[Show abstract][Hide abstract] ABSTRACT: Previous experiments have shown that the insulin receptor (IR) is expressed in mammalian rods and contributes to the protection of photoreceptors during bright-light exposure. The role of the insulin receptor in the production of the light response is however unknown. We have used suction-electrode recording to examine the responses of rods after conditionally knocking down the insulin receptor. Our results show that these IR knock-down rods have an accelerated decay of the light response and a small decrease in sensitivity by comparison to littermate WT rods. Our results indicate that the insulin receptor may have some role in controlling the rate of rod response decay, but they exclude a major role of the insulin receptor pathway in phototransduction.
[Show abstract][Hide abstract] ABSTRACT: Previous experiments have indicated that growth factor receptor-bound protein 14 (Grb14) may modulate rod photoreceptor cyclic guanosine-monophosphate (cGMP) gated channels by decreasing channel affinity for cGMP; however, the function of Grb14 in rod physiology is not known. In this study we examined the role of Grb14 by recording electrical responses from rods in which the gene for the Grb14 protein had been deleted. Suction-electrode recordings from single mouse rods showed that responses of dark-adapted Grb14-/- mice to brief flashes decayed more rapidly than strain-controlled wild-type (WT) rods, with decreased values of both integration time and the exponential time course of decay (τREC). This result is consistent with an increase in channel affinity for cGMP produced by deletion of Grb14. However Grb14-/- mouse rods also showed little change in dark current and a large and significant decrease in the limiting time constant τD, which are not consistent with an effect on channel affinity but seem rather to indicate modulation of the rate of inactivation of cyclic nucleotide phosphodiesterase 6 (PDE6). Grb14 has been reported to translocate from the inner to the outer segment in bright light, but we saw effects on response time course even in dark-adapted rods, though the effects were somewhat greater after rods had been adapted by exposure to bleaching illumination. Our results indicate that the mechanism of Grb14 action may be more complex than previously realized.
[Show abstract][Hide abstract] ABSTRACT: Amphibian and mammalian rods can both detect single photons of light even though they differ greatly in physical dimensions, mammalian rods being much smaller in diameter than amphibian rods. To understand the changes in physiology and biochemistry required by such large differences in outer segment geometry, we developed a computational approach, taking into account the spatial organization of the outer segment divided into compartments, together with molecular dynamics simulations of the signaling cascade. We generated simulations of the single-photon response together with intrinsic background fluctuations in toad and mouse rods. Combining this computational approach with electrophysiological data from mouse rods, we determined key biochemical parameters. On average around one phosphodiesterase (PDE) molecule is spontaneously active per mouse compartment, similar to the value for toad, which is unexpected due to the much smaller diameter in mouse. A larger number of spontaneously active PDEs decreases dark noise, thereby improving detection of single photons; it also increases cGMP turnover, which accelerates the decay of the light response. These constraints explain the higher PDE density in mammalian compared with amphibian rods that compensates for the much smaller diameter of mammalian disks. We further find that the rate of cGMP hydrolysis by light-activated PDE is diffusion limited, which is not the case for spontaneously activated PDE. As a consequence, in the small outer segment of a mouse rod only a few activated PDEs are sufficient to generate a signal that overcomes noise, which permits a shorter lifetime of activated rhodopsin and greater temporal resolution.
