Review

Signal flow in visual transduction

Diabetic retinopathy (DR) is a leading cause of blindness. It has long been regarded as vascular disease, but work in the past years has shown abnormalities also in the neural retina. Unfortunately, research on the vascular and neural abnormalities have remained largely separate, instead of being integrated into a comprehensive view of DR that includes both the neural and vascular components. Recent evidence suggests that the most predominant neural cell in the retina (photoreceptors) and the adjacent retinal pigment epithelium (RPE) play an important role in the development of vascular lesions characteristic of DR. This review summarizes evidence that the outer retina is altered in diabetes, and that photoreceptors and RPE contribute to retinal vascular alterations in the early stages of the retinopathy. The possible molecular mechanisms by which cells of the outer retina might contribute to retinal vascular damage in diabetes also are discussed. Diabetes-induced alterations in the outer retina represent a novel therapeutic target to inhibit DR.

Biological systems must sense and adapt to changes in their environment. Molecular networks capable of such adaptation belong to two well-known classes, feedforward and feedback structures, but the fundamental limitations and tradeoffs of these two classes remain unknown. Here we study the advantages and limitations of the feedforward class using three-node circuits representative of these architectures. The feedforward model we investigate displays a tradeoff between the sensitivity of the response (its peak response) and its precision (its error in its return to steady-state). We suggest two ways in which this tradeoff can be alleviated: (1) by introducing a nonlinearity in the production of a specific node in the network, and (2) by adding a feedback loop to the input. We present analytical and numerical examples to support our findings.

The thermal properties of rhodopsin, which set the threshold of our vision, have long been investigated, but the chemical kinetics of the thermal decay of rhodopsin has not been revealed in detail. To understand thermal decay quantitatively, we propose a kinetic model consisting of two pathways: 1) thermal isomerization of 11-cis-retinal followed by hydrolysis of Schiff base (SB) and 2) hydrolysis of SB in dark state rhodopsin followed by opsin-catalyzed isomerization of free 11-cis-retinal. We solve the kinetic model mathematically and use it to analyze kinetic data from four experiments that we designed to assay thermal decay, isomerization, hydrolysis of SB using dark state rhodopsin, and hydrolysis of SB using photoactivated rhodopsin. We apply the model to WT rhodopsin and E181Q and S186A mutants at 55 °C, as well as WT rhodopsin in H₂O and D₂O at 59 °C. The results show that the hydrogen-bonding network strongly restrains thermal isomerization but is less important in opsin and activated rhodopsin. Furthermore, the ability to obtain individual rate constants allows comparison of thermal processes under various conditions. Our kinetic model and experiments reveal two unusual energetic properties: the steep temperature dependence of the rates of thermal isomerization and SB hydrolysis in the dark state and a strong deuterium isotope effect on dark state SB hydrolysis. These findings can be applied to study pathogenic rhodopsin mutants and other visual pigments.

Rhodopsin has developed mechanisms to optimize its sensitivity to light by suppressing dark noise and enhancing quantum yield. We propose that an intramolecular hydrogen-bonding network formed by ∼20 water molecules, the hydrophilic residues, and peptide backbones in the transmembrane region is essential to restrain thermal isomerization, the source of dark noise. We studied the thermal stability of rhodopsin at 55 °C with single point mutations (E181Q and S186A) that perturb the hydrogen-bonding network at the active site. We found that the rate of thermal isomerization increased by 1–2 orders of magnitude in the mutants. Our results illustrate the importance of the intact hydrogen-bonding network for dim-light detection, revealing the functional roles of water molecules in rhodopsin. We also show that thermal isomerization of 11-cis-retinal in solution can be catalyzed by wild-type opsin and that this catalytic property is not affected by the mutations. We characterize the catalytic effect and propose that it is due to steric interactions in the retinal-binding site and increases quantum yield by predetermining the trajectory of photoisomerization. Thus, our studies reveal a balancing act between dark noise and quantum yield, which have opposite effects on the thermal isomerization rate. The acquisition of the hydrogen-bonding network and the tuning of the steric interactions at the retinal-binding site are two important factors in the development of dim-light vision.

Rod outer segment disk membranes are densely packed with rhodopsin. The recent notion of raft or microdomain structures in disk membranes suggests that the local density of rhodopsin in disk membranes could be much higher than the average density corresponding to the lipid/protein ratio. Little is known about the effect of high packing density of rhodopsin on the structure and function of rhodopsin and lipid membranes. Here we examined the role of rhodopsin packing density on membrane dynamic properties, membrane acyl chain packing, and the structural stability and function of rhodopsin using a combination of biophysical and biochemical techniques. We reconstituted rhodopsin into large unilamellar vesicles consisting of polyunsaturated 18:0,22:6n3PC, which approximates the polyunsaturated nature of phospholipids in disk membranes, with rhodopsin/lipid ratios ranging from 1:422 to 1:40. Our results showed that increased rhodopsin packing density led to reduced membrane dynamics revealed by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene, increased phospholipid acyl chain packing, and reduced rhodopsin activation, yet it had minimal impact on the structural stability of rhodopsin. These observations imply that densely packed rhodopsin may impede the diffusion and conformational changes of rhodopsin, which could reduce the speed of visual transduction.

A population of cerebrospinal fluid-contacting neurons (CSFcn) in the lateral septal organ (LSO) may serve as encephalic photoreceptors (EPRs) functioning to signal the onset of seasonal reproductive development in birds. Previous studies on CSFcn in the LSO have focused on identification of retinal protein components in fixed brain tissue. In order to understand better the mechanisms underlying the light-induced photosexual response in birds, a physiological characterization is required. In this study, changes of intracellular free calcium concentration ([Ca²⁺]_i) were monitored during light stimulation of CSFcn in the LSO in live brain slices from embryonic chicks. Using the fluorescent calcium indicator fluo-4, a reduced [Ca²⁺]_i within CSFcn was recorded in response to photostimulation, which is consistent with what has been demonstrated in rods and cones following illumination. Results support the hypothesis that CSFcn in the LSO function as EPRs in the avian brain.