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Reproducibility of the Single-Photon Response (SPR) of Retinal Rods: Rigorous Tests and Predictions of a Multiple Phosphorylation (MPn) Model

¹ Retinal Computational Modeling, Smith-Kettlewell Eye Res Inst, SF, CA, United States
² Dept. Biol. & Courant Inst. Mathematical Sciences, NYU, NY, NY, United States

Commercial Relationships: R.D. Hamer, None; S.C. Nicholas, None; D. Tranchina, None.

Grant Identification: NIH Grant EY115-13; SK Fund 2109-0200

Abstract

Purpose: An enduring problem in rod physiology has been to explain SPR reproducibility in terms of inherently variable underlying biochemistry. We developed a biochemical model with stochastic "front-end" reactions (through PDE^* inactivation). SPR reproducibility is achieved via graded inactivation of R^* by multiple steps of phosphorylation (Pn) with final quench by arrestin (Arr). We test the model against a broad array of data, including genetic knockout (KO) data. We present a new theoretical framework for evaluating SPR variability, and use simulations and existing data to rule out entire classes of models.
Methods: Monte-Carlo simulations of dim-flash responses, assuming: Poisson arrival of photons; G protein (G), R^* kinase (RK) & Arr compete to bind with R^*, with Pn-dependent binding affinities; R^* activity is quenched upon Arr-binding; Ca⁺⁺-modulation of guanylate cyclase is the only feedback.
Results: (1) The MPn model accounts for 4 measures of reproducibility: low coefficients of variation of SPR amplitude (CV_ampl=0.2-0.25) and SPR area (CV_area=0.3-0.4); ~proportionality between mean² and variance of dim-flash responses; low SPR variance that peaks much later, and is broader than the mean² SPR. (2) The MPn model reproduces transduction gain experiments of Rieke & Baylor (1998). When [GTP] is lowered, response amplitude decreases with almost no change in kinetics unless [ATP] is also lowered (to slow Pn). (3) The MPn model reproduces KO data from Arr-/-, RK-/-, GCAPs-/- and RGS9-/- rods. (4) Theory shows that CV_area is a preferred gauge of SPR variability. Classical measures (CV_ampl or CV_duration) overestimate the number of underlying R^* inactivation steps. There is a tradeoff between CV_ampl and CV_dur that depends on the relative inactivation kinetics of R^* vs downstream reactions. (5) Simulations and KO data rule out local depletion of G or PDE as the main mechanism for SPR reproducibility, and any model in which R^* activity is fixed until quenching.
Conclusions: (I) Result (2) argues against Rieke & Baylor’s idea that Pn can only account for a small fraction of R^* inactivation. (II) A 1-step R^* shutoff with Ca⁺⁺-feedback onto R^* lifetime (Whitlock & Lamb, 1999; Pugh & Lamb, 2001) cannot account for SPR behavior (Result 5). (III) Experimental manipulations that selectively slow downstream reactions will increase CV_ampl, decrease CV_dur, with no effect on CV_area. Slowing R^* inactivation will shift the balance towards duration variability, but may increase CV_area (Result 4).

Keywords: retina: distal(photoreceptors, horizontal cell • phosphorylation • computational modeling

© 2003, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any part of this abstract, contact the ARVO Office at arvo{at}arvo.org.