Proceedings of the National Academy of Sciences 11/2013; 110. DOI:10.1073/pnas.1314030110 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Light isomerizes 11-cis-retinal in a retinal rod and produces an active form of rhodopsin (Rh*) that binds to the G-protein transducin and activates the phototransduction cascade. Rh* is turned off by phosphorylation by rhodopsin kinase [G-protein-coupled receptor kinase 1 (GRK1)] and subsequent binding of arrestin. To evaluate the role of GRK1 in rod light response decay, we have generated the transgenic mouse RKS561L in which GRK1, which is normally present at only 2-3% of rhodopsin, is overexpressed by ∼12-fold. Overexpression of GRK1 increases the rate of Rh* phosphorylation and reduces the exponential decay constant of the response (τ(REC)) and the limiting time constant (τ(D)) both by ∼30%; these decreases are highly significant. Similar decreases are produced in Rv(-/-) rods, in which the GRK1-binding protein recoverin has been genetically deleted. These changes in response decay are produced by acceleration of light-activated phosphodiesterase (PDE*) decay rather than Rh* decay, because light-activated PDE* decay remains rate limiting for response decay in both RKS561L and Rv(-/-) rods. A model incorporating an effect of GRK1 on light-activated PDE* decay rate can satisfactorily account for the changes in response amplitude and waveform. Modulation of response decay in background light is nearly eliminated by deletion of recoverin. Our experiments indicate that rhodopsin kinase and recoverin, in addition to their well-known role in regulating the turning off of Rh*, can also modulate the decay of light-activated PDE*, and the effects of these proteins on light-activated PDE* decay may be responsible for the quickening of response recovery in background light.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 11/2012; 32(45):15998-16006. DOI:10.1523/JNEUROSCI.1639-12.2012 · 6.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Caveolin-1 (Cav-1), an integral component of caveolar membrane domains, is expressed in several retinal cell types, including photoreceptors, retinal vascular endothelial cells, Müller glia, and retinal pigment epithelium (RPE) cells. Recent evidence links Cav-1 to ocular diseases, including autoimmune uveitis, diabetic retinopathy, and primary open angle glaucoma, but its role in normal vision is largely undetermined. In this report, we show that ablation of Cav-1 results in reduced inner and outer retinal function as measured, in vivo, by electroretinography and manganese-enhanced MRI. Somewhat surprisingly, dark current and light sensitivity were normal in individual rods (recorded with suction electrode methods) from Cav-1 knock-out (KO) mice. Although photoreceptor function was largely normal, in vitro, the apparent K(+) affinity of the RPE-expressed α1-Na(+)/K(+)-ATPase was decreased in Cav-1 KO mice. Cav-1 KO retinas also displayed unusually tight adhesion with the RPE, which could be resolved by brief treatment with hyperosmotic medium, suggesting alterations in outer retinal fluid homeostasis. Collectively, these findings demonstrate that reduced retinal function resulting from Cav-1 ablation is not photoreceptor-intrinsic but rather involves impaired subretinal and/or RPE ion/fluid homeostasis.
[Show abstract][Hide abstract] ABSTRACT: When a substantial fraction of rhodopsin in a rod photoreceptor is exposed to bright light, the rod is desensitized by a process known as bleaching adaptation. Experiments on isolated photoreceptors in amphibians have revealed many of the features of bleaching adaptation, but such experiments have not so far been possible in mammals. We now describe a method for making microspectrophotometric measurements of pigment concentration and suction-electrode recording of electrical responses over a wide range of bleaching exposures from isolated mouse rods or pieces of mouse retina. We show that if pigment is bleached at a low rate in the presence of bovine serum albumin (BSA), and intermediate photoproducts are allowed to decay, mouse rods are stably desensitized; subsequent treatment with exogenous 11-cis retinal results in pigment regeneration and substantial recovery of sensitivity to the dark-adapted value. Stably bleached wild-type (WT) rods show a decrease in circulating current and acceleration of the time course of decay, much as in steady background light; similar effects are seen in guanylyl cyclase-activating protein knockout (GCAPs(-/-)) rods, indicating that regulation of guanylyl cyclase is not necessary for at least a part of the adaptation produced by bleaching. Our experiments demonstrate that in mammalian rods, as in amphibian rods, steady-state desensitization after bleaching is produced by two components: (1) a reduction in the probability of photon absorption produced by a decrease in rhodopsin concentration; and (2) an equivalent background light whose intensity is proportional to the fraction of bleached pigment, and which adapts the rod like real background light. These two mechanisms together fully account for the ‘log-linear' relationship in mammalian retina between sensitivity and per cent bleach, which can be measured in the steady state following exposure to bright light. Our methods will now make possible an examination of bleaching adaptation and pigment regeneration in mouse animal lines with mutations or other alterations in the proteins of transduction.
The Journal of Physiology 03/2012; 590(Pt 10):2353-64. DOI:10.1113/jphysiol.2012.228627 · 4.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: All sensory receptors adapt. As the mean level of light or sound or odor is altered, the sensitivity of the receptor is adjusted to permit the cell to function over as wide a range of ambient stimulation as possible. In a rod photoreceptor, adaptation to maintained background light produces a decrease (or "sag") in the response to the prolonged illumination, as well as an acceleration in response decay time and a Weber-Fechner-like decrease in sensitivity. Earlier work on salamander indicated that adaptation is controlled by the intracellular concentration of Ca(2+). Three Ca(2+)-dependent mechanisms were subsequently identified, namely, regulation of guanylyl cyclase, modulation of activated rhodopsin lifetime, and alteration of channel opening probability, with the contribution of the cyclase thought to be the most important. Later experiments on mouse that exploit the powerful techniques of molecular genetics have shown that cyclase does indeed play a significant role in mammalian rods, but that much of adaptation remains even when regulation of cyclase and both of the other proposed pathways have been genetically deleted. The identity of the missing mechanism or mechanisms is unclear, but recent speculation has focused on direct modulation of spontaneous and light-activated phosphodiesterase.
