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Skitsko, Dominic

University of British Columbia

GNAT1 is the α-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 cell. Mutations in human GNAT1 cause Congenital Stationary Night Blindness. Experiments in knock-out GNAT1-/- mice have observed an absent rod ERG with largely unaffected morphology (Calvert et al., 2000). We sought to generate GNAT1-KO Xenopus laevis, taking advantage of their genetic similarity to humans and large photoreceptor size to study anatomically normal but non-functional photoreceptors. We have previously generated F0 GNAT1-KO animals with CRISPR technology, but these animals are genetically chimeric. Therefore, we interbred these F0 animals to produce F1 animals of defined genotypes. Investigation of F1 animals via western blot, histology, electroretinography (ERG), and DNA sequencing has identified true and functional knockouts, a relationship between GNAT1 expression and phototransduction kinetics, and mutations that alter trafficking. X. laevis tadpoles with nonsense mutations in all four GNAT1 alleles presented absent scotopic ERGs. Histology and western blot confirmed the absence of GNAT1 protein, indicating full knockout. Reduction in the level of GNAT1 protein led to a suppression of the ERG, however the relationship was not as direct as expected. We also noted a delay in the timing of the A and B-waves. Greater reduction in GNAT1 protein was associated with greater suppression and delay of the ERG, indicating a second functional role of GNAT1 in addition to signal transduction. We identified two distinct mutations tied to unexpected phenotypes. GNAT1.L-Q184L conferred an ERG phenotype similar to full knockout animals but with minimally reduced GNAT1 protein. A three base pair deletion (T183,Q184→K183) in GNAT1.S caused abnormal rod morphology at the connecting cilium, diminished ERG response, altered return of GNAT1 to the outer segment following light exposure, and defective dark adaptation. The morphological and electrophysiological effects of the Δ3-GNAT1.S mutation were dominantly inherited and required a population of wildtype GNAT1 present to exert their maximum effect.

Text

2024-05-21

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/88339

Experimental Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/