Role of guanylyl cyclase modulation in mouse cone phototransduction

A negative phototransduction feedback in rods and cones is critical for the timely termination of their light responses and for extending their function to a wide range of light intensities. The calcium feedback mechanisms that modulate phototransduction in rods have been studied extensively. However, the corresponding modulation mechanisms that enable cones to terminate rapidly their light responses and to adapt in bright light, properties critical for our daytime vision, are still not understood. In cones, calcium feedback to guanylyl cyclase is potentially a key step in phototransduction modulation. The guanylyl cyclase activity is modulated by the calcium-binding guanylyl cyclase activating proteins (GCAP1 and GCAP2). Here, we used single-cell and transretinal recordings from mouse to determine how GCAPs modulate dark-adapted responses as well as light adaptation in mammalian cones. Deletion of GCAPs increased threefold the amplitude and dramatically prolonged the light responses in dark-adapted mouse cones. It also reduced the operating range of mouse cones in background illumination and severely impaired their light adaptation. Thus, GCAPs exert powerful modulation on the mammalian cone phototransduction cascade and play an important role in setting the functional properties of cones in darkness and during light adaptation. Surprisingly, despite their better adaptation capacity and wider calcium dynamic range, mammalian cones were modulated by GCAPs to a lesser extent than mammalian rods. We conclude that a disparity in the strength of GCAP modulation cannot explain the differences in the dark-adapted properties or in the operating ranges of mammalian rods and cones

Investigating the Ca2+-dependent and Ca2+-independent mechanisms for mammalian cone light adaptation

Abstract Vision is mediated by two types of photoreceptors: rods, enabling vision in dim light; and cones, which function in bright light. Despite many similarities in the components of their respective phototransduction cascades, rods and cones have distinct sensitivity, response kinetics, and adaptation capacity. Cones are less sensitive and have faster responses than rods. In addition, cones can function over a wide range of light conditions whereas rods saturate in moderately bright light. Calcium plays an important role in regulating phototransduction and light adaptation of rods and cones. Notably, the two dominant Ca2+-feedbacks in rods and cones are driven by the identical calcium-binding proteins: guanylyl cyclase activating proteins 1 and 2 (GCAPs), which upregulate the production of cGMP; and recoverin, which regulates the inactivation of visual pigment. Thus, the mechanisms producing the difference in adaptation capacity between rods and cones have remained poorly understood. Using GCAPs/recoverin-deficient mice, we show that mammalian cones possess another Ca2+-dependent mechanism promoting light adaptation. Surprisingly, we also find that, unlike in mouse rods, a unique Ca2+-independent mechanism contributes to cone light adaptation. Our findings point to two novel adaptation mechanisms in mouse cones that likely contribute to the great adaptation capacity of cones over rods

The retina visual cycle is driven by cis retinol oxidation in the outer segments of cones

Vertebrate rod and cone photoreceptors require continuous supply of chromophore for regenerating their visual pigments after photoactivation. Cones, which mediate our daytime vision, demand a particularly rapid supply of 11-cis retinal chromophore in order to maintain their function in bright light. An important contribution to this process is thought to be the chromophore precursor 11-cis retinol, which is supplied to cones from Müller cells in the retina and subsequently oxidized to 11-cis retinal as part of the retina visual cycle. However, the molecular identity of the cis retinol oxidase in cones remains unclear. Here, as a first step in characterizing this enzymatic reaction, we sought to determine the subcellular localization of this activity in salamander red cones. We found that the onset of dark adaptation of isolated salamander red cones was substantially faster when exposing directly their outer vs. their inner segment to 9-cis retinol, an analogue of 11-cis retinol. In contrast, this difference was not observed when treating the outer vs. inner segment with 9-cis retinal, a chromophore analogue which can directly support pigment regeneration. These results suggest, surprisingly, that the cis-retinol oxidation occurs in the outer segments of cone photoreceptors. Confirming this notion, pigment regeneration with exogenously added 9-cis retinol was directly observed in the truncated outer segments of cones, but not in rods. We conclude that the enzymatic machinery required for the oxidation of recycled cis retinol as part of the retina visual cycle is present in the outer segments of cones

