Justin Berry

Effect of Rhodopsin Phosphorylation on Dark Adaptation in Mouse Rods

Rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that is activated when its 11-cis-retinal moiety is photoisomerized to all-trans retinal. This step initiates a cascade of reactions by which rods signal changes in light intensity. Like other GPCRs, rhodopsin is deactivated through receptor phosphorylation and arrestin binding. Full recovery of receptor sensitivity is then achieved when rhodopsin is regenerated through a series of steps that return the receptor to its ground state. Here, we show that dephosphorylation of the opsin moiety of rhodopsin is an extremely slow but requisite step in the restoration of the visual pigment to its ground state. We make use of a novel observation: isolated mouse retinae kept in standard media for routine physiologic recordings display blunted dephosphorylation of rhodopsin. Isoelectric focusing followed by Western blot analysis of bleached isolated retinae showed little dephosphorylation of rhodopsin for up to 4 h in darkness, even under conditions when rhodopsin was completely regenerated. Microspectrophotometeric determinations of rhodopsin spectra show that regenerated phospho-rhodopsin has the same molecular photosensitivity as unphosphorylated rhodopsin and that flash responses measured by trans-retinal electroretinogram or single-cell suction electrode recording displayed dark-adapted kinetics. Single quantal responses displayed normal dark-adapted kinetics, but rods were only half as sensitive as those containing exclusively unphosphorylated rhodopsin. We propose a model in which light-exposed retinae contain a mixed population of phosphorylated and unphosphorylated rhodopsin. Moreover, complete dark adaptation can only occur when all rhodopsin has been dephosphorylated, a process that requires >3 h in complete darkness. SIGNIFICANCE STATEMENT G-protein-coupled receptors (GPCRs) constitute the largest superfamily of proteins that compose ∼4% of the mammalian genome whose members share a common membrane topology. Signaling by GPCRs regulate a wide variety of physiological processes, including taste, smell, hearing, vision, and cardiovascular, endocrine, and reproductive homeostasis. An important feature of GPCR signaling is its timely termination. This normally occurs when, after their activation, GPCRs are rapidly phosphorylated by specific receptor kinases and subsequently bound by cognate arrestins. Recovery of receptor sensitivity to the ground state then requires dephosphorylation of the receptor and unbinding of arrestin, processes that are poorly understood. Here we investigate in mouse rod photoreceptors the relationship between rhodopsin dephosphorylation and recovery of visual sensitivity.

Rhodopsin kinase and arrestin binding control the decay of photoactivated rhodopsin and dark adaptation of mouse rods

G-protein receptor kinase and arrestin 1 are required for inactivation of photoactivated vertebrate rhodopsin. Frederiksen et al. show that they additionally regulate the subsequent decay of inactive rhodopsin into opsin and all-trans retinal and therefore dark adaptation.

First Observation of Dynamics in Lipid Multilayers using X-ray Photon Correlation Spectroscopy (XPCS)

Collective modes of layer undulations in lipid multilayers are of considerable interest because they can be used to measure the elastic moduli and viscosity of the lipid bilayers as a function of interlamellar spacing, properties of the interlayer aqueous channels, and temperature. These are fundamental quantities required to calculate the configurations and fluctuations of lipid membranes, relevant in modeling many biomembrane functionalities (e.g., intermembrane interactions and polyvalent ligand recognition) that depend on elasticity and dynamics of membrane phases. However, only relatively few investigations have been made of lipid systems, with e.g. dynamical light scattering, neutron spin echo and inelastic neutron scattering. There remains a gap in time scales and length scales which the technique of x-ray photon correlation spectroscopy (XPCS) can fill.We present here the first XPCS measurements of the dynamics of 1,2-dioleoyl-sn-glycero-3-phtosphocholine (DOPC) multilayers at relative humidity of 99% and temperature of 28°C. The measurements were done at and near the 1st Bragg peak of the multilayer, in which range the intensity-intensity autocorrelation function includes heterodyne oscillations [1] due to a large static component as well as homodyne oscillations predominated at q-values off the Bragg peak. According to de Jeus theory [2], there are two different modes of relaxation time in the system: a slow and a fast one. Our experiment reveals the existence of the slow mode, which exhibits a plateau in relaxation time over a range of q (10e-5 A-1∼10e-4 A-1). The results of the analysis of these correlation functions according to the model of de Jeu et al. will be presented.1. C. Gutt et al, Phys. Rev. Lett. 91, 076104 (2003)2. W. H. de Jue et al, Rev. Mod. Phys., 75, 181-235 (2003)