The ΔTsat shift was +180 ms (upward) for Grk1+/− responses compared to wild-type, and −250 ms (downward) for Grk1S561 responses. Assuming downstream signaling is the same in WT, Grk1+/−, and Grk1S561L rods, the lifetimes of R∗(τReff) can be calculated from these ΔTsat values ( Gross and Burns, 2010): equation(1) τReff=[1τE+(1τReff,wt−1τE)e−ΔTsat/τE]−1. Assuming the lifetime of R∗ in normal rods (τReff,wt) is 40 ms (Gross and Burns, 2010), the values

of τReff for Grk1+/− and Grk1S561L rods calculated with Equation 1 are 76 ms and 15 ms, respectively. Thus, modifying the expression level or catalytic activity of rhodopsin kinase tunes the effective lifetime of R∗ and the vertical offset of the Tsat relation, while the slower, G∗-E∗ deactivation governs the

slope of the relation. To examine the consequences Ribociclib cell line of shorter and longer R∗ effective lifetimes for the SPR, we recorded the responses of Grk1+/− and Grk1S561L rods to very dim flashes and found little change in the amplitude of the SPR ( Figure 1C; Table 1). Grk1+/− rods with ∼2-fold longer effective R∗ lifetime (τReff = 76 ms versus 40 ms for WT rods) had only a modest, 23% increase in SPR amplitude. Rods expressing transgenic Grk1S561L, with a more than 2-fold shorter effective R∗ lifetime (τReff = 15 ms), had only a 24% decrease in SPR amplitude. Overall, while the effective R∗ lifetimes of the three genetic lines span a 5-fold range with ratios of about 1:2.7:5, the normalized average SPR amplitudes span a much smaller selleck inhibitor whatever range, with ratios of 1:1.3:1.6. These results establish that SPR amplitude does not vary in proportion to R∗ lifetime. In principle, R∗ molecules with longer lifetimes should activate more PDE molecules on the disc membrane and result in larger decreases in cGMP, locally closing a greater fraction of CNG channels. Because the density of PDE is only about 150 holoenzymes per disc face (1:300 ratio to rhodopsin, Pentia et al., 2006), it is conceivable that the rate of G∗-E∗ production may decrease as available PDE

molecules are depleted by longer-lived R∗ molecules. Thus, we calculated the average number of G∗-E∗ molecules active during the SPR and compared this to the total number of PDE molecules on the disc (see Experimental Procedures). Assuming a maximal rate of 300 s−1 for R∗ activation of the G protein (Leskov et al., 2000; Heck and Hofmann, 2001) and our measured R∗ and G∗-E∗ lifetimes, only ∼7 G∗-E∗ complexes are predicted at the peak of the SPR in normal rods (τReff = 40 ms). For Grk1+/− rods (τReff = 76 ms), the maximum number of G∗-E∗ units active during the SPR is only ∼10 (7% of the total number; Figure 2A, dashed line plotted against righthand ordinate). Thus, even if the maximal rate of G protein activation is 2-fold higher than current estimates, PDE depletion makes negligible contribution to the SPR amplitude stability over the range of R∗ lifetimes extending well beyond 76 ms.