[Show abstract][Hide abstract] ABSTRACT: The light-dependent decrease in cyclic guanosine monophosphate (cGMP) in the rod outer segment is produced by a phosphodiesterase (PDE6), consisting of catalytic α and β subunits and two inhibitory γ subunits. The molecular mechanism of PDE6γ regulation of the catalytic subunits is uncertain. To study this mechanism in vivo, we introduced a modified Pde6g gene for PDE6γ into a line of Pde6g(tm1)/Pde6g(tm1) mice that do not express PDE6γ. The resulting ILE86TER mice have a PDE6γ that lacks the two final carboxyl-terminal Ile(86) and Ile(87) residues, a mutation previously shown in vitro to reduce inhibition by PDE6γ. ILE86TER rods showed a decreased sensitivity and rate of activation, probably the result of a decreased level of expression of PDE6 in ILE86TER rods. More importantly, they showed a decreased rate of decay of the photoresponse, consistent with decreased inhibition of PDE6 α and β by PDE6γ. Furthermore, ILE86TER rods had a higher rate of spontaneous activation of PDE6 than WT rods. Circulating current in ILE86TER rods that also lacked both guanylyl cyclase activating proteins (GCAPs) could be increased several fold by perfusion with 100μM of the PDE6 inhibitor 3-isobutyl-1-methylxanthine (IBMX), consistent with a higher rate of dark PDE6 activity in the mutant photoreceptors. In contrast, IBMX had little effect on the circulating current of WT rods, unlike previous results from amphibians. Our results show for the first time that the Ile(86) and Ile(87) residues are necessary for normal inhibition of PDE6 catalytic activity in vivo, and that increased basal activity of PDE can be partially compensated by GCAP-dependent regulation of guanylyl cyclase.
[Show abstract][Hide abstract] ABSTRACT: Vertebrate photoreceptors are thought to adapt to light by a change in Ca(2+), which is postulated to mediate modulation of (1) excited rhodopsin (Rh*) by Ca(2+)-dependent binding of recoverin, (2) guanylyl cyclase activity via Ca(2+)-dependent GCAP proteins, and (3) cyclic nucleotide-gated channels by binding of Ca(2+)-calmodulin. Previous experiments genetically deleted recoverin and the GCAPs and showed that significant regulation of sensitivity survives removal of (1) and (2). We genetically deleted the channel Ca(2+)-calmodulin binding site in the mouse Mus musculus and found that removal of (3) alters response waveform, but removal of (3) or of (2) and (3) together still leaves much of adaptation intact. These experiments demonstrate that an important additional mechanism is required, which other experiments indicate may be regulation of phosphodiesterase 6 (PDE6). We therefore constructed a kinetic model in which light produces a Ca(2+)-mediated decrease in PDE6 decay rate, with the novel feature that both spontaneously activated and light-activated PDE6 are modulated. This model, together with Ca(2+)-dependent acceleration of guanylyl cyclase, can successfully account for changes in sensitivity and response waveform in background light.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 12/2010; 30(48):16232-40. DOI:10.1523/JNEUROSCI.2868-10.2010 · 6.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cone vision is less sensitive than rod vision. Much of this difference can be attributed to the photoreceptors themselves, but the reason why the cones are less sensitive is still unknown. Recent recordings indicate that one important factor may be a difference in the rate of activation of cone transduction; that is, the rising phase of the cone response per bleached rhodopsin molecule (Rh*) has a smaller slope than the rising phase of the rod response per Rh*, perhaps because some step between Rh* and activation of the phosphodiesterase 6 (PDE6) effector molecule occurs with less gain. Since rods and cones have different G-protein alpha subunits, and since this subunit (Talpha) plays a key role both in the interaction of G-protein with Rh* and the activation of PDE6, we investigated the mechanism of the amplification difference by expressing cone Talpha in rod Talpha-knockout rods to produce so-called GNAT2C mice. We show that rods in GNAT2C mice have decreased sensitivity and a rate of activation half that of wild-type (WT) mouse rods. Furthermore, GNAT2C responses recover more rapidly than WT responses with kinetic parameters resembling those of native mouse cones. Our results show for the first time that part of the difference in sensitivity and response kinetics between rods and cones may be the result of a difference in the G-protein alpha subunit. They also indicate more generally that the molecular nature of G-protein alpha may play an important role in the kinetics of G-protein cascades for metabotropic receptors throughout the body.
