58 research outputs found
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Membrane guanylyl cyclase complexes shape the photoresponses of retinal rods and cones
In vertebrate rods and cones, photon capture by rhodopsin leads to the destruction of cyclic GMP (cGMP) and the subsequent closure of cyclic nucleotide gated ion channels in the outer segment plasma membrane. Replenishment of cGMP and reopening of the channels limit the growth of the photon response and are requisite for its recovery. In different vertebrate retinas, there may be as many as four types of membrane guanylyl cyclases (GCs) for cGMP synthesis. Ten neuronal Ca2+ sensor proteins could potentially modulate their activities. The mouse is proving to be an effective model for characterizing the roles of individual components because its relative simplicity can be reduced further by genetic engineering. There are two types of GC activating proteins (GCAPs) and two types of GCs in mouse rods, whereas cones express one type of GCAP and one type of GC. Mutant mouse rods and cones bereft of both GCAPs have large, long lasting photon responses. Thus, GCAPs normally mediate negative feedback tied to the light-induced decline in intracellular Ca2+ that accelerates GC activity to curtail the growth and duration of the photon response. Rods from other mutant mice that express a single GCAP type reveal how the two GCAPs normally work together as a team. Because of its lower Ca2+ affinity, GCAP1 is the first responder that senses the initial decrease in Ca2+ following photon absorption and acts to limit response amplitude. GCAP2, with a higher Ca2+ affinity, is recruited later during the course of the photon response as Ca2+ levels continue to decline further. The main role of GCAP2 is to provide for a timely response recovery and it is particularly important after exposure to very bright light. The multiplicity of GC isozymes and GCAP homologs in the retinas of other vertebrates confers greater flexibility in shaping the photon responses in order to tune visual sensitivity, dynamic range and frequency response
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ROS-GC interlocked Ca2+-sensor S100B protein signaling in cone photoreceptors: review
Photoreceptor rod outer segment membrane guanylate cyclase (ROS-GC) is central to visual transduction; it generates cyclic GMP, the second messenger of the photon signal. Photoexcited rhodopsin initiates a biochemical cascade that leads to a drop in the intracellular level of cyclic GMP and closure of cyclic nucleotide gated ion channels. Recovery of the photoresponse requires resynthesis of cyclic GMP, typically by a pair of ROS-GCs, 1 and 2. In rods, ROS-GCs exist as complexes with guanylate cyclase activating proteins (GCAPs), which are Ca2+-sensing elements. There is a light-induced fall in intracellular Ca2+. As Ca2+ dissociates from GCAPs in the 20β200 nM range, ROS-GC activity rises to quicken the photoresponse recovery. GCAPs then progressively turn down ROS-GC activity as Ca2+ and cyclic GMP levels return to baseline. To date, GCAPs mediate the only known mechanism of ROS-GC regulation in the photoreceptors. However, in mammalian cone outer segments, cone synapses and ON bipolar cells, another Ca2+ sensor protein, S100B, complexes with ROS-GC1 and senses the Ca2+ signal with a K1/2 of 400 nM. Unlike GCAPs, S100B stimulates ROS-GC activity when Ca2+ is bound. Thus, the ROS-GC system in cones functions as a Ca2+ bimodal switch; with rising intracellular Ca2+, its activity is first turned down by GCAPs and then turned up by S100B. This presentation provides a historical perspective on the role of S100B in the photoreceptors, offers a pictorial model for the βbimodalβ operation of the ROS-GC switch and projects future tasks that are needed to understand its operation. Some accounts of this review have been adopted from the original publications of these authors
Recoverin Regulates Light-dependent Phosphodiesterase Activity in Retinal Rods
