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.
Highlights
Cyclic nucleotide-gated (CNG) ion channels are important non-selective cation channels that show increased gating with cyclic nucleotides; they function as cellular transducers in signaling pathways of sensory and CNS neurons as well as in non-neuronal cells [1, 2]
In the visual system, rodmediated phototransduction utilizes outer segment membrane CNG channels assembled from three CNGA1 subunits and one CNGB1, whereas the cone-specific CNG channels are composed of CNGA3 and CNGB3 subunits, arranged in a proposed stoichiometric ratio of 3:1, respectively [3,4,5], and see [6]
We report the first spontaneous canine ACHM2 disease models caused by R424W and V644del mutations in the dog CNGA3 ortholog
Summary
Cyclic nucleotide-gated (CNG) ion channels are important non-selective cation channels that show increased gating with cyclic nucleotides; they function as cellular transducers in signaling pathways of sensory and CNS neurons as well as in non-neuronal cells [1, 2]. Homozygous or compound heterozygous mutations in any of five critical genes involved in the cone phototransduction cascade have been causally attributed to functional abnormalities known as complete and incomplete achromatopsia (ACHM). ACHM refers to a group of cone-selective retinal disorders characterized by loss of photopic vision, photophobia, poor visual acuity, pendular nystagmus, rod monochromacy and anomalous cone photoreceptormediated function caused directly by mutations in genes disrupting cone phototransduction pathway [7, 8]. Mutations associated with the CNGB3 subunit are most common in European subjects (ACHM3; OMIM# 262300) and account for 50% of all known cases worldwide, whereas the CNGA3-associated achromatopsia (ACHM2; OMIM# 216900) accounts for ~30% of all diagnosed cases far. There is no treatment available that restores cone function in achromatopsia patients
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