The Journal of Physiology 09/2010; 588(Pt 17):3231-41. DOI:10.1113/jphysiol.2010.191221 · 4.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Callimachus' Hymns were written in the tradition of the rhapsodic Homeric Hymns and make constant, though usually oblique and often amusing, reference to these archaic poems. This allusion is particularly extensive in the Hymn to Artemis, the only Callimachus Hymn to proceed like most longer Homeric Hymns, with brief introduction and prolonged narration. Then, just as the poem seems to end, it begins anew, not once but over and over again. The middle of the poem is clearly based on the Homeric Hymn to Apollo, which also begins anew at the Delian/Pythian transition. The correspondences are so great that Callimachus must have had this poem in much the form we do. The choice of the Apollo Hymn as model reflects the theme of sibling rivalry that pervades the Artemis Hymn: even in her own poem, Artemis can't escape the influence of her brother.
[Show abstract][Hide abstract] ABSTRACT: Photoreceptors in metazoans can be grouped into two classes, with their photoreceptive membrane derived either from cilia or microvilli. Both classes use some form of the visual pigment protein opsin, which together with 11-cis retinaldehyde absorbs light and activates a G-protein cascade, resulting in the opening or closing of ion channels. Considerable attention has recently been given to the molecular evolution of the opsins and other photoreceptor proteins; much is also known about transduction in the various photoreceptor types. Here we combine this knowledge in an attempt to understand why certain photoreceptors might have conferred particular selective advantages during evolution. We suggest that microvillar photoreceptors became predominant in most invertebrate species because of their single-photon sensitivity, high temporal resolution, and large dynamic range, and that rods and a duplex retina provided primitive chordates and vertebrates with similar sensitivity and dynamic range, but with a smaller expenditure of ATP.
Current biology: CB 02/2010; 20(3):R114-24. DOI:10.1016/j.cub.2009.12.006 · 9.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Ca(2+)-binding protein recoverin is thought to regulate rhodopsin kinase and to modulate the lifetime of the photoexcited state of rhodopsin (Rh*), the visual pigment of vertebrate rods. Recoverin has been postulated to inhibit the kinase in darkness, when Ca(2+) is high, and to be released from the disk membrane in light when Ca(2+) is low, accelerating rhodopsin phosphorylation and shortening the lifetime of Rh*. This proposal has remained controversial, in part because the normally rapid turnoff of Rh* has made Rh* modulation difficult to study in an intact rod. To circumvent this problem, we have made mice that underexpress rhodopsin kinase so that Rh* turnoff is rate limiting for the decay of the rod light response. We show that background light speeds the decay of Rh* turnoff, and that this no longer occurs in mice that have had recoverin knocked out. This is the first demonstration in an intact rod that light accelerates Rh* inactivation and that the Ca(2+)-binding protein recoverin may be required for the light-dependent modulation of Rh* lifetime.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 01/2010; 30(4):1213-20. DOI:10.1523/JNEUROSCI.4353-09.2010 · 6.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The visual pigment in vertebrate photoreceptors is a G protein-coupled receptor that consists of a protein, opsin, covalently attached to a chromophore, 11-cis-retinal. Activation of the visual pigment by light triggers a transduction cascade that produces experimentally measurable electrical responses in photoreceptors. The interactions between opsin and chromophore can be investigated with electrophysiologial recordings in intact amphibian and mouse rod and cone photoreceptor cells. Here we describe methods for substituting the native chromophore with various chromophore analogs to investigate how specific parts of the chromophore affect the signaling properties of the visual pigment and the function of photoreceptors. We also describe methods for genetically substituting the native rod opsin gene with cone opsins or with mutant rod opsins to investigate and compare their signaling properties. These methods are useful not only for understanding the relation between the properties of visual pigments and the function of photoreceptors but also for understanding the mechanisms by which mutations in rod opsin produce night blindness and other visual disorders.
