Bringing the Ca2+ sensitivity of myristoylated recoverin into the physiological range.

Valerio Marino,Davide Zamboni,Daniele Dell'Orco,Karl-Wilhelm Koch,Matteo Riva

doi:10.1098/rsob.200346

Valerio Marino, Davide Zamboni + Show 3 more

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https://doi.org/10.1098/rsob.200346

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Abstract

The prototypical Ca2+-sensor protein recoverin (Rec) is thought to regulate the activity of rhodopsin kinase (GRK1) in photoreceptors by switching from a relaxed (R) disc membrane-bound conformation in the dark to a more compact, cytosol-diffusing tense (T) conformation upon cell illumination. However, the apparent affinity for Ca2+ of its physiologically relevant form (myristoylated recoverin) is almost two orders of magnitude too low to support this mechanism in vivo. In this work, we compared the individual and synergistic roles of the myristic moiety, the GRK1 target and the disc membrane in modulating the calcium sensitivity of Rec. We show that the sole presence of the target or the disc membrane alone are not sufficient to achieve a physiological response to changes in intracellular [Ca2+]. Instead, the simultaneous presence of GRK1 and membrane allows the T to R transition to occur in a physiological range of [Ca2+] with high cooperativity via a conformational selection mechanism that drives the structural transitions of Rec in the presence of multiple ligands. Our conclusions may apply to other sensory transduction systems involving protein complexes and biological membranes.

Highlights

Recoverin (Rec) is a 23 kDa neuronal calcium sensor (NCS) belonging to the EF-hand superfamily of Ca2+-binding proteins [1]. It is mostly expressed in vertebrate photoreceptors, where it contributes to the regulation of the phototransduction cascade by responding to the transient drop in the concentration of intracellular Ca2+ that follows the light-activation of rhodopsin [2,3]
Several in vitro studies reported that the half-maximal inhibition of GRK1 by Rec occurs between 1.5 and 6 μM free Ca2+ [6,12,35], which is an order of magnitude higher than the physiological range of cytoplasmic Ca2+
No experimental tests of these hypotheses to reconcile experimental data with the physiological framework existed before our study, except one approach that showed Rec and calmodulin acting synergistically on GRK1, which can affect the Ca2+ sensitivity [35]

Summary

Introduction

Recoverin (Rec) is a 23 kDa neuronal calcium sensor (NCS) belonging to the EF-hand superfamily of Ca2+-binding proteins [1]. It is mostly expressed in vertebrate photoreceptors, where it contributes to the regulation of the phototransduction cascade by responding to the transient drop in the concentration of intracellular Ca2+ that follows the light-activation of rhodopsin [2,3]. Several lines of evidence suggest that Rec and its orthologues inhibit rhodopsin kinase (GRK1) at high Ca2+, in the dark state of the rod photoreceptor [4,5,6,7]. The myristoyl moiety is sequestered into a hydrophobic groove in the T state (figure 1a), while it becomes solvent-exposed upon Ca2+ binding, when the royalsocietypublishing.org/journal/rsob Open Biol. 10: 200346

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