Abstract
Purpose:
Deactivation of rhodopsin (R*) requires multiple phosphorylation of its COOH-terminal serine and threonine residues by Grk1, followed by the binding of arrestin-1 (Arr1). Single cell recordings from rods that under-express Arr1 have suggested that R* lifetime is normally brief and dictated largely by phosphorylation (Gross and Burns, 2010). Here, we apply a recently developed model of phototransduction to single photon responses (SPRs) of rods lacking either phosphorylation or Arr1 binding, providing new quantitative estimates of the in situ kinetics of R* deactivation.
Methods:
Suction electrode recordings from intact rod outer segments were performed as previously described. Measured SPRs were compared to simulations produced by a spatiotemporal phototransduction model (Gross et al 2012a). The model input was a time series representing R* activity, generated assuming a Markov deactivation process in which Arr1 binds with high affinity after three phosphorylations.
Results:
The Grk1-/- SPR is approximately a step function (Chen et al., 1999), whose ~2-fold larger amplitude constrains the maximal R* activity in the context of the downstream model, and whose 3-4 s mean duration sets the upper limit for the rate of Arr1 binding to unphosphorylated R*. In contrast, Arr1-/- SPRs exhibit peak amplitudes similar to wild-type SPRs and have long-lasting tail currents with amplitudes ~50% of the peak (e.g. Xu et al., 1997). Simulated SPRs revealed that this tail current can be explained by a relatively small fraction (~5%) of maximal R* activity that persists after extensive phosphorylation of R*. In order to achieve this low level of R* activity on the time scale of the SPR, phosphorylation of R* must proceed at a maximal, initial rate of 100/s.
Conclusions:
The deduced R* deactivation kinetics predict that 3-4 G protein/PDE (G*-E*) complexes are active during the plateau tail phase of the Arr1-/- SPR, a number that corresponds to ~50% of the G*-E* level at the peak of the wild-type SPR. In contrast, ~ 60 G*-E* complexes are predicted for the Grk1-/- SPR. The gross disproportionality between the number of G*-E* complexes and the response plateau amplitudes reveals the consequences of cascade saturation and the disproportionate role of calcium feedback to cGMP synthesis on larger responses (Gross et al., 2012b).
Keywords: 648 photoreceptors