosomal Recessive Retinitis Pigmentosa Leads to the CNGA1 Protein Reduction on Membrane.","date":"2019","source":"Biochemical 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letters","url":"https://pubmed.ncbi.nlm.nih.gov/27561605","citation_count":7,"is_preprint":false},{"pmid":"2370846","id":"PMC_2370846","title":"Overproduction and translational regulation of rp49 ribosomal protein mRNA in transgenic Drosophila carrying extra copies of the gene.","date":"1990","source":"Molecular & general genetics : MGG","url":"https://pubmed.ncbi.nlm.nih.gov/2370846","citation_count":6,"is_preprint":false},{"pmid":"32705276","id":"PMC_32705276","title":"Identification of a novel homozygous variant in the CNGA1 gene in a Chinese family with autosomal recessive retinitis pigmentosa.","date":"2020","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/32705276","citation_count":4,"is_preprint":false},{"pmid":"38340451","id":"PMC_38340451","title":"Establishment of the induced pluripotent stem cell line SJTUGHi002-A from a CNGA1-related recessive retinitis pigmentosa patient.","date":"2024","source":"Stem cell research","url":"https://pubmed.ncbi.nlm.nih.gov/38340451","citation_count":1,"is_preprint":false},{"pmid":"40897815","id":"PMC_40897815","title":"Gene augmentation therapy restores vision and preserves photoreceptors in a mouse model of CNGA1-retinitis pigmentosa.","date":"2025","source":"Communications medicine","url":"https://pubmed.ncbi.nlm.nih.gov/40897815","citation_count":0,"is_preprint":false},{"pmid":"40795580","id":"PMC_40795580","title":"Generation of a human iPSC line (BTHBIOi001-A) from a retinitis pigmentosa patient with CNGA1 gene mutation.","date":"2025","source":"Stem cell research","url":"https://pubmed.ncbi.nlm.nih.gov/40795580","citation_count":0,"is_preprint":false},{"pmid":"42192302","id":"PMC_42192302","title":"Systematic functional evaluation of CNGA1 missense variants associated with retinitis pigmentosa.","date":"2026","source":"Molecular medicine (Cambridge, Mass.)","url":"https://pubmed.ncbi.nlm.nih.gov/42192302","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.12.19.629380","title":"Atomistic Mechanism of Cation Permeation and Pore Voltage Sensitivity in Cyclic Nucleotide-Gated CNGA1 Ion Channel","date":"2024-12-21","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.19.629380","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":17697,"output_tokens":4396,"usd":0.059515,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12374,"output_tokens":4267,"usd":0.084273,"stage2_stop_reason":"end_turn"},"total_usd":0.143788,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structures of human CNGA1 in both apo closed and cGMP-bound open states reveal that CNGA1 forms a central channel gate in the middle of the membrane (occluding the central cavity), distinct from most other voltage-gated tetrameric cation channels. Structural analysis of the wild-type channel and the E365Q selectivity filter mutant defined two Ca2+ binding sites inside the selectivity filter, providing a structural basis for Ca2+ blockage and permeation. The E365Q mutant also revealed two alternative side-chain conformations at Q365, explaining voltage-dependent gating acquired upon E365 mutation.\",\n      \"method\": \"Cryo-EM structure determination of apo and cGMP-bound states; E365Q active-site mutagenesis; ion binding profile analysis\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structures in two functional states combined with mutagenesis and ion binding analysis in a single rigorous study\",\n      \"pmids\": [\"33651975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structure of the native heterotetrameric bovine rod CNG channel (3 CNGA1 + 1 CNGB1) in the closed state shows that three CNGA1 subunits are tethered at their C-termini by a coiled-coil region. The CNGB1 subunit has a differently oriented C-helix in its CNBD compared to the three CNGA1 subunits, and CNGB1 residue R994 reaches into the ionic pathway to introduce an additional pore gate distinct from the central hydrophobic gate of homomeric CNGA1 channels.\",\n      \"method\": \"Cryo-EM structure of native bovine retinal rod CNG channel; subunit-resolved structural analysis\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM of native heterotetrameric channel with subunit-resolved structural detail\",\n      \"pmids\": [\"34969975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Two salt bridges in the C-linker region of CNGA1 (an intersubunit bridge between neighboring C-linkers, and an intrasubunit bridge between the C-linker and its CNBD) stabilize a closed channel conformation. Breaking these salt bridges by mutation increases the favorability of channel opening; swapping the charged residues restores wild-type behavior. This indicates that in CNGA1, the CNBD can be in a ligand-activated configuration while the C-linker remains in the resting configuration, suggesting a modular gating scheme.