Effect of the ILE86TER mutation in the γ subunit of cGMP phosphodiesterase (PDE6) on rod photoreceptor signaling (original) (raw)
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Determination of basal phosphodiesterase activity in mouse rod photoreceptors with cGMP clamp
Scientific Reports
Light regulates cGMp concentration in the photoreceptor cytoplasm by activating phosphodiesterase (pDe) molecules through a G-protein signalling cascade. spontaneous pDe activity is present in rod outer segments even in darkness. this basal pDe activity (β dark) has not been determined in wild type mammalian photoreceptor cells although it plays a key role in setting the sensitivity and recovery kinetics of rod responses. We present a novel method for determination of β dark using local electroretinography (LeRG) from isolated mouse retinas. the method is based on the ability of pDe inhibitors to decrease β dark , which can be counterbalanced by increasing pDe activity with light. this procedure clamps cytoplasmic cGMp to its dark value. β dark can be calculated based on the amount of light needed for the "cGMp clamp" and information extracted from the registered rod photoresponses. Here we apply this method to determine β dark values for the first time in the mammalian rods and obtain the following estimates for different mouse models: 3.9 s −1 for wild type, 4.5 s −1 for guanylate cyclase activating proteins (GCAPs) knockout, and 4.4 s −1 for GCAps and recoverin double knockout mice. our results suggest that depletion of GCAPs or recoverin do not affect β dark. Photoreceptor cells convert light information to sensory signals in a process called phototransduction. When a photon is absorbed in a rhodopsin molecule in the rod outer segment disk membrane, the rhodopsin activates G-proteins, transducins, and the activated transducins bind to phosphodiesterase-6 molecules (PDE) forming enzyme complexes, which hydrolyse cyclic guanosine monophosphate (cGMP) at nearly a diffusion limited rate 1. A rapid drop in the cytoplasmic cGMP concentration leads to the closure of the cyclic nucleotide gated (CNG) channels in the outer segment plasma membrane, hyperpolarization of the cell membrane, change in the release rate of glutamate in the rod terminal and transmission of the light-generated signal to the inner retina (see e.g. 2,3). Thermal energy causes spontaneous activations of phototransduction molecules, which leads to fluctuations in the cytoplasmic level of cGMP. These fluctuations make up the main part the dark noise of photoreceptors 4. The dark noise consists mainly of three components: discrete spontaneous activations of rhodopsin, high frequency noise from fluctuations in the CNG channel conductance, and continuous noise from thermal activations of PDE 4. The amount of active PDE in darkness determines the rate constant for spontaneous cGMP hydrolysis, i.e. the basal PDE activity (β dark), which sets the steady state level and the turnover rate of cGMP. Hence, it is one of the main factors in setting the kinetics of photoresponse deactivation and spatial propagation of cGMP concentration drop during photoresponses 5. The basal PDE activity has been determined earlier for amphibian rod photoreceptors by abruptly blocking the activity of either PDE or guanylate cyclase 6-10. In the method, single photoreceptor outer segment is exposed to rapid solution changes while recording photoreceptor circulating dark current. However, this has turned out to be challenging with the fragile mammalian photoreceptors, and until now, no one has determined the β dark of wild type mammalian photoreceptors. Gross et al. (2012) demonstrated that when the calcium mediated feedback to guanylate cyclase is abolished by knocking out the guanylate cyclase activating proteins (GCAPs) and the lifetime of activated PDE is decreased by overexpressing RGS9, the basal PDE activity becomes the dominant factor determining the light response deactivation kinetics 5. In these circumstances, the late recovery of a single-photon response allows the determination of β dark. However, it is not known
Biochemical and Biophysical Research Communications, 2009
The γ subunit of rod-specific cGMP phosphodiesterase 6 (PDE6γ), an effector of the G-protein GNAT1, is a key regulator of phototransduction. The results of several in vitro biochemical reconstitution experiments conducted to examine the effects of phosphorylation of PDE6γ on its ability to regulate the PDE6 catalytic core have been inconsistent, showing that phosphorylation of PDE6γ may increase or decrease the ability of PDE6γ to deactivate phototransduction. To resolve role of phosphorylation of PDE6γ in living photoreceptors, we generated transgenic mice in which either one or both Threonine (T) sites in PDE6γ (T22 and T35), which are candidates for putative regulatory phosphorylation, were substituted with alanine (A). Phosphorylation of these sites was examined as a function of light exposure. We found that phosphorylation of T22 increases with light exposure in intact mouse rods while constitutive phosphorylation of T35 is unaffected by light in intact mouse rods and cones. Phosphorylation of the cone isoform of PDE6γ, PDE6H, is constitutively phosphorylated at the T20 residue. Light-induced T22 phosphorylation was lost in T35A transgenic rods, and T35 phosphorylation was extinguished in T22A transgenic rods. The interdependency of phosphorylation of T22 and T35 suggests that light-induced, post-translational modification of PDE6γ is essential for the regulation of G-protein signaling.
