Abstract
Abstract: :
Purpose: To identify the separate functional roles and biophysical mechanisms of rhodopsin (R) phosphorylation and arrestin binding contributing to uniformity of rod single photon responses (USPR). Methods: Rod disk membranes were phosphorylated to various P/R ratios, regenerated with 11-cis retinal, stripped of peripheral proteins and recombined with G protein (G) or arrestin (Arr). Metarhodopsin II (MII) and extra MII due to G or Arr binding were measured by dual wavelength weak, multi flash bleach titration spectroscopy. Reactions were deconvolved and binding constants determined using a Simplex reaction model-fitting algorithm. The same data provided kinetic constants. Results: Linear increase in R phosphorylation causes exponential DECREASE in G affinity and linear INCREASE in Arr affinity. These effects on G and Arr activity are NOT kinetically simultaneous. Rather, rate constants for all decreased G affinity states are ca. 100-fold faster than those for arrestin binding. Moreover, arrestin binding, though strong, is quite slow at P/R=1. Its speed increases linearly with P/R. G and Arr binding are powerfully and oppositely affected by the electrostatics of phosphorylation. Conclusions: Graded loss of R* G interaction during progress of R* multi phosphorylation serves to initiate replacement of undesirable single step exponential variability in R* lifetime with the multi step Gaussian statistical averaging needed for reliable single photon detection (USPR). Exponential variability is the inevitable undesirable consequence of terminating receptor molecule activity through any single step reaction such as attachment of a single phosphate, blocking by a single arrestin molecule or spontaneous R* decay. Breaking R* activity decay into multiple steps obviates the futility of the frequent rapid termination events that characterize exponential lifetime distributions (decay statistics). Such brief events are inconsistent with the known USPR of rods (Baylor et.al., 1979). They would make reliable single photon detection impossible. Exponential distributions also have a relatively high frequency of long-lived R* single photon events that would be confused with effects of multiple photons. Supported by EY00012, EY01583 and personal funding.
Keywords: 517 photoreceptors • 515 phosphorylation • 580 signal transduction