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.

Concepts and Strategies in Retinal Gene Therapy

“…These models represent bona fide human disease homologues where the disease phenotype in model and man are the same. Selected examples include RPE65 -LCA, 3 , 5 , 29 BEST1 -BVMD, 30 – 32 CNGB3 - and CNGA3 -achromatopsia, 33 , 34 RHO -ADRP, 35 RPGR -XLRP, 32 , 36 – 38 and NPHP5 -LCA. 39 …”

Large Animal Models of Inherited Retinal Degenerations: A Review

“…Protein modeling of the canine R424W mutation found the disruption of this salt bridge plays an important role in protein folding, subunit assembly and channel gating. In vitro expression studies of the R424W mutation showed increased mislocalization of the mutant CNGA3 protein and the mutant channels did not produce cyclic nucleotide-activated currents [82].…”

“…The mutations, p.Arg424Trp and p.Val644del (R424W and V644del, respectively), provided intriguing mutation sites for in vitro testing. Modeling of these mutations lead to further insights into CNG channel gating and subunit interactions [82]. The residue R424 is located in the S6 transmembrane helix and forms a salt bridge with the residue E306 in the S4-S5 linker.…”

“…The one amino acid deletion shifts all subsequent residue interactions within the domain. In vitro studies in a heterologous expression system showed evidence of mislocalization and a decrease in the total number of active channels, but some mutant channels (~60%) reached the cell membrane and had normal cyclic nucleotide-activated currents [82].…”

Electroretinographic findings in day-blind dogs

“…For selected canine breeds, the diagnosis of cd is aided by genetic tests. As far as the aetiology of cd is concerned, CNGA3 (Cyclic Nucleotide Gated Channel Alpha 3) gene mutation in German Shepherd (R424W mutation) and Labrador Retriever (V644del mutation) (Tanaka et al, 2015), CNGB3 (Cyclic Nucleotide Gated Channel Beta 3) gene mutations in German Shorthaired Pointers (D262N mutation) (Sidjanin et al, 2002) and CNGB3 gene deletion in Alaskan Malamute (Sidjanin et al, 2002), Miniature Australian Shepherd, Alaskan Sled Dogs and Siberian Husky (Yeh et al, 2013) have been discussed. All these genes mutations are specifically expressed in the cone photoreceptors leading to achromatopsia.…”