Abstract
Purpose: :
The deactivation of transducin has been shown to rate-limits the recovery of rod phototransduction. Using a gain of function approach by transgenic overexpression of transducin GAP complex or a G-protein coupled receptor kinase GRK7, we hope to determine the rate-limiting step of cone phototransduction recovery.
Methods: :
R9AP and human GRK7 (hGRK7) are cloned downstream of a blue or green opsin promoter, followed by a mouse protamine polyA signal, to drive cone specific expression in mouse retina. Immunocytochemistry (ICC) and confocal microscopy are used to determine the level of transgene expression. Recovery of cone phototransduction are determined by double flash electroretinogram (ERG) protocol, with interstimulus intervals (ISI) ranging from 100 to 2000 ms and under the presence of a white background light. The waveforms are normalized to the maximum recovery and fitted with a single exponential function.
Results: :
ICC studies shows that GRK1 is expressed in the outer segments of rod and cone photoreceptors. Pixel density analysis reveals that the level of GRK1 in cone photoreceptor is ~1.6 fold higher than that of rod using two independent antibodies. So far we have characterized four transgenic lines (MCV7, 8, 9, and 10) and studied their cone recovery kinetics. In MCV7 and MCV8 lines (hGRK7 behind blue and green opsin promoters, respectively), hGRK7 are expressed and localized to the outer segment (OS) of mouse cones. When crossed into the GRK1 knockout (GRK1-/-) background, transgenic hGRK7 can restore cone-derived ERG B-wave in both MCV7 and MCV8 lines, indicating that hGRK7 can substitute for the loss of GRK1 in cones. Not surprisingly, in MCV9 and MCV10 lines (R9AP behind blue and green opsin promoters, respectively), RGS9-1 are overexpressed in cones only. Both MCV9 and MCV10, when compared to their WT siblings, have accelerated cone recovery.
Conclusions: :
The higher concentration of GRK1 in cones than in rods indicates that cone opsin phosphorylation by GRK1 might not rate-limit the recovery of cone phototransduction. Akin to what was reported in rod, overexpression of R9AP alone is sufficient to elevate the concentration of RGS9-1/Gβ5-L/R9AP in cones. When the GAP complex is overexpressed, the recovery of cone-derived ERG responses accelerates in two independent transgenic lines. These data suggest that transducin deactivation constitutes the slowest step required for the normal recovery of both rod and cone photoreceptors in mice.
Keywords: photoreceptors • opsins • transgenics/knock-outs