\",\n      \"method\": \"Site-directed mutagenesis of salt bridge residues in CNGA1 (and HCN2) expressed in Xenopus oocytes; electrophysiology\",\n      \"journal\": \"The Journal of general physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — charge-swap mutagenesis with functional rescue, two orthogonal channel systems tested, rigorous controls\",\n      \"pmids\": [\"15572346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Mutations restricted to the region around the ion selectivity filter of CNGA1 render channels essentially fully voltage-gated even in saturating cGMP, with channels remaining mostly closed at physiological voltages. This voltage-dependent gating resembles the selectivity filter-based mechanism of KcsA K+ channels. The mutations loosen the attachment of the selectivity filter to its surrounding structure, shifting gating equilibrium toward closed states. The wild-type structure surrounding the selectivity filter has evolved to suppress inherent voltage-dependent gating, ensuring cGMP binding alone suffices to open the channel.\",\n      \"method\": \"Site-directed mutagenesis of selectivity filter region; patch-clamp electrophysiology in Xenopus oocytes\",\n      \"journal\": \"The Journal of general physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis combined with detailed electrophysiological characterization; mechanistic model supported by multiple mutants\",\n      \"pmids\": [\"19635856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"In homotetrameric CNGA1 channels, at saturating cGMP concentrations, current levels distribute around a single mean with long open times. At low cGMP, current levels become heterogeneous (smaller, equal, or larger than saturating cGMP levels) with short open times. Ions that stay longer in the pore (generating larger single-channel currents) increase heterogeneity of current levels and decrease open probability and open times, suggesting that cGMP and permeating ions jointly modulate the extent and frequency of conformational fluctuations of the channel pore.\",\n      \"method\": \"Single-channel and macroscopic patch-clamp recordings from CNGA1 channels expressed in Xenopus oocytes; varying cGMP concentrations and permeant ion species\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct electrophysiological assay with multiple ionic conditions; single lab but orthogonal conditions tested\",\n      \"pmids\": [\"15308684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Using tail and gating current measurements from homotetrameric CNGA1 channels expressed in Xenopus oocytes, two distinct mechanisms underlie rectification: (1) single-channel current rectification in symmetrical Rb+ and Cs+ dominating macroscopic currents, and (2) voltage-dependent gating becoming prominent with larger organic cations (ethylammonium, dimethylammonium). Isochronal tail current analysis revealed at least two voltage-dependent transitions; only the first is sensitive to S4 helix charge mutations. The number of elementary charges per channel moving across the membrane is less than 2 (vs ~12 in K+ channels), indicating a restricted S4 motion with inefficient coupling to the channel gate that renders CNGA1 poorly sensitive to voltage under physiological conditions.\",\n      \"method\": \"Tail and gating current recordings from CNGA1 in Xenopus oocytes; S4 charge mutagenesis; multiple permeant ion species\",\n      \"journal\": \"Physiological reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gating current measurements combined with S4 mutagenesis and multiple ionic conditions; single lab\",\n      \"pmids\": [\"24400150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Dequalinium (an organic divalent cation) blocks CNGA1 channels from the intracellular side with K1/2 ~190 nM (at 0 mV) in a state-independent, voltage-dependent manner (zdelta ~1), occupying a binding site within the ion-conducting pathway. At the single-channel level, dequalinium acts as a slow blocker without changing unitary conductance. CNGA1+CNGB1 heteromeric channels show reduced sensitivity (K1/2 ~385 nM). Extracellular dequalinium also blocks but with voltage dependence suggesting it crosses the membrane to act from the inside.