Removal of phosphorylation sites of subunit of phosphodiesterase 6 alters rod light response
The Journal of Physiology, 2006
The phosphodiesterase 6 γ (PDE6γ) inhibitory subunit of the rod PDE6 effector enzyme plays a central role in the turning on and off of the visual transduction cascade, since binding of PDE6γ to the transducin α subunit (Tα) initiates the hydrolysis of the second messenger cGMP, and PDE6γ in association with RGS9-1 and the other GAP complex proteins (Gβ5, R9AP) accelerates the conversion of TαGTP to TαGDP, the rate-limiting step in the decay of the rod light response. Several studies have shown that PDE6γ can be phosphorylated at two threonines, T22 and T35, and have proposed that phosphorylation plays some role in the physiology of the rod. We have examined this possibility by constructing mice in which T22 and/or T35 were replaced with alanines. Our results show that T35A rod responses rise and decay more slowly and are less sensitive to light than wild-type (WT). T22A responses show no significant difference in initial time course with WT but decay more rapidly, especially at dimmer intensities. When the T22A mutation is added to T35A, double mutant rods no longer showed the prolonged decay of T35A rods but remained slower than WT in initial time course. Our experiments suggest that the polycationic domain of PDE6γ containing these two phosphorylation sites can influence the rate of PDE6 activation and deactivation and raise the possibility that phosphorylation or dephosphorylation of PDE6γ could modify the time course of transduction, thereby influencing the wave form of the light response.
Cellular Signalling, 2011
The inhibitory subunit of rod cyclic guanosine monophosphate (cGMP) phosphodiesterase, PDE6γ, is a major component of rod transduction and is required to support photoreceptor integrity. The N74A allele of PDE6γ has previously been shown in experiments carried out in vitro to reduce the regulatory inhibition on the PDE6 catalytic core subunits, PDE6αβ. This should, in intact rods, lead to an increase in basal (dark) PDE6 activity producing a state equivalent to light adaptation in the rods and we have examined this possibility using ERG and suction-electrode measurements. The murine opsin promoter was used to drive the expression of a mutant N74A and a wild-type PDE6γ control transgene in the photoreceptors of +/Pde6g tm1 mice. This transgenic line was crossed with Pde6g tm1 /Pde6g tm1 mice to generate animals able to synthesize only the transgenic mutant PDE6γ. We find that the N74A mutation did not produce a significant decrease in circulating current, a decrease in sensitivity or affect the kinetics of the light response, all hallmarks of the light-adapted state. In an in vitro assay of the PDE purified from the N74A transgenic mice and control mice we could find no increase in basal activity of the mutant PDE6. Both the results from the physiology and the biochemistry experiments are consistent with the interpretation that the mutation causes a much milder phenotype in vivo than was predicted from observations made using a cell-free assay system. The in vivo regulation of PDE6γ on PDE6αβ may be more dynamic and context-dependent than was replicated in vitro.
Functional modulation of phosphodiesterase-6 by calcium in mouse rod photoreceptors
Scientific Reports, 2021
Phosphodiesterase-6 (PDE6) is a key protein in the G-protein cascade converting photon information to bioelectrical signals in vertebrate photoreceptor cells. Here, we demonstrate that PDE6 is regulated by calcium, contrary to the common view that PDE1 is the unique PDE class whose activity is modulated by intracellular Ca2+. To broaden the operating range of photoreceptors, mammalian rod photoresponse recovery is accelerated mainly by two calcium sensor proteins: recoverin, modulating the lifetime of activated rhodopsin, and guanylate cyclase-activating proteins (GCAPs), regulating the cGMP synthesis. We found that decreasing rod intracellular Ca2+concentration accelerates the flash response recovery and increases the basal PDE6 activity (βdark) maximally by ~ 30% when recording local electroretinography across the rod outer segment layer from GCAPs−/−recoverin−/−mice. Our modeling shows that a similar elevation in βdarkcan fully explain the observed acceleration of flash response ...