The Ca2+-binding protein recoverin may regulate visual transduction in retinal rods and cones, but its functional role and mechanism of action remain controversial. We compared the photoresponses of rods from control mice and from mice in which the recoverin gene was knocked out. Our analysis indicates that Ca2+-recoverin prolongs the dark-adapted flash response and increases the rod's sensitivity to dim steady light. Knockout rods had faster Ca2+ dynamics, indicating that recoverin is a significant Ca2+ buffer in the outer segment, but incorporation of exogenous buffer did not restore wild-type behavior. We infer that Ca2+-recoverin potentiates light-triggered phosphodiesterase activity, probably by effectively prolonging the catalytic activity of photoexcited rhodopsin
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Enzymatic Relay Mechanism Stimulates Cyclic GMP Synthesis in Rod Photoresponse: Biochemical and Physiological Study in Guanylyl Cyclase Activating Protein 1 Knockout Mice
Regulation of cGMP synthesis by retinal membrane guanylyl cyclase isozymes (RetGC1 and RetGC2) in rod and cone photoreceptors by calcium-sensitive guanylyl cyclase activating proteins (GCAP1 and GCAP2) is one of the key molecular mechanisms affecting the response to light and is involved in congenital retinal diseases. The objective of this study was to identify the physiological sequence of events underlying RetGC activation in vivo, by studying the electrophysiological and biochemical properties of mouse rods in a new genetic model lacking GCAP1. The GCAP1β/β retinas expressed normal levels of RetGC isozymes and other phototransduction proteins, with the exception of GCAP2, whose expression was elevated in a compensatory fashion. RetGC activity in GCAP1β/β retinas became more sensitive to Ca2+ and slightly increased. The bright flash response in electroretinogram (ERG) recordings recovered quickly in GCAP1β/β, as well as in RetGC1β/βGCAP1β/β, and RetGC2β/βGCAP1β/β hybrid rods, indicating that GCAP2 activates both RetGC isozymes in vivo. Individual GCAP1β/β rod responses varied in size and shape, likely reflecting variable endogenous GCAP2 levels between different cells, but single-photon response (SPR) amplitude and time-to-peak were typically increased, while recovery kinetics remained faster than in wild type. Recovery from bright flashes in GCAP1β/β was prominently biphasic, because rare, aberrant SPRs producing the slower tail component were magnified. These data provide strong physiological evidence that rod photoresponse recovery is shaped by the sequential recruitment of RetGC isozyme activation by GCAPs according to the different GCAP sensitivities for Ca2+ and specificities toward RetGC isozymes. GCAP1 is the βfirst-responseβ sensor protein that stimulates RetGC1 early in the response and thus limits the SPR amplitude, followed by activation of GCAP2 that adds stimulation of both RetGC1 and RetGC2 to speed-up photoreceptor recovery
Increased Light Exposure Alleviates One Form of Photoreceptor Degeneration Marked by Elevated Calcium in the Dark
Background: In one group of gene mutations that cause photoreceptor degeneration in human patients, guanylyl cyclase is overactive in the dark. The ensuing excess opening of cGMP-gated cation channels causes intracellular calcium to rise to toxic levels. The Y99C mutation in guanylate cyclase-activating protein 1 (GCAP1) has been shown to act this way. We determined whether prolonged light exposure, which lowers cGMP levels through activation of phototransduction, might protect photoreceptors in a line of transgenic mice carrying the GCAP1-Y99C. Methodology/Principal Findings: We reared cohorts of GCAP1-Y99C transgenic mice under standard cyclic, constant dark and constant light conditions. Mouse eyes were analyzed by histology and by immunofluorescence for GFAP upregulation, a non-specific marker for photoreceptor degeneration. Full-field electroretinograms (ERGs) were recorded to assess retinal function. Consistent with our hypothesis, constant darkness accelerated disease, while continuous lighting arrested photoreceptor degeneration. Conclusions/Significance: In contrast to most forms of retinal degeneration, which are exacerbated by increased exposure to ambient light, a subset with mutations that cause overly active guanylyl cyclase and high intracellular calcium benefitted from prolonged light exposure. These findings may have therapeutic implications for patients with these types of genetic defects
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Rhodopsin Expression Level Affects Rod Outer Segment Morphology and Photoresponse Kinetics
