Proceedings of The Physiological Society
University of Cambridge (2008) Proc Physiol Soc 11, C77
Background light modulates activated rhodopsin lifetime in mouse rods
C. Chen1, M. L. Woodruff2, F. S. Chen1, D. Chen3, G. L. Fain2,4
1. Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia, USA. 2. Physiological Science, UCLA, Los Angeles, California, USA. 3. Tulane University, New Orleans, Louisiana, USA. 4. Ophthalmology, UCLA, Los Angeles, California, USA.
Retinal rods adapt to steady background light by acceleration of response decay and a decrease in sensitivity. Recent experiments (1) have shown that in mouse, response decay quickens largely from modulation of turn-off of cyclic GMP phosphodiesterase (PDE). This process also decreases sensitivity, but experiments on salamander rods suggest that a Ca-dependent change in activated rhodopsin (R*) lifetime may also make an important contribution (2). Changes in R* lifetime are difficult to study directly, since it is normally so short that PDE turnoff is rate limiting for the decay of the light response. We therefore made suction-electrode recordings from isolated rods (as in ref. 1) of mice genetically engineered to make PDE turnoff much more rapid than normal, and R* turnoff slower, so that rod responses would decay only as R* activity was extinguished. We used R9AP95 mice in which the GTPase activating (GAP) proteins are over-expressed by about 6-fold. Since the GAP proteins are obligate activators of transducin alpha GTP hydrolysis, they regulate the rate of PDE turnoff, and over-expression of these proteins greatly speeds the kinetics of PDE deactivation (3). We then mated the R9AP95 mice with animals in which rhodopsin kinase (RK) activity had been reduced either to about 40% (in RK+/-) or about 15% (in RKux), in order to slow the rate of rhodopsin phosphorylation and turnoff of R*. We quantified the rate of turnoff by fitting the waveform of response recovery to a single exponential with time constant τ_REC. Previous experiments showed that τ_REC in animals that are R9AP95 alone is less than 80 ms and much more rapid than in WT animals (3), indicating that R9AP95 alone greatly accelerates PDE turnoff, which is normally rate-limiting. The value of τ_REC, however, was progressively slowed to 112 ± 16 (SE, n = 14) in R9AP95;RK+/- and 415 ± 70 (n = 7) in R9AP95;RKux. This shows that decreasing rhodopsin kinase activity with RK+/- and RKux slows the rate of rhodopsin phosphorylation sufficiently, so that R* lifetime becomes rate-limiting for response decay. When these rods were then exposed to background light, flash response recovery was accelerated. This could only have occurred if the R* lifetime was shortened by the background. This is the first direct physiological demonstration that R* lifetime is modulated during light adaptation. Our results also indicate that response recovery can be accelerated even in the absence of a background simply by increasing the flash intensity. Since increasing intensities produce progressively larger and longer reductions in circulating current and decreases in outer segment Ca, our results are consistent with a mechanism in which background light lowers Ca, which in turn decreases R* lifetime probably by modulating the rate of rhodopsin phosphorylation.
Where applicable, experiments conform with Society ethical requirements