Molecular Mechanism of Canine CNGA3-associated Channelopathies

Abstract

Purpose: Homozygous or compound heterozygous mutations in CNGA3 in man underlie the severe and currently untreatable cone-specific disorder of complete and incomplete achromatopsia (ACHM2). We assessed the molecular and functional consequences of two natural canine models of CNGA3-associated channelopathy: a newly discovered V644del mutation and the previously reported R424W transition occurring in the German shepherd dog.

Methods: A combined strategy of phenotype-directed candidate gene analysis and homozygosity mapping was used to identify the genetic basis of ACHM. A set of in vitro experiments and molecular dynamics simulations (NAMD software) were used to assess the mutational effects on CNGA3 subunits.

Results: Both the CNGA3-R424W and -V644del mutations lead to complete achromatopsia, and absence of cone photoreceptor function in all affected dogs. The mutational in vitro electrophysiological analysis demonstrated that the R424 residue in S6 is essential for stabilizing the channel open state through a salt bridge interaction with a residue on the S4-S5 linker. The in silico modeling provided additional insights into the nature of changes in the pore of R424W-mutant channel. The V644del mutation alters the stability of trimeric coiled-coil interactions in the C-terminus impacting channel assembly. Moreover, the canine disease shares clinical phenotype and molecular pathology with human patients carrying corresponding mutations in CNGA3 ortholog that cluster in the C-terminal mutational hotspot of the subunit, a critical part for channel gating and tetramerization.

Conclusions: We propose R424W and V644del as valuable disease models for studying CNG channel malfunction and its consequences on the cone-specific signal transduction pathway. Based on our findings, the two novel canine models of CNGA3-associated channelopathy represent prime candidates for rAAV-mediated gene augmentation therapy, and will both complement and validate the ongoing proof-of-concept studies.