\",\n      \"method\": \"Inside-out and outside-out patch clamp from Xenopus oocytes expressing CNGA1 or CNGA1+CNGB1; single-channel recordings; concentration-response analysis\",\n      \"journal\": \"The Journal of general physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct electrophysiological characterization with single-channel resolution; single lab with multiple approaches\",\n      \"pmids\": [\"12508052\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"A missense mutation c.1537G>A (p.G513R) in CNGA1 causes the mutant protein to be largely retained inside the cell rather than being targeted to the plasma membrane, as shown by in vitro expression in HEK293 cells. This suggests that loss of CNGA1 from the plasma membrane underlies the pathogenic mechanism in this form of autosomal recessive retinitis pigmentosa.\",\n      \"method\": \"In vitro expression of Flag-tagged wild-type and mutant CNGA1 in HEK293 cells; western blot; immunofluorescence localization\",\n      \"journal\": \"Bioscience reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — two orthogonal methods (western blot + immunofluorescence) in heterologous cells; single lab\",\n      \"pmids\": [\"26802146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The missense mutation c.622G>A (p.D208N) in CNGA1, predicted to eliminate interactions between transmembrane segments S2 and S4, significantly reduces expression of CNGA1 protein, particularly at the plasma membrane of transfected HEK293T cells.\",\n      \"method\": \"Expression of Flag-tagged wild-type and G622A mutant CNGA1 in HEK293T cells; western blot; immunofluorescence\",\n      \"journal\": \"Biochemical genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single set of heterologous cell experiments with two detection methods but no functional electrophysiology\",\n      \"pmids\": [\"30652268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CNGA1 (but not CNGA2 or CNGA3) is expressed principally in rat alveolar type I cells at the protein level. Activation of CNG channels with 8Br-cGMP stimulates lung liquid absorption ~1.8-fold in a rat in situ lung liquid clearance assay; this stimulation is abolished by the CNGA1-specific inhibitor pseudechetoxin (PsTx) or L-cis-diltiazem, but not by amiloride. Patch-clamp confirmed CNGA1 channels in HEK293 cells are not inhibited by amiloride and ENaC is not inhibited by PsTx. Thus CNGA1 channels in alveolar type I cells underlie the amiloride-insensitive component of lung liquid reabsorption.\",\n      \"method\": \"RT-PCR and protein expression profiling; in situ rat lung liquid clearance assay with pharmacological inhibitors; patch-clamp of recombinant CNGA1 and ENaC in HEK293 cells\",\n      \"journal\": \"Pflugers Archiv : European journal of physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in situ functional assay combined with pharmacological specificity controls and patch-clamp validation; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"21559843\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CNG-A1 is localized at the basolateral membrane of proximal and distal tubules of rat renal cortex. Ovariectomy decreases CNG-A1 expression in the renal cortex, and 17β-estradiol benzoate replacement restores CNG-A1 expression to control levels, whereas progesterone replacement does not. This indicates estrogen, but not progesterone, modulates CNGA1 expression in renal cortex.\",\n      \"method\": \"Immunofluorescence localization in rat kidney; western blot of ovariectomized and hormone-replaced rats\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — localization and expression modulation by hormone shown by immunofluorescence and western blot in a single study; no functional channel assay\",\n      \"pmids\": [\"22759964\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CNGA1 knockout mice (generated by CRISPR/Cas9) show progressive retinal thinning, rod and cone degeneration, and functional deficits consistent with retinitis pigmentosa. RNA-sequencing of retinae revealed down-regulated synaptic transmission and phototransduction pathways as early as postnatal day 9, down-regulation of the PI3K-AKT-mTOR pathway indicating upregulation of autophagic processes, and chaperone-mediated autophagy coinciding with the time course of photoreceptor death.\",\n      \"method\": \"CRISPR/Cas9 knockout mouse model; ERG; immunofluorescence; TUNEL staining; RNA-sequencing; pathway analysis\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean knockout with multiple orthogonal readouts (functional, histological, transcriptomic); single lab\",\n      \"pmids\": [\"34418172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"AAV8-mediated subretinal delivery of mCnga1 driven by the rhodopsin promoter in Cnga1-/- mice results in correctly localized CNGA1 protein expression, sustained rescue of rod-mediated retinal function, long-term photoreceptor preservation for at least 9 months, improved vision-guided behavior, and upregulation of phototransduction-associated genes by RNA-seq. This demonstrates that CNGA1 restoration is sufficient to rescue rod photoreceptor function and survival.