Background: The retinal rod outer segment is a sensory cilium that is specialized for the conversion of light into an electrical signal. Within the cilium, up to several thousand membranous disks contain as many as a billion copies of rhodopsin for efficient photon capture. Disks are continually turned over, requiring the daily synthesis of a prodigious amount of rhodopsin. To promote axial diffusion in the aqueous cytoplasm, the disks have one or more incisures. Across vertebrates, the range of disk diameters spans an order of magnitude, and the number and length of the incisures vary considerably, but the mechanisms controlling disk architecture are not well understood. The finding that transgenic mice overexpressing rhodopsin have enlarged disks lacking an incisure prompted us to test whether lowered rhodopsin levels constrain disk assembly. Methodology/Principal Findings: The structure and function of rods from hemizygous rhodopsin knockout (R+/β) mice with decreased rhodopsin expression were analyzed by transmission electron microscopy and single cell recording. R+/β rods were structurally altered in three ways: disk shape changed from circular to elliptical, disk surface area decreased, and the single incisure lengthened to divide the disk into two sections. Photocurrent responses to flashes recovered more rapidly than normal. A spatially resolved model of phototransduction indicated that changes in the packing densities of rhodopsin and other transduction proteins were responsible. The decrease in aqueous outer segment volume and the lengthened incisure had only minor effects on photon response amplitude and kinetics. Conclusions/Significance: Rhodopsin availability limits disk assembly and outer segment girth in normal rods. The incisure may buffer the supply of structural proteins needed to form larger disks. Decreased rhodopsin level accelerated photoresponse kinetics by increasing the rates of molecular collisions on the membrane. Faster responses, together with fewer rhodopsins, combine to lower overall sensitivity of R+/β rods to light
Cyclic Nucleotide-gated Ion Channels in Rod Photoreceptors Are Protected from Retinoid Inhibition
In vertebrate rods, photoisomerization of the 11-cis retinal chromophore of rhodopsin to the all-trans conformation initiates a biochemical cascade that closes cGMP-gated channels and hyperpolarizes the cell. All-trans retinal is reduced to retinol and then removed to the pigment epithelium. The pigment epithelium supplies fresh 11-cis retinal to regenerate rhodopsin. The recent discovery that tens of nanomolar retinal inhibits cloned cGMP-gated channels at low [cGMP] raised the question of whether retinoid traffic across the plasma membrane of the rod might participate in the signaling of light. Native channels in excised patches from rods were very sensitive to retinoid inhibition. Perfusion of intact rods with exogenous 9- or 11-cis retinal closed cGMP-gated channels but required higher than expected concentrations. Channels reopened after perfusing the rod with cellular retinoid binding protein II. PDE activity, flash response kinetics, and relative sensitivity were unchanged, ruling out pharmacological activation of the phototransduction cascade. Bleaching of rhodopsin to create all-trans retinal and retinol inside the rod did not produce any measurable channel inhibition. Exposure of a bleached rod to 9- or 11-cis retinal did not elicit channel inhibition during the period of rhodopsin regeneration. Microspectrophotometric measurements showed that exogenous 9- or 11-cis retinal rapidly cross the plasma membrane of bleached rods and regenerate their rhodopsin. Although dark-adapted rods could also take up large quantities of 9-cis retinal, which they converted to retinol, the time course was slow. Apparently cGMP-gated channels in intact rods are protected from the inhibitory effects of retinoids that cross the plasma membrane by a large-capacity buffer. Opsin, with its chromophore binding pocket occupied (rhodopsin) or vacant, may be an important component. Exceptionally high retinoid levels, e.g., associated with some retinal degenerations, could overcome the buffer, however, and impair sensitivity or delay the recovery after exposure to bright light
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