Journal of Biological Chemistry, 2010
The central enzyme of the visual transduction cascade, cGMP phosphodiesterase (PDE6), is regulated by its ␥-subunit (P␥), whose inhibitory constraint is released upon binding of activated transducin. It is generally believed that the last four or five C-terminal amino acid residues of P␥ are responsible for blocking catalysis. In this paper, we showed that the last 10 C-terminal residues (P␥78 -87) are the minimum required to completely block catalysis. The kinetic mechanism of inhibition by the P␥ C terminus depends on which substrate is undergoing catalysis. We also discovered a second mechanism of P␥ inhibition that does not require this C-terminal region and that is capable of inhibiting up to 80% of the maximal cGMP hydrolytic rate. Furthermore, amino acids 63-70 and/or the intact ␣2 helix of P␥ stabilize binding of C-terminal P␥ peptides by 100-fold. When PDE6 catalytic subunits were reconstituted with portions of the P␥ molecule and tested for activation by transducin, we found that the C-terminal region (P␥63-87) by itself could not be displaced but that transducin could relieve inhibition of certain P␥ truncation mutants. Our results are consistent with two distinct mechanisms of P␥ inhibition of PDE6. One involves direct interaction of the C-terminal residues with the catalytic site. A second regulatory mechanism may involve binding of other regions of P␥ to the catalytic domain, thereby allosterically reducing the catalytic rate. Transducin activation of PDE6 appears to require interaction with both the C terminus and other regions of P␥ to effectively relieve its inhibitory constraint. . 2 The abbreviations used are: PDE6, photoreceptor cyclic nucleotide phosphodiesterase; P␣, catalytic dimer of PDE6 ␣and -subunits; P␥, inhibitory ␥ subunit of PDE6; T␣*, activated transducin ␣-subunit; GTP␥S, guanosine 5Ј-O-(thiotriphosphate).
Modulation of Phosphodiesterase6 Turnoff during Background Illumination in Mouse Rod Photoreceptors
Journal of Neuroscience, 2008
In rod photoreceptors of wild-type mice, background light produces an acceleration of the decay of responses to brief flashes, accompanied by a decrease in the rate-limiting time constant for response decay. In rods in which phosphodiesterase gamma (PDEgamma) lacks one of its sites of phosphorylation (T35A rods), both the waveform of response decay and the rate-limiting time constant are nearly unaffected by backgrounds. These effects are not the result of the removal of the phosphorylation site per se, because rods lacking both of the phosphorylation sites of PDEgamma (T22A/T35A rods) adapt to light in a nearly normal manner. Because PDEgamma is one of the proteins of the GTPase activating protein (GAP) complex, our experiments argue for a novel mechanism of photoreceptor light adaptation produced by modulation of GAP-dependent hydrolysis of transducin alpha GTP. In PDEgamma T35A rods, a change in the conformation of the PDEgamma subunit may hinder or mask this mechanism, which in mammals appears to be primarily responsible for the quickening of the temporal resolution of the rod response in backgrounds. Modulation of PDE turnoff also helps to prevent premature saturation of the rod in bright backgrounds, thus making an important contribution to light adaptation. Our experiments provide evidence for modulation of GAP protein-dependent response turnoff, which may also play a role in controlling signal duration at hormone receptors and synapses in the CNS.
Excised inside-out patches of vertebrate rod outer segment can support phototransduction. I have examined how ionic and metabolic conditions influence the functional properties of light-sensitive patches from Gekko gekko. I find that such patches retain a variable level of basal phosphodiesterase activity, which lowers the cyclic guanosine monophosphate (cGMP) concentration reaching the channels and reduces the dark current. The dose/response relationship for channel opening by cGMP varies among patches and this variability is only reduced by working in darkness with the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX), suggesting that it is only partially due to phosphodiesterase activity. MgATP or MgGTP, but not Mg or ATP separately, increase this activity but a kinase does not appear to be involved. Intracellular monovalent cations also influence dark current intensity and light response kinetics. With 5 mM MgGTP, 1 mM IBMX, and 144 mM Li+, Na+, K+, or Rb+, dark current intensity and recovery time follow the respective sequences K+ > Rb+ > Na+ > Li+ and K+ < Rb+ < Li+ < Na+. Without IBMX, a dark current develops with K+ but not with Na+. These effects are not due to altered channel permeability (P) [PLi+:Na+:K+:Rb+:guanidinium)/PNa+ = 0.84:1.00:1.01:1.09:0.42], or differential Mg2+ block, but to modulation of guanylate cyclase, which overcomes phosphodiesterase when the major cation is K+ but not when it is Na+