Age-related deterioration of rod vision in mice

Even in healthy individuals, aging leads to deterioration in visual acuity, contrast sensitivity, visual field, and dark adaptation. Little is known about the neural mechanisms that drive the age-related changes of the retina and, more specifically, photoreceptors. According to one hypothesis, the age-related deterioration in rod function is due to the limited availability of 11-cis-retinal for rod pigment formation. To determine how aging affects rod photoreceptors and to test the retinoid-deficiency hypothesis, we compared the morphological and functional properties of rods of adult and aged B6D2F1/J mice. We found that the number of rods and the length of their outer segments were significantly reduced in 2.5-year-old mice compared with 4-month-old animals. Aging also resulted in a twofold reduction in the total level of opsin in the retina. Behavioral tests revealed that scotopic visual acuity and contrast sensitivity were decreased by twofold in aged mice, and rod ERG recordings demonstrated reduced amplitudes of both a- and b-waves. Sensitivity of aged rods determined from single-cell recordings was also decreased by 1.5-fold, corresponding to not more than 1% free opsin in these photoreceptors, and kinetic parameters of dim flash response were not altered. Notably, the rate of rod dark adaptation was unaffected by age. Thus, our results argue against age-related deficiency of 11-cis-retinal in the B6D2F1/J mouse rod visual cycle. Surprisingly, the level of cellular dark noise was increased in aged rods, providing an alternative mechanism for their desensitization

Role of Noncovalent Binding of 11-cis-Retinal to Opsin in Dark Adaptation of Rod and Cone Photoreceptors

AbstractRegeneration of visual pigments of vertebrate rod and cone photoreceptors occurs by the initial noncovalent binding of 11-cis-retinal to opsin, followed by the formation of a covalent bond between the ligand and the protein. Here, we show that the noncovalent interaction between 11-cis-retinal and opsin affects the rate of dark adaptation. In rods, 11-cis-retinal produces a transient activation of the phototransduction cascade that precedes sensitivity recovery, thus slowing dark adaptation. In cones, 11-cis-retinal immediately deactivates phototransduction. Thus, the initial binding of the same ligand to two very similar G protein receptors, the rod and cone opsins, activates one and deactivates the other, contributing to the remarkable difference in the rates of rod and cone dark adaptation

Examining the role of cone-expressed RPE65 in mouse cone function

Abstract Efficient chromophore supply is paramount for the continuous function of vertebrate cone photoreceptors. It is well established that isomerization of all-trans- to 11-cis- retinoid in the retinal pigmented epithelium by RPE65 is a key reaction in this process. Mutations in RPE65 result in a disrupted chromophore supply, retinal degeneration, and blindness. Interestingly, RPE65 has recently been found to also be expressed in cone photoreceptors in several species, including mouse and human. However, the functional role of cone-expressed RPE65 has remained unknown. Here, we used loss and gain of function approaches to investigate this issue. First, we compared the function of cones from control and RPE65-deficient mice. Although we found that deletion of RPE65 partially suppressed cone dark adaptation, the interpretation of this result was complicated by the abnormal cone structure and function caused by the chromophore deficiency in the absence of RPE65 in the pigmented epithelium. As an alternative approach, we generated transgenic mice to express human RPE65 in the cones of mice where RPE65 expression is normally restricted to the pigmented epithelium. Comparison of control (RPE65-deficient) and transgenic (RPE65-expressing) cones revealed no morphological or functional changes, with only a slight delay in dark adaptation, possibly caused by the buffering of retinoids by RPE65. Together, our results do not provide any evidence for a functional role of RPE65 in mouse cones. Future studies will have to determine whether cone-expressed RPE65 plays a role in maintaining the long-term homeostasis of retinoids in cones and their function and survival, particularly in humans

