Zebrafish Recoverin Isoforms Display Differences in Calcium Switch Mechanisms

Abstract

Primary steps in vertebrate vision occur in rod and cone cells of the retina and require precise molecular switches in excitation, recovery, and adaptation. In particular, recovery of the photoresponse and light adaptation processes are under control of neuronal Ca2+ sensor (NCS) proteins. Among them, the Ca2+ sensor recoverin undergoes a pronounced Ca2+-dependent conformational change, a prototypical so-called Ca2+-myristoyl switch, which allows selective targeting of G protein-coupled receptor kinase. Zebrafish (Danio rerio) has gained attention as a model organism in vision research. It expresses four different recoverin isoforms (zRec1a, zRec1b, zRec2a, and zRec2b) that are orthologs to the one known mammalian variant. The expression pattern of the four isoforms cover both rod and cone cells, but the differential distribution in cones points to versatile functions of recoverin in these cell types. Initial functional studies on zebrafish larvae indicate different Ca2+-sensitive working modes for zebrafish recoverins, but experimental evidence is lacking so far. The aims of the present study are (1) to measure specific Ca2+-sensing properties of the different recoverin isoforms, (2) to ask whether switch mechanisms triggered by Ca2+ resemble that one observed with mammalian recoverin, and (3) to investigate a possible impact of an attached myristoyl moiety. For addressing these questions, we employ fluorescence spectroscopy, surface plasmon resonance (SPR), dynamic light scattering, and equilibrium centrifugation. Exposure of hydrophobic amino acids, due to the myristoyl switch, differed among isoforms and depended also on the myristoylation state of the particular recoverin. Ca2+-induced rearrangement of the protein-water shell was for all variants less pronounced than for the bovine ortholog indicating either a modified Ca2+-myristoyl switch or no switch. Our results have implications for a step-by-step response of recoverin isoforms to changing intracellular Ca2+ during illumination.

Calcium affects CHP1 and CHP2 conformation and their interaction with sodium/proton exchanger 1

“…37 8-Anilinonaphthalene-1-sulfonic acid (ANS) and its derivatives are widely used to detect Ca 2+induced conformational changes of EFCaBPs. 38 Yet, ANS binds to proteins not only by hydrophobic but also by electrostatic interactions. 39,40 Therefore, we used SYPRO® Orange to detect Ca 2+ -induced hydrophobicity changes.…”

“…The fluorescence of this dye is quenched in hydrophilic and enhanced in hydrophobic environment . 8‐Anilinonaphthalene‐1‐sulfonic acid (ANS) and its derivatives are widely used to detect Ca 2+ ‐induced conformational changes of EFCaBPs . Yet, ANS binds to proteins not only by hydrophobic but also by electrostatic interactions .…”

Biochemistry and physiology of zebrafish photoreceptors

“…This implies different Ca 2+ sensitivities for these RCVs, since intracellular Ca 2+ concentration correlates with light levels [ 158 ]. Indeed, a recent biochemical work demonstrated distinct Ca 2+ affinities, Ca 2+ -dependent membrane binding, and Ca 2+ -induced conformational changes among zebrafish isoforms [ 45 ]. Furthermore, salamander cone photoresponse, but not rod response, is also dominated by a Ca 2+ -sensitive mechanism [ 121 , 216 ].…”

The Binding Properties and Physiological Functions of Recoverin

“…ERG response recovery accelerates in Rcv(s) knockdown animals and some Rcvs operate at varying light intensities, suggesting different Ca 2+ sensitivity and (or) GRK affinity among Rcvs. Indeed, a recent biochemical work shows different Ca 2+ affinity and Ca 2+ -induced conformational changes among zebrafish Rcv isoforms and bovine Rcv (Elbers et al, 2018). Interestingly, the amino acids at the critical positions of Ca 2+ binding sites are completely conserved across species with the exception of one zebrafish Rcv, where only three out of four positions are conserved in EF2 (Lamb and Hunt, 2018).…”