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O. P. Gross, M. E. Burns; Arrestin Availability Directly Affects Rhodopsin Lifetime. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5831.
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Although arrestin is known to play an essential role in the deactivation of photoexcited rhodopsin, previous studies in mouse rods have shown that low arrestin expression has little effect on photoresponses. This has been interpreted to mean that arrestin binding does not rate-limit photoresponse recovery (Xu et al., 1997). More recently, the rate-liming step for recovery has been shown to be T/PDE deactivation, with rhodopsin phosphorylation and arrestin binding occurring rapidly, on the time scale of the peak of the dim flash response (Krispel et al., 2006). Thus, changes in arrestin concentration would be expected primarily to affect the rising phase and peak amplitude of responses. We have re-examined the role of arrestin binding in rhodopsin shutoff by carefully analyzing the single photon responses of rods that underexpress arrestin, both in the wild-type background and in rods in which T/PDE deactivation has been accelerated so that the light responses more closely follow the time course of rhodopsin activity.
Quantitative western blot analysis was performed to assess the expression of rod arrestin in purified rod outer segments (ROS) of wild-type (c57Bl/6) and Arr+/- mice. Arrestin knockout mice (Xu et al., 1997) were bred with R9AP transgenic rods expressing 4-fold more RGS9 complex than normal (Krispel et al., 2006) in order to generate Arr+/-RGS9ox mice. Suction electrode recordings and single photon response analysis were performed as previously described (Krispel et al., 2006).
Quantitative western blot analysis of purified ROS from wildtype and Arr+/- retinas revealed a greater than 50% reduction in arrestin expression in the hemizygous ROS. A clear phenotype was observed in rods that underexpressed arrestin compared to those with normal arrestin expression. In rods with fast T/PDE deactivation, underexpression of arrestin caused a slower time to peak, slower time constant for recovery, and larger amplitude for elementary responses. Consistent with previous work (Xu et. al., 1997), this trend was considerably more modest when T/PDE deactivation was rate-limiting.
Decreased arrestin concentration in the ROS led to slower rhodopsin shutoff. The larger magnitude of the effects observed when T/PDE deactivation was fast confirms that rhodopsin deactivation via arrestin binding is normally faster than deactivation of downstream deactivation processes. This study constitutes the first step towards development of an electrophysiological assay for the amount of arrestin in the ROS.
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