Having established how GCAPs-mediated feedback stabilizes the amplitude of the average

SPR across genotypes with differing selleck chemical average R∗ lifetimes, we now consider whether it also contributes to reduction of the trial-to-trial variability of SPR amplitudes in an individual rod, i.e., contributes to SPR reproducibility. SPR reproducibility has long been deemed something of a biophysical mystery: despite being driven by individual stochastically deactivating R∗ molecules, SPRs have highly invariant amplitudes, with coefficient of variation (c.v.; standard deviation divided by the mean) of ∼0.2 in amphibian rods (Baylor et al., 1979; Rieke and Baylor, 1998) and ∼0.3 in mammalian rods (Baylor et al., 1984; Figure 6F). In recent years, empirical and theoretical studies have led to general agreement that multiple phosphorylations of R∗ smooth its stochastic deactivation (Rieke and Baylor, 1998; Mendez et al., 2000; Field and Rieke, 2002; Hamer et al., 2003; Doan et al., 2006). Theoretical simulations

have suggested that stochastic R∗ shutoff is nonetheless the primary source of SPR variability and also that the limited diffusion of cGMP acts to suppress the variability associated with R∗ deactivation (Bisegna et al., 2008; Caruso et al., 2010, 2011). These same theoretical studies have also concluded that calcium-mediated feedback plays little role in the reproducibility of the SPR (Caruso et al., 2011), a conclusion at odds with what might now be expected, given our current VX-809 solubility dmso results with GCAPs-mediated feedback and SPR amplitude stability. To directly assess whether calcium feedback to cGMP synthesis contributes to SPR reproducibility, we recorded hundreds of dim flash responses from wild-type (Figure 6A)

and GCAPs−/− (Figure 6B) rods and calculated the mean and time-dependent Bay 11-7085 standard deviation of the ensembles of isolated SPRs (“singletons”; gray and pink traces, Figures 6C–6D; Experimental Procedures). In addition to being larger, the response peaks of isolated GCAPs−/− singletons were more variable in amplitude and were more broadly distributed in time. As a result, the time-dependent standard deviation of GCAPs−/− singletons had a larger, broader peak than that of WT singletons (note difference in both x- and y-scaling, Figures 6C–6D). The increase in the GCAPs−/− singleton standard deviation relative to that of WT was larger than the relative increase in singleton mean amplitude, resulting in a larger c.v. of the response amplitude (c.v. = 0.34 ± 0.01, n = 5 for WT and 0.42 ± 0.02, n = 4 for GCAPs−/− rods; p = 0.02; Figure 6F, solid green and blue bars). Thus, although R∗ and G∗-E∗ deactivation are the same for WT and GCAPs−/− rods, reproducibility is impaired in the absence of GCAPs-mediated feedback.