The Na+/Ca2+, K+ exchanger 2 modulates mammalian cone phototransduction

Calcium ions (Ca(2+)) modulate the phototransduction cascade of vertebrate cone photoreceptors to tune gain, inactivation, and light adaptation. In darkness, the continuous current entering the cone outer segment through cGMP-gated (CNG) channels is carried in part by Ca(2+), which is then extruded back to the extracellular space. The mechanism of Ca(2+) extrusion from mammalian cones is not understood. The dominant view has been that the cone-specific isoform of the Na(+)/Ca(2+), K(+) exchanger, NCKX2, is responsible for removing Ca(2+) from their outer segments. However, indirect evaluation of cone function in NCKX2-deficient (Nckx2(−/−)) mice by electroretinogram recordings revealed normal photopic b-wave responses. This unexpected result suggested that NCKX2 may not be involved in the Ca(2+) homeostasis of mammalian cones. To address this controversy, we examined the expression of NCKX2 in mouse cones and performed transretinal recordings from Nckx2(−/−) mice to determine the effect of NCKX2 deletion on cone function directly. We found that Nckx2(−/−) cones exhibit compromised phototransduction inactivation, slower response recovery and delayed background adaptation. We conclude that NCKX2 is required for the maintenance of efficient Ca(2+) extrusion from mouse cones. However, surprisingly, Nckx2(−/−) cones adapted normally in steady background light, indicating the existence of additional Ca(2+)-extruding mechanisms in mammalian cones

Interphotoreceptor retinoid-binding protein as the physiologically relevant carrier of 11-cis-retinol in the cone visual cycle

Cones function in constant light and are responsible for mediating daytime human vision. Like rods, cones utilize the photo-sensitive molecule 11-cis retinal to detect light, and in constant illumination, a continuous supply of 11-cis retinal is needed. A retina visual cycle is thought to provide a privileged supply of 11-cis retinal to cones by utilizing 11-cis retinol generated in Muller cells. In the cycle, 11-cis retinol is transported from Muller cells to cone inner segments, where it is oxidized to 11-cis retinal. This oxidation step is only carried out in cones, thus rendering the cycle cone-specific. IRBP is a retinoid-binding protein in the sub-retinal space that binds 11-cis retinol endogenously. Cones in Irbp(−/−) mice are retinoid-deficient under photopic conditions, and it is possible that 11-cis retinol supplies are disrupted in the absence of IRBP. We tested the hypothesis that IRBP facilitates the delivery of 11-cis retinol to cones by preserving the isomeric state of 11-cis retinol in light. With electrophysiology, we show that the cone-like photoreceptors of Nrl(−/−) mice utilize the cone visual cycle similarly to wild-type cones. Then, using oxidation assays in isolated Nrl(−/−)Rpe65(−/−) retinas, we show that IRBP delivers 11-cis retinol for oxidation in cones and improves the efficiency of the oxidation reaction. Finally, we show that IRBP protects the isomeric state of 11-cis retinol in the presence of light. Together, these findings suggest that IRBP plays an important role in the delivery of 11-cis retinol to cones and can facilitate cone function in the presence of light

Circadian and light-driven regulation of rod dark adaptation

Continuous visual perception and the dark adaptation of vertebrate photoreceptors after bright light exposure require recycling of their visual chromophore through a series of reactions in the retinal pigmented epithelium (RPE visual cycle). Light-driven chromophore consumption by photoreceptors is greater in daytime vs. nighttime, suggesting that correspondingly higher activity of the visual cycle may be required. However, as rod photoreceptors are saturated in bright light, the continuous turnover of their chromophore by the visual cycle throughout the day would not contribute to vision. Whether the recycling of chromophore that drives rod dark adaptation is regulated by the circadian clock and light exposure is unknown. Here, we demonstrate that mouse rod dark adaptation is slower during the day or after light pre-exposure. This surprising daytime suppression of the RPE visual cycle was accompanied by light-driven reduction in expression of Rpe65, a key enzyme of the RPE visual cycle. Notably, only rods in melatonin-proficient mice were affected by this daily visual cycle modulation. Our results demonstrate that the circadian clock and light exposure regulate the recycling of chromophore in the RPE visual cycle. This daily melatonin-driven modulation of rod dark adaptation could potentially protect the retina from light-induced damage during the day