Satoru Kawamura

Rod and cone photoreceptors: molecular basis of the difference in their physiology.

Vertebrate retinal photoreceptors consist of two types of cells, the rods and cones. Rods are highly light-sensitive but their flash response time course is slow, so that they can detect a single photon in the dark but are not good at detecting an object moving quickly. Cones are less light-sensitive and their flash response time course is fast, so that cones mediate daylight vision and are more suitable to detect a moving object than rods. The phototransduction mechanism was virtually known by the mid 80s, and detailed mechanisms of the generation of a light response are now understood in a highly quantitative manner at the molecular level. However, most of these studies were performed in rods, but not in cones. Therefore, the mechanisms of low light-sensitivity or fast flash response time course in cones have not been known. The major reason for this slow progress in the study of cone phototransduction was due to the inability of getting a large quantity of purified cones to study them biochemically. We succeeded in its purification using carp retina, and have shown that each step responsible for generation of a light response is less effective in cones and that the reactions responsible for termination of a light response are faster in cones. Based on these findings, we speculated a possible mechanism of evolution of rods that diverged from cones.

Low amplification and fast visual pigment phosphorylation as mechanisms characterizing cone photoresponses

Vertebrate cone photoreceptors are known to show lower light sensitivity and briefer photoresponses than rod photoreceptors. To understand the molecular mechanisms characterizing cone photoresponses, we compared some of the reactions in the phototransduction cascade between rods and cones. For this purpose, rods and cones were obtained in quantities large enough to do biochemical studies. The cells were purified from the retina of carp (Cyprinus carpio) with a stepwise Percoll gradient. The purified rod fraction contained almost no other kinds of cells besides rods, and the purified cone fraction contained a mixture of red-, green-, and blue-sensitive cones in the ratio 3:≈1:≈1. We prepared membrane preparations from the rod and the cone fraction, and in these membranes, we measured activation efficiencies of the reactions in the phototransduction cascade. The results showed that the signal amplification is lower in the cone membranes, which accounts for the lower light sensitivity in cones. Furthermore, we measured the time courses of visual pigment phosphorylation. The result showed that the phosphorylation is much faster in the cone membranes, which also explains the lower light sensitivity and, in addition, the briefer photoresponse in cones.

Purification and characterization of S-modulin, a calcium-dependent regulator on cGMP phosphodiesterase in frog rod photoreceptors.

S-modulin is a 26 kDa protein that regulates light sensitivity of cGMP phosphodiesterase in a Ca(2+)-dependent manner in frog rod outer segments (ROSs). In the present study, we purified S-modulin by taking advantage of a hydrophobic interaction between Phenyl Sepharose and S-modulin at high Ca2+ concentrations. The yield was greater than 90%. 45Ca(2+)-binding experiment showed that S-modulin is a Ca(2+)-binding protein. At high Ca2+ concentrations, S-modulin binds to ROS membranes. The binding target of the Ca2+/S-modulin complex is possibly a ROS membrane lipid(s), but it was difficult to identify. The binding was observed mainly at greater than 1 microM Ca2+. The amino acid sequence deduced from proteolytic fragments of S-modulin was approximately 80% and 60% identical to those of recovering and visinin, respectively.

Recoverin Improves Rod-Mediated Vision by Enhancing Signal Transmission in the Mouse Retina

Vision in dim light requires that photons absorbed by rod photoreceptors evoke signals that reliably propagate through the retina. We investigated how a perturbation in rod physiology affects propagation of those signals in the retina and ultimately visual sensitivity. Recoverin is a protein in rods that prolongs phototransduction and enhances visual sensitivity. It is not present in neurons postsynaptic to rods, yet we found that light-evoked responses of rod bipolar and ganglion cells were shortened when measured in recoverin-deficient retinas. Unexpectedly, the effect of recoverin on postsynaptic signals could not be explained by its effect on phototransduction. Instead, it is an effect of recoverin downstream of phototransduction in rods that prolongs signal transmission and enhances visual sensitivity. An important implication of our findings is that the recovery phase of the rod photoresponse does not contribute significantly to visual sensitivity near absolute threshold.

Averageness or symmetry: Which is more important for facial attractiveness?

Effects of averageness and symmetry on the judgment of facial attractiveness were investigated using a generalized Procrustes method and multiple regression analyses. Participants (n=114) rated attractiveness of 96 photographs of faces with neutral expressions. Through a generalized Procrustes method, the faces and their mirror-reversed versions were represented as points on a hyperplane. Both averageness and symmetry of each individual were defined as distances on the plane. A multiple regression analysis was performed to examine the effect of symmetry and averageness for each gender. For male faces, both symmetry and averageness affected attractiveness ratings positively , and there was no difference between the effects of averageness and symmetry. On the other hand, for female faces only averageness affected attractiveness, whereas symmetry did not. However, these effects were not large.