\",\n      \"method\": \"AAV8 gene augmentation in Cnga1-/- mice; ERG; immunofluorescence; vision-guided behavior assay; RNA-seq\",\n      \"journal\": \"Communications medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo rescue experiment with multiple functional and structural readouts; single lab, single study\",\n      \"pmids\": [\"40897815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"A medium-throughput aequorin-based functional assay of 86 CNGA1 missense variants showed that 72.1% significantly impaired CNG channel function (classified as functionally abnormal) and 23.3% were functionally normal. Two putative missense variants were shown by in vitro minigene splice assays to induce missplicing rather than amino acid change. AlphaMissense and CPT-1 in silico predictors best correlated with functional outcomes. Functional data enabled reclassification of 62.1% of VUS as likely pathogenic or likely benign.\",\n      \"method\": \"Aequorin-based medium-throughput cation flux assay for CNGA1 channel function; in vitro minigene splice assays; correlation with in silico predictors\",\n      \"journal\": \"Molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — medium-throughput functional assay covering 86 variants with two orthogonal methods (flux assay + splice assay); single lab\",\n      \"pmids\": [\"42192302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Large-scale atomistic MD simulations of homotetrameric CNGA1 under transmembrane voltages reveal that monovalent cation permeation involves hydrated cations in the selectivity filter. Hydration fluctuations in the central gate region, driven by pore flexibility, are proposed as the mechanism for excess single-channel conductance noise and characteristic flickering. An allosteric coupling between the selectivity filter and the central cavity gate is proposed as the mechanism for intrinsic voltage sensitivity of the pore.\",\n      \"method\": \"Atomistic molecular dynamics simulations under transmembrane voltage; analysis of cation hydration and pore dynamics\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational simulation only, no experimental validation reported; preprint\",\n      \"pmids\": [\"bio_10.1101_2024.12.19.629380\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CNGA1 is the principal alpha subunit of the rod photoreceptor cyclic nucleotide-gated (CNG) channel: it forms a homotetramer (or 3:1 heterotetrameric complex with CNGB1 in native rods) whose pore gate is located in the middle of the membrane, opens upon cGMP binding to its C-terminal cyclic nucleotide-binding domain via rearrangement of the C-linker region (stabilized in the closed state by intersubunit and intrasubunit salt bridges), has a selectivity filter that defines Ca2+ binding sites and suppresses inherent voltage-dependent gating so that cGMP binding alone suffices to open the channel at physiological voltages, and whose loss causes progressive rod photoreceptor degeneration that can be rescued by AAV-mediated gene augmentation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CNGA1 is the principal pore-forming alpha subunit of the rod photoreceptor cyclic nucleotide-gated (CNG) cation channel, the effector that transduces light-driven changes in cGMP into membrane current [#0, #11]. It assembles as a homotetramer in heterologous systems and as a 3:1 heterotetramer with CNGB1 in native rods, where the three CNGA1 subunits are tethered at their C-termini through a coiled-coil region while the incorporated CNGB1 subunit contributes an additional pore gate (via residue R994) distinct from the central hydrophobic gate of homomeric channels [#1]. Unlike most voltage-gated tetrameric cation channels, CNGA1 positions its activation gate in the middle of the membrane, occluding the central cavity, and opens upon cGMP binding to its C-terminal cyclic nucleotide-binding domain [#0]. Gating is modular: intersubunit and intrasubunit salt bridges in the C-linker stabilize the closed state, so that the CNBD can adopt a ligand-bound configuration while the C-linker remains at rest, and breaking these bridges favors opening [#2]. The selectivity filter defines two intrapore Ca2+ binding sites underlying Ca2+ block and permeation, and the wild-type filter architecture has evolved to suppress an inherent voltage-dependent gating mode so that cGMP binding alone suffices to open the channel at physiological voltages [#0, #3]. Consistent with this, CNGA1 moves fewer than two elementary gating charges across the membrane and is poorly voltage-sensitive under physiological conditions [#5]. Loss of CNGA1 causes progressive rod and cone degeneration: knockout mice develop a retinitis pigmentosa-like phenotype with down-regulated phototransduction and synaptic transmission and engagement of autophagic pathways [#11], and pathogenic missense variants act by retaining the protein intracellularly and depleting it from the plasma membrane [#7]. AAV-mediated CNGA1 gene augmentation restores correctly localized protein, rod function, and photoreceptor survival, establishing that CNGA1 restoration is sufficient for rescue [#12]. A distinct pool of CNGA1 functions outside the retina, mediating the amiloride-insensitive component of cGMP-stimulated lung liquid reabsorption in alveolar type I cells [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Establishing where blockers bind within the conduction pathway clarified the architecture of the open pore and provided a pharmacological tool to distinguish homomeric from heteromeric channels.\",\n      \"evidence\": \"Inside-out/outside-out patch clamp with dequalinium block of CNGA1 and CNGA1+CNGB1 in Xenopus oocytes\",\n      \"pmids\": [\"12508052\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Blocker binding site not resolved at atomic resolution\", \"Functional consequence of differential CNGB1 sensitivity in native rods not addressed\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identifying C-linker salt bridges that stabilize the closed state revealed that ligand binding and gate opening are mechanically separable, defining a modular allosteric gating scheme.\",\n      \"evidence\": \"Charge-swap mutagenesis with functional rescue in CNGA1 and HCN2 expressed in Xenopus oocytes; electrophysiology\",\n      \"pmids\": [\"15572346\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural transition coupling CNBD to the membrane gate\", \"Quantitative energetic contribution of each bridge not fully partitioned\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Single-channel analysis showed cGMP and permeant ions jointly shape pore conformational fluctuations, linking ligand occupancy and ion occupancy to gating behavior.\",\n      \"evidence\": \"Single-channel and macroscopic patch-clamp from CNGA1 in Xenopus oocytes varying cGMP and permeant ion species\",\n      \"pmids\": [\"15308684\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of ion-dependent fluctuations not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Mutations around the selectivity filter that rendered the channel fully voltage-gated demonstrated that the wild-type filter architecture actively suppresses intrinsic voltage sensitivity, explaining why cGMP alone opens the channel physiologically.\",\n      \"evidence\": \"Selectivity filter region mutagenesis with patch-clamp in Xenopus oocytes\",\n      \"pmids\": [\"19635856\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not provide the atomic structure of the filter in open and closed states\", \"Coupling between filter and central gate not directly observed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identifying CNGA1 in alveolar type I cells and its pharmacological control of fluid clearance extended its role beyond phototransduction to epithelial liquid transport.\",\n      \"evidence\": \"Protein expression profiling, in situ rat lung liquid clearance with PsTx/L-cis-diltiazem/amiloride, and patch-clamp validation in HEK293 cells\",\n      \"pmids\": [\"21559843\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"cGMP source and upstream signaling in alveolar cells not defined\", \"Physiological relevance to human lung not tested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Gating current measurements quantified that CNGA1 moves fewer than two charges with weak S4-to-gate coupling, mechanistically explaining its poor physiological voltage sensitivity.\",\n      \"evidence\": \"Tail and gating current recordings with S4 charge mutagenesis and multiple permeant ions in Xenopus oocytes\",\n      \"pmids\": [\"24400150\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural motion of S4 not visualized\", \"Two voltage-dependent transitions not fully assigned to structural elements\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrating that a pathogenic missense variant traps CNGA1 intracellularly established loss of plasma membrane targeting as a disease mechanism in autosomal recessive retinitis pigmentosa.