Abstract
Purpose :
In the vertebrate retina, termination of the photoresponse is essential for sustained visual function and requires phosphorylation of photoactivated visual pigments in rods and cones by the G protein-coupled receptor kinases, GRK1 and GRK7. To determine the contribution of these GRKs to visual signaling in cones, we generated zebrafish lines in which Grk1b, Grk7a, or both have been selectively deleted. Also, we determined that both Grk1 and Grk7 are phosphorylated in the dark by cAMP-dependent protein kinase (PKA) in vertebrates. To understand how changes in cAMP levels may influence the cone photoresponse via phosphorylation of the cone-specific Grks, we incubated larvae in forskolin, an activator of adenylyl cyclase, prior to ERG analysis.
Methods :
TALEN genome editing was used to knock out Grk1b or Grk7a in zebrafish. A Grk1b/Grk7a double knockout (DKO) line was generated by crossing the single knockout lines. Recovery was evaluated by ERG in response to white light using a dual flash paradigm with increasing interstimulus intervals. To evaluate the effect of elevated cAMP on the rate of recovery in vivo, wildtype and knockout larvae were incubated in forskolin or vehicle (DMSO). All larvae were treated with APB prior to ERG analysis to isolate the cone mass receptor potential.
Results :
Deletion of grk1b or grk7a results in a significant decrease in recovery to successive stimuli compared to wild type larvae. This decrease was similar in grk1b-/- and grk7a-/- zebrafish larvae. Interestingly, the DKO larvae exhibit a faster recovery to successive stimuli compared to wildtype. After forskolin treatment, only wildtype larvae display a significantly decreased rate of recovery compared to DMSO-treated larvae.
Conclusions :
While these results suggest that grk1b, as well as grk7a, plays a significant role in cone visual signal termination, the rate of signal termination in DKO larval zebrafish cones may be regulated by the native rate of decay of photoactivated cone opsins. Increased levels of intracellular cAMP in wildtype zebrafish cones results in a significantly decreased recovery to successive stimuli compared to vehicle-treated larvae. However, this decrease is partially attenuated in grk1b-/-, grk7a-/- and DKO larvae. This observation suggests that cone photoresponse recovery in vertebrates is influenced by cAMP in vivo. This regulation may be mediated by phosphorylation of the cone-specific Grks.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.