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C.M. Krispel, C.K. Chen, D. Chen, Y.J. Chen, N. Calero, M.E. Burns; The Rate Limiting Step in the Deactivation of the Phototransduction Cascade . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4628.
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Purpose: The phototransduction cascade of retinal rods converts absorbed photons into an electrical neural response. The single exponential recovery phase of this light response reflects the rate of a single biochemical step in the deactivation of the phototransduction cascade. The identity of this rate–limiting step is unknown, but must be either the deactivation of the receptor protein rhodopsin (Rh), or the deactivation of the G protein /effector complex (T/PDE) (Lyubarsky et al 1996). The purpose of this study was to determine which of these deactivation steps rate limits the recovery of the light response. Methods: To determine the rate–limiting step, we sought to speed up Rh and T/PDE deactivation by overexpression of the relevant enzymes. Rh deactivation requires phosphorylation by rhodopsin kinase (RK), while timely deactivation of T/PDE requires GTP hydrolysis catalyzed by the RGS9 protein complex. Mice expressing different levels of these proteins in their photoreceptors were generated by standard transgenic methods. For each mouse, we used suction electrodes to record flash responses from individual rod photoreceptors from one retina, and determined the protein expression levels by quantitative western blots from the second retina. We then compared response kinetics to the expression levels of RK and RGS9 in each of the transgenic lines. Results: Five lines of mice with varying expression levels of RGS9 and four lines of mice with varying expression levels of RK were examined. We found that response recovery rate increased with increasing expression of the RGS9 complex. Lines with the highest expression levels (2–3 fold overexpression) showed the fastest responses, which recovered with a time constant of 70 ms. In contrast, increased expression of RK did not accelerate response recovery. Conclusions: These results show that overexpression of the RGS9 complex speeds the time course of the rod’s light response, while overexpression of RK does not. We conclude that GTP hydrolysis by transducin normally rate–limits the recovery of the flash response in rods. Thus, the control of RGS9 expression and activity is likely very important for controlling signal duration in photoreceptors. The second–slowest step in response recovery is significantly faster, and appears to have a time constant of 70 ms.
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