\",\n      \"evidence\": \"Heterologous expression of WT and p.G513R CNGA1 in HEK293 cells; western blot and immunofluorescence\",\n      \"pmids\": [\"26802146\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No electrophysiology to confirm channel function loss\", \"Trafficking defect studied in heterologous cells, not rods\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"A second trafficking-defective missense variant reinforced reduced plasma-membrane expression as a recurring pathogenic theme.\",\n      \"evidence\": \"Expression of WT and p.D208N CNGA1 in HEK293T cells; western blot and immunofluorescence\",\n      \"pmids\": [\"30652268\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No functional electrophysiology\", \"Single lab, single heterologous system\", \"S2-S4 interaction loss inferred, not structurally confirmed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Cryo-EM of human CNGA1 in apo-closed and cGMP-bound open states defined a mid-membrane central gate and two intrapore Ca2+ sites, providing the structural framework for ligand gating and ion permeation.\",\n      \"evidence\": \"Cryo-EM of apo and cGMP-bound CNGA1 with E365Q selectivity filter mutagenesis and ion binding analysis\",\n      \"pmids\": [\"33651975\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics of the gating transition not captured\", \"Structures of homomeric channel may differ from native heteromer\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Cryo-EM of the native bovine 3:1 CNGA1-CNGB1 channel revealed C-terminal coiled-coil tethering and a CNGB1-specific second pore gate, defining how the native rod channel differs from the homomer.\",\n      \"evidence\": \"Cryo-EM of native bovine retinal rod CNG channel with subunit-resolved analysis\",\n      \"pmids\": [\"34969975\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Open-state structure of the heteromer not resolved\", \"Functional role of the CNGB1 R994 gate not directly tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A CRISPR knockout mouse causally linked CNGA1 loss to progressive photoreceptor degeneration and identified early down-regulated phototransduction and autophagy engagement as molecular correlates of cell death.\",\n      \"evidence\": \"CRISPR/Cas9 Cnga1 knockout mice with ERG, immunofluorescence, TUNEL, and RNA-seq\",\n      \"pmids\": [\"34418172\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal role of autophagy/PI3K-AKT-mTOR changes in death not tested\", \"Cone degeneration mechanism downstream of rod loss not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"AAV8 gene augmentation rescued rod function and photoreceptor survival in knockout mice, establishing that restoring CNGA1 alone is sufficient and providing therapeutic proof of concept.\",\n      \"evidence\": \"AAV8 subretinal delivery of mCnga1 in Cnga1-/- mice; ERG, immunofluorescence, vision-guided behavior, RNA-seq\",\n      \"pmids\": [\"40897815\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Durability beyond 9 months and large-animal/human efficacy not shown\", \"Optimal therapeutic window relative to degeneration stage not defined\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"A medium-throughput functional assay across 86 missense variants enabled large-scale variant classification and revealed that some apparent missense changes act through missplicing.\",\n      \"evidence\": \"Aequorin-based cation flux assay, minigene splice assays, and in silico predictor correlation\",\n      \"pmids\": [\"42192302\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Assay measures function in heterologous cells, not native rods\", \"Trafficking versus gating defects not distinguished for each variant\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How allosteric coupling between the selectivity filter and the central cavity gate generates the channel's characteristic flicker and residual voltage sensitivity awaits direct experimental confirmation.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"MD-predicted hydration fluctuations at the gate not experimentally validated\", \"No time-resolved structural data on the gating transition\", \"Coupling mechanism inferred computationally\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [0, 4, 9]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 2, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [7, 9, 10]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [11, 12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9709957\", \"supporting_discovery_ids\": [11, 12]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"complexes\": [\"Rod CNG channel (CNGA1/CNGB1 heterotetramer)\"],\n    \"partners\": [\"CNGB1\"],\n    \"other_free_text\": []\n  }\n}\n```","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}