[Show abstract][Hide abstract] ABSTRACT: The performance of sensory systems in many cases is limited by the physical nature of the stimulus. For vision, the quantal nature of light limits detection by dark-adapted observers; only now are we beginning to be aware of the subtleties in the biophysical mechanisms underlying this exquisite sensitivity.
[Show abstract][Hide abstract] ABSTRACT: Ion flow into the rod photoreceptor outer segment (ROS) is regulated by a member of the cyclic-nucleotide-gated cation-channel family; this channel consists of two subunit types, alpha and beta. In the rod cells, the Cngb1 locus encodes the channel beta-subunit and two related glutamic-acid-rich proteins (GARPs). Despite intensive research, it is still unclear why the beta-subunit and GARPs are coexpressed and what function these proteins serve. We hypothesized a role for the proteins in the maintenance of ROS structural integrity. To test this hypothesis, we created a Cngb1 5'-knockout photoreceptor null (Cngb1-X1). Morphologically, ROSs were shorter and, in most rods that were examined, some disks were misaligned, misshapen and abnormally elongated at periods when stratification was still apparent and degeneration was limited. Additionally, a marked reduction in the level of channel alpha-subunit, guanylate cyclase I (GC1) and ATP-binding cassette transporter (ABCA4) was observed without affecting levels of other ROS proteins, consistent with a requirement for the beta-subunit in channel assembly or targeting of select proteins to ROS. Remarkably, phototransduction still occurred when only trace levels of homomeric alpha-subunit channels were present, although rod sensitivity and response amplitude were both substantially reduced. Our results demonstrate that the beta-subunit and GARPs are necessary not only to maintain ROS structural integrity but also for normal disk morphogenesis, and that the beta-subunit is required for normal light sensitivity of the rods.
[Show abstract][Hide abstract] ABSTRACT: The G90D rhodopsin mutation is known to produce congenital night blindness in humans. This mutation produces a similar condition in mice, because rods of animals heterozygous (D+) or homozygous (D+/+) for this mutation have decreased dark current and sensitivity, reduced Ca(2+), and accelerated values of tau(REC) and tau(D), similar to light-adapted wild-type (WT) rods. Our experiments indicate that G90D pigment activates the cascade, producing an equivalent background light of approximately 130 Rh* rod(-1) for D+ and 890 Rh* rod(-1) for D+/+. The active species of the G90D pigment could be unregenerated G90D opsin or G90D rhodopsin, either spontaneously activated (as Rh*) or in some other form. Addition of 11-cis-retinal in lipid vesicles, which produces regeneration of both WT and G90D opsin in intact rods and ROS membranes, had no effect on the waveform or sensitivity of dark-adapted G90D responses, indicating that the active species is not G90D opsin. The noise spectra of dark-adapted G90D and WT rods are similar, and the G90D noise variance is much less than of a WT rod exposed to background light of about the same intensity as the G90D equivalent light, indicating that Rh* is not the active species. We hypothesize that G90D rhodopsin undergoes spontaneous changes in molecular conformation which activate the transduction cascade with low gain. Our experiments provide the first indication that a mutant form of the rhodopsin molecule bound to its 11-cis-chromophore can stimulate the visual cascade spontaneously at a rate large enough to produce visual dysfunction.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 12/2008; 28(45):11662-72. DOI:10.1523/JNEUROSCI.4006-08.2008 · 6.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Why do vertebrates use rods and cones that hyperpolarize, when in insect eyes a single depolarizing photoreceptor can function at all light levels? We answer this question at least in part with a comprehensive assessment of ATP consumption for mammalian rods from voltages and currents and recently published physiological and biochemical data. In darkness, rods consume 10(8) ATP s(-1), about the same as Drosophila photoreceptors. Ion fluxes associated with phototransduction and synaptic transmission dominate; as in CNS, the contribution of enzymes of the second-messenger cascade is surprisingly small. Suppression of rod responses in daylight closes light-gated channels and reduces total energy consumption by >75%, but in Drosophila light opens channels and increases consumption 5-fold. Rods therefore provide an energy-efficient mechanism not present in rhabdomeric photoreceptors. Rods are metabolically less "costly" than cones, because cones do not saturate in bright light and use more ATP s(-1) for transducin activation and rhodopsin phosphorylation. This helps to explain why the vertebrate retina is duplex, and why some diurnal animals like primates have a small number of cones, concentrated in a region of high acuity.
Current biology: CB 12/2008; 18(24):1917-21. DOI:10.1016/j.cub.2008.10.029 · 9.92 Impact Factor