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Calcium feedback to cGMP synthesis strongly attenuates single-photon responses driven by long rhodopsin lifetimes.

ABSTRACT: Rod photoreceptors generate amplified, reproducible responses to single photons via a G protein signaling cascade. Surprisingly, genetic perturbations that dramatically alter the deactivation of the principal signal amplifier, the GPCR rhodopsin (R?), do not much alter the amplitude of single-photon responses (SPRs). These same perturbations, when crossed into a line lacking calcium feedback regulation of cGMP synthesis, produced much larger alterations in SPR amplitudes. Analysis of SPRs from rods with and without feedback reveal that the consequences of trial-to-trial fluctuations in R? lifetime in normal rods are also dampened by feedback regulation of cGMP synthesis. Thus, calcium feedback trumps the mechanisms of R? deactivation in determining the SPR amplitude, attenuating responses arising from longer R? lifetimes to a greater extent than those arising from shorter ones. As a result, rod SPRs achieve a more stereotyped amplitude, a characteristic considered important for reliable transmission through the visual system.

SUBMITTER: Gross OP

PROVIDER: S-EPMC3594095 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Calcium feedback to cGMP synthesis strongly attenuates single-photon responses driven by long rhodopsin lifetimes.

Gross Owen P OP Pugh Edward N EN Burns Marie E ME

Neuron 20121017 2

Rod photoreceptors generate amplified, reproducible responses to single photons via a G protein signaling cascade. Surprisingly, genetic perturbations that dramatically alter the deactivation of the principal signal amplifier, the GPCR rhodopsin (R∗), do not much alter the amplitude of single-photon responses (SPRs). These same perturbations, when crossed into a line lacking calcium feedback regulation of cGMP synthesis, produced much larger alterations in SPR amplitudes. Analysis of SPRs from r ...[more]

PMID: 23083739

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Project description:Bistable opsins are photopigments expressed in both invertebrates and vertebrates. These light-sensitive G-protein-coupled receptors undergo a reversible reaction upon illumination. A first photon initiates the cis to trans isomerization of the retinal chromophore-attached to the protein through a protonated Schiff base-and a series of transition states that eventually results in the formation of the thermally stable and active Meta state. Excitation by a second photon reverts this process to recover the original ground state. On the other hand, monostable opsins (e.g., bovine rhodopsin) lose their chromophore during the decay of the Meta II state (i.e., they bleach). Spectroscopic studies on the molecular details of the two-photon cycle in bistable opsins are limited. Here, we describe the successful expression and purification of recombinant rhodopsin-1 from the jumping spider Hasarius adansoni (JSR1). In its natural configuration, spectroscopic characterization of JSR1 is hampered by the similar absorption spectra in the visible spectrum of the inactive and active states. We solved this issue by separating their absorption spectra by replacing the endogenous 11-cis retinal chromophore with the blue-shifted 9-cis JSiR1. With this system, we used time-resolved ultraviolet-visible spectroscopy after pulsed laser excitation to obtain kinetic details of the rise and decay of the photocycle intermediates. We also used resonance Raman spectroscopy to elucidate structural changes of the retinal chromophore upon illumination. Our data clearly indicate that the protonated Schiff base is stable throughout the entire photoreaction. We additionally show that the accompanying conformational changes in the protein are different from those of monostable rhodopsin, as recorded by light-induced FTIR difference spectroscopy. Thus, we envisage JSR1 as becoming a model system for future studies on the reaction mechanisms of bistable opsins, e.g., by time-resolved x-ray crystallography.

Dataset Information

Calcium feedback to cGMP synthesis strongly attenuates single-photon responses driven by long rhodopsin lifetimes.

Publications

Calcium feedback to cGMP synthesis strongly attenuates single-photon responses driven by long rhodopsin lifetimes.

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