title: Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment.
abstract: Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivo and channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivo channels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients.
authors.affiliation: Department of Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Naoto
authors.initials: N
authors.lastname: Tanaka
authors.affiliation: Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors.forename: Emily V
authors.initials: EV
authors.lastname: Dutrow
authors.affiliation: Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors.forename: Keiko
authors.initials: K
authors.lastname: Miyadera
authors.affiliation: Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Lucie
authors.initials: L
authors.lastname: Delemotte
authors.affiliation: Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: MacDermaid
authors.affiliation: Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors.forename: Shelby L
authors.initials: SL
authors.lastname: Reinstein
authors.affiliation: Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors.forename: William R
authors.initials: WR
authors.lastname: Crumley
authors.affiliation: Veterinary Vision, Penrith, Cumbria, CA11 9FJ, United Kingdom.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Dixon
authors.affiliation: Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors.forename: Margret L
authors.initials: ML
authors.lastname: Casal
authors.affiliation: Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Klein
authors.affiliation: Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors.forename: Gustavo D
authors.initials: GD
authors.lastname: Aguirre
authors.affiliation: Department of Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Jacqueline C
authors.initials: JC
authors.lastname: Tanaka
authors.affiliation: Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors.forename: Karina E
authors.initials: KE
authors.lastname: Guziewicz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D053447:Channelopathies / Q000175:diagnosis / Q000235:genetics* / Q000662:veterinary; D003117:Color Vision Defects / Q000175:diagnosis / Q000235:genetics* / Q000662:veterinary; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004285:Dogs; D006801:Humans; D015640:Ion Channel Gating; D016350:Leucine Zippers; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels