Inherited macular degeneration-associated mutations in CNGB3 increase the ligand sensitivity and spontaneous open probability of cone cyclic nucleotide-gated channels

Abstract

Cyclic nucleotide gated (CNG) channels are a critical component of the visual transduction cascade in the vertebrate retina. Mutations in the genes encoding these channels have been associated with a spectrum of inherited retinal disorders. To gain insight into their pathophysiological mechanisms, we have investigated the functional consequences of several CNGB3 mutations, previously associated with macular degeneration (Y469D and L595F) or complete achromatopsia (S156F, P309L, and G558C), by expressing these subunits in combination with wild-type CNGA3 in Xenopus oocytes and characterizing them using patch-clamp recordings in the inside-out configuration. These mutations did not prevent the formation of functional heteromeric channels, as indicated by sensitivity to block by L-cis-diltiazem. With the exception of S156F, each of the mutant channels displayed electrophysiological properties reflecting enhanced channel activity at physiological concentrations of cGMP (i.e., a gain-of-function phenotype). The increased channel activity produced by these mutations resulted from either increased functional expression levels, or increased sensitivity to cyclic nucleotides. Furthermore, L595F increased the spontaneous open probability in the absence of activating ligand, signifying a ligand independent gain-of-function change. In addition to the CNGB3 disease-associate mutations, we characterized the effects of several common CNGB3 and CNGA3 single-nucleotide polymorphisms (SNPs) on heteromeric CNGA3+CNGB3 channel function. Two of the SNPs examined (A3-T153M, and B3-W234C) produced decreased ligand sensitivity for heteromeric CNG channels. These changes may contribute to background disease susceptibility when combined with other genetic or non-genetic factors. Together, these studies help to define the underlying molecular phenotype for mutations relating to CNG channel disease pathogenesis.

Achromatopsia: Genetics and Gene Therapy

“…Folding, intracellular processing, and transport are thought to be impaired [29]. The effects of individual CNGA3 amino acid substitutions on CNG channel function have mainly been studied in vitro [21,[30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46]. Some insights have been gained, but the exact mechanisms linking CNGA3 amino acid substitutions to cone photoreceptor dysfunction and eventual degeneration are still not well understood.…”

The Effect of A2E on the Ca2+-PKC Signaling Pathway in Human RPE Cells Exposed to Blue Light

“…AMD is a progressive blinding disease with no cure and is the main cause of visual disability in industrialized countries [10][11][12]. Increasing evidence has shown that mitochondrial and RPE cells damage plays important roles in the pathogenesis of AMD [13].…”

Comprehensive variant spectrum of the CNGA3 gene in patients affected by achromatopsia

“…Therefore, functional studies are needed to validate the possible pathogenic effect of the respective variants. Commonly used methods to characterize the functionality of mutant CNG channels involve calcium imaging and patch‐clamp recordings of heterologously expressed mutant channels (Burkard et al, 2018; Dai & Varnum, 2013; Koeppen et al, 2008, 2010; Kuniyoshi et al, 2016; Liu & Varnum, 2005; Matveev et al, 2010; Meighan et al, 2015; Muraki‐Oda et al, 2007; Patel et al, 2005; Reuter et al, 2008; Shaikh et al, 2015; Täger et al, 2018; Tränkner et al, 2004). Furthermore, a recently developed functional assay proposes a fluorescence‐based, high‐throughput calcium influx‐based approach for analyzing mutant CNGA3 channels (Jacobson et al, 2019).…”

“…Following functional characterization of heterologously expressed mutant channels in previously conducted studies, 27 out of 54 missense variants completely abolished formation of functional channels (Koeppen et al, 2008, 2010; Liu & Varnum, 2005; Matveev et al, 2010; Muraki‐Oda et al, 2007; Patel et al, 2005; Shaikh et al, 2015; Tränkner et al, 2004). Seventeen previously analyzed missense substitutions allowed the formation of functional channels but with impaired/altered functionality such as impaired protein folding and/or channel trafficking or altered biophysical properties, for example, altered apparent cGMP‐sensitivity or ion selectivity (Burkard et al, 2018; Dai & Varnum, 2013; Koeppen et al, 2008; Liu & Varnum, 2005; Meighan et al, 2015; Muraki‐Oda et al, 2007; Patel et al, 2005; Reuter et al, 2008; Tränkner et al, 2004). Furthermore, an improvement in the mutant channel functionality was achieved in a total of ten missense variants by using chemical chaperones or co‐expressing the CNGB3 subunit and/or reducing the cell cultivation temperature from the standard 37°C to 27/28°C, a procedure that is known to enhance protein folding and trafficking (Koeppen et al, 2010; Kuniyoshi et al, 2016; Patel et al, 2005; Reuter et al, 2008).…”

“…Nonsense and frameshift variants likely result in the loss of the CNGA3 subunit, while some missense variants in the CNGA3 subunit have been shown to impair or even abolish CNG channel function and/or protein folding and trafficking leading to impaired phototransduction and impaired or lost cone function (Burkard et al, 2018; Dai & Varnum, 2013; Duricka et al, 2012; Koeppen et al, 2008, 2010; Kuniyoshi et al, 2016; Liu & Varnum, 2005; Matveev et al, 2010; Meighan et al, 2015; Michalakis et al, 2017; Muraki‐Oda et al, 2007; Patel et al, 2005; Reuter et al, 2008; Shaikh et al, 2015; Täger et al, 2018; Tränkner et al, 2004).…”