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Published on: August 24, 2022
Disease-associated mutations in CNGB3 promote cytotoxicity in photoreceptor-derived cells
Chunming Liu1, Tshering Sherpa, Michael D Varnum
1College of Optometry, Western University of Health Sciences, Pomona, CA, USA.
Purpose:
To determine if achromatopsia associated F525N and T383fsX mutations in the CNGB3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels increases susceptibility to cell death in photoreceptor-derived cells.
Methods:
Photoreceptor-derived 661W cells were transfected with cDNA encoding wild-type (WT) CNGA3 subunits plus WT or mutant CNGB3 subunits, and incubated with the membrane-permeable CNG channel activators 8-(4-chlorophenylthio) guanosine 3',5'-cyclic monophosphate (CPT-cGMP) or CPT-adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Cell viability under these conditions was determined by measuring lactate dehydrogenase release. Channel ligand sensitivity was calibrated by patch-clamp recording after expression of WT or mutant channels in Xenopus oocytes.
Results:
Coexpression of CNGA3 with CNGB3 subunits containing F525N or T383fsX mutations produced channels exhibiting increased apparent affinity for CPT-cGMP compared to WT channels. Consistent with these effects, cytotoxicity in the presence of 0.1 μM CPT-cGMP was enhanced relative to WT channels, and the increase in cell death was more pronounced for the mutation with the largest gain-of-function effect on channel gating, F525N. Increased susceptibility to cell death was prevented by application of the CNG channel blocker L-cis-diltiazem. Increased cytotoxicity was also found to be dependent on the presence of extracellular calcium.
Conclusions:
These results indicate a connection between disease-associated mutations in cone CNG channel subunits, altered CNG channel-activation properties, and photoreceptor cytotoxicity. The rescue of cell viability via CNG channel block or removal of extracellular calcium suggests that cytotoxicity in this model depends on calcium entry through hyperactive CNG channels.
Insights
Achromatopsia mutations in cone cyclic nucleotide-gated (CNG) channels increase cell death in photoreceptor cells. Blocking these hyperactive channels or removing extracellular calcium rescues cell viability.
Area of Science:
- Ophthalmology
- Cell Biology
- Molecular Genetics
Background:
- Achromatopsia is a severe inherited retinal disorder characterized by reduced visual acuity, photophobia, and absence of color vision.
- Cone photoreceptor dysfunction is central to achromatopsia, often linked to mutations in genes encoding cyclic nucleotide-gated (CNG) channels.
Purpose of the Study:
- To investigate if specific achromatopsia-associated mutations (F525N, T383fsX) in the CNGB3 subunit of cone CNG channels enhance photoreceptor cell death.
- To elucidate the mechanism by which these mutations affect channel function and contribute to cytotoxicity.
Main Methods:
- Photoreceptor-derived 661W cells were transfected with wild-type (WT) or mutant CNGB3 subunits alongside CNGA3.
- Cells were treated with CNG channel activators (CPT-cGMP, CPT-cAMP), and cell viability was assessed via lactate dehydrogenase release.
- Channel activity was characterized using patch-clamp recordings in Xenopus oocytes.
Main Results:
- Mutant CNGB3 subunits (F525N, T383fsX) resulted in CNG channels with increased apparent affinity for CPT-cGMP.
- Cytotoxicity was significantly enhanced in cells expressing mutant channels, particularly with the F525N mutation.
- Cell death was preventable by L-cis-diltiazem (a CNG channel blocker) and dependent on extracellular calcium.
Conclusions:
- Disease-associated mutations in cone CNG channel subunits alter channel gating properties, leading to photoreceptor cytotoxicity.
- Hyperactivation of CNG channels and subsequent calcium influx appear to mediate cell death.
- Targeting CNG channel activity or calcium entry may offer therapeutic strategies for achromatopsia.
