Abstract
Purpose: :
Rods adapt to background light with a quickening of response decay and decrease in sensitivity. Acceleration of response decay is largely the result of modulation of turn-off of PDE, and this also contributes to the decrease in sensitivity; but other experiments suggest that a Ca2+-dependent change in activated rhodopsin (R*) lifetime may also make an important contribution. R* lifetime is difficult to study directly, since it is normally so short that PDE turnoff is rate limiting. We therefore genetically engineered mice to make PDE turnoff more rapid than normal, and R* turnoff slower, so that rod responses would decay only as R* activity was extinguished.
Methods: :
We made suction-electrode recordings from single mouse rods by conventional techniques.
Results: :
We used R9AP95 mice in which the GAP proteins are over-expressed by about 6-fold, greatly speeding the kinetics of PDE deactivation. R9AP95 mice were mated to animals in which rhodopsin kinase (RK) activity had been reduced either to about 40% (in RK+/-) or about 15% (in RKux, a chimera of RK and BARK1), in order to slow the rate of Rh phosphorylation. We quantified the rate of turnoff by fitting the waveform of recovery to a single exponential (tauREC). The tauREC in animals that are R9AP95 alone is less than 80 ms and much more rapid than in WT animals; it was little affected in R9AP95;RK+/- animals, indicating that a 40% reduction in RK expression was insufficient to make R* decay rate limiting. But tauREC was nearly 400 ms in R9AP95;RKux and almost as slow as in RKux alone, indicating that decreasing rhodopsin kinase activity to only about 15% slowed the rate of phosphorylation sufficiently so R* lifetime became rate-limiting. When these rods were exposed to background illumination, flash response recovery was markedly accelerated; tauREC declined by more than a factor of 3.
Conclusions: :
The acceleration of response decay in the R9AP95;RKux rods exposed to background light would only occur if R* lifetime were shortened in backgrounds. Modulation of R* lifetime during background exposure would reduce the number of TalphaGTPs produced per R* and may contribute to the decrease in sensitivity in backgrounds. Our experiments provide the first direct physiological demonstration that R* lifetime is modulated during light adaptation.
Keywords: photoreceptors • opsins • retina: distal (photoreceptors, horizontal cells, bipolar cells)