Alterations in phototransduction timing, dark adaptation and GNAT1 trafficking in GNAT1-edited Xenopus laevis.
- 1The University of British Columbia
Abstract
GNAT1 (also called -transducin) is the -subunit of a heterotrimeric G-protein present in rod photoreceptors that plays a critical role in phototransduction. Photoactivated rhodopsin binds GNAT1, which stimulates phosphodiesterase to hydrolyze cGMP, closing ion channels and hyperpolarizing the rod cell. To examine the effects of GNAT1 ablation on light-induced autophagy and light-exacerbated retinal degeneration, we previously generated genetically chimeric, GNAT1-edited Xenopus laevis using CRISPR technology. Here, we interbred these F0 animals to produce F1 animals with defined genotypes, including full knockouts, and characterized the F1 generation by Western blot, histology, and electroretinography (ERG). In X. laevis tadpoles with nonsense mutations in all four GNAT1 alleles, the GNAT1 protein was absent, and the scotopic ERG was absent, indicating full knockout. In animals with reduced GNAT1 expression, we observed increased A- and B-wave implicit times. Greater GNAT1 protein reduction was associated with greater ERG delay, demonstrating a role for GNAT1 in the timing of phototransduction. We also identified two unexpected phenotypes linked to distinct gene edits. The Q184L-GNAT1.L mutation conferred an ERG phenotype similar to that of a full knockout, but with minimal reduction in GNAT1 protein levels, consistent with loss of function. This mutation is also present in the human population. A three-base-pair deletion (T183,Q184[->]K183) in GNAT1.S caused dominantly inherited diminished ERG response and defective dark adaptation, and was associated with photoreceptor structural abnormalities and GNAT1 accumulation at the connecting cilium, consistent with a dominant-negative effect on GNAT1 trafficking.
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BibTeX
@misc{skitsko2026alterations,
title = {{Alterations in phototransduction timing, dark adaptation and GNAT1 trafficking in GNAT1-edited Xenopus laevis.}},
author = {D. Skitsko and B. M. Tam and C. N. Chiu and P. Burns and O. L. Moritz},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.26.761424},
url = {https://www.biorxiv.org/content/10.64898/2026.10.26.761424v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Alterations in phototransduction timing, dark adaptation and GNAT1 trafficking in GNAT1-edited Xenopus laevis.
AU - D. Skitsko
AU - B. M. Tam
AU - C. N. Chiu
AU - P. Burns
AU - O. L. Moritz
PY - 2026
DA - 2026/10/26
PB - bioRxiv
DO - 10.64898/2026.10.26.761424
UR - https://www.biorxiv.org/content/10.64898/2026.10.26.761424v1
AB - GNAT1 (also called -transducin) is the -subunit of a heterotrimeric G-protein present in rod photoreceptors that plays a critical role in phototransduction. Photoactivated rhodopsin binds GNAT1, which stimulates phosphodiesterase to hydrolyze cGMP, closing ion channels and hyperpolarizing the rod cell. To examine the effects of GNAT1 ablation on light-induced autophagy and light-exacerbated retinal degeneration, we previously generated genetically chimeric, GNAT1-edited Xenopus laevis using CRISPR technology. Here, we interbred these F0 animals to produce F1 animals with defined genotypes, including full knockouts, and characterized the F1 generation by Western blot, histology, and electroretinography (ERG). In X. laevis tadpoles with nonsense mutations in all four GNAT1 alleles, the GNAT1 protein was absent, and the scotopic ERG was absent, indicating full knockout. In animals with reduced GNAT1 expression, we observed increased A- and B-wave implicit times. Greater GNAT1 protein reduction was associated with greater ERG delay, demonstrating a role for GNAT1 in the timing of phototransduction. We also identified two unexpected phenotypes linked to distinct gene edits. The Q184L-GNAT1.L mutation conferred an ERG phenotype similar to that of a full knockout, but with minimal reduction in GNAT1 protein levels, consistent with loss of function. This mutation is also present in the human population. A three-base-pair deletion (T183,Q184[->]K183) in GNAT1.S caused dominantly inherited diminished ERG response and defective dark adaptation, and was associated with photoreceptor structural abnormalities and GNAT1 accumulation at the connecting cilium, consistent with a dominant-negative effect on GNAT1 trafficking.
ER -