An essential role of Rab5 in uniformity of synaptic vesicle size

Rab5 small GTPase is a famous regulator of endocytic vesicular transport from plasma membrane to early endosomes. In neurons, Rab5 is found not only on endocytic vesicles in cell bodies but also on synaptic vesicles in nerve terminals. However, the function of Rab5 on synaptic vesicles remains unclear. Here, we elucidate the function of Rab5 on synaptic vesicles with in vivo and in vitro experiments using Drosophila photoreceptor cells. Functional inhibition of Rab5 with Rab5N142I, a dominant negative version of Drosophila Rab5, induced enlargement of synaptic vesicles. This enlargement was, however, suppressed by enhancing synaptic vesicle recycling under light illumination. In addition, synaptic vesicles prepared from Rab5N142I-expressing flies exhibited homotypic fusion in vitro. These results indicate that Rab5 functions to keep the size of synaptic vesicles uniform by preventing their homotypic fusion. By contrast, Rab5 was not involved in the endocytic reformation of synaptic vesicles, contrary to expectation from its conventional function. Furthermore, we electrophysiologically and behaviourally showed that the function of Rab5 is essential for efficient signal transmission across synapses.

Photoreceptor light-adaptation mediated by S-modulin, a member of a possible regulatory protein family of protein phosphorylation in signal transduction

In vertebrate retinal photoreceptors, cytoplasmic [Ca2+] decreases upon exposure to light. A Ca(2+)-binding protein, S-modulin, detects this [Ca2+] decrease and reduces the light-sensitivity of the cell to induce light-adaptation. The reduction of the sensitivity is attained by disinhibition or facilitation of rhodopsin phosphorylation, a quenching mechanism of light-activated rhodopsin. S-modulin-like proteins are found in the brain as well. Several of these proteins show similar S-modulin effects, suggesting that these proteins also participate in the regulation of protein phosphorylation in the signal transduction in their host cells.

Involvement of ATP in activation and inactivation sequence of phosphodiesterase in frog rod outer segments

Cyclic GMP phosphodiesterase in frog rod outer segments is activated after flash illumination and is inactivated when left in the dark. ATP reduces the initial peak activity caused by dim flashes (with 50 microM ATP being required for a half-maximal effect) and also accelerates inactivation (with 2 microM ATP being required for a half-maximal effect). An acceleration of inactivation caused by ATP addition is 3-7-fold, depending on the preparation, and ATP effect can be observed even 1 min after a dim flash is given. The accelerated inactivation is also flash intensity-dependent. A low intensity of light causes more rapid inactivation than does a high intensity of light. ATP appears to control phosphodiesterase activity in various ways.

LIGHT‐SENSITIVITY MODULATING PROTEIN IN FROG RODS

Abstract— Cyclic GMP is the second messenger in the phototransduction mechanism in rod photo‐receptors. Light‐induced activation of cGMP phosphodiesterase (PDE), the hydrolyzing enzyme of cGMP, reduces cytoplasmic cGMP concentration to close the cGMP‐activated channel and thereby causes a hyperpolarizing light response. Ca2+ concentration decreases during light‐adaptation and this decrease is thought to be at least one of the underlying mechanisms of light‐adaptation. Our previous electrophysiological work suggested that PDE in frog rod photoreceptors is regulated by this Ca2+ concentration decrease. In the present work, we isolated a protein that binds to disk membranes at high Ca2+ concentrations. In the presence of this protein (a 26 kDa protein), PDE light sensitivity becomes high at high Ca2+ concentrations. The effect was observed at physiological ranges of Ca2+ concentrations. Thus we could explain high light‐sensitivity of photoreceptors under the dark‐adapted condition. According to its function, we termed the 26 kDa protein ‘sensitivity‐modulating protein’ or ‘S‐modulin’. During the purification we noticed that there are additional mechanisms present that may contribute to light‐adaptation in frog rod photoreceptors.

High cGMP synthetic activity in carp cones

Cones show briefer light responses than rods and do not saturate even under very bright light. Using purified rod and cone homogenates, we measured the activity of guanylate cyclase (GC), an enzyme responsible for cGMP synthesis and therefore recovery of a light response. The basal GC activity was 36 times higher in cones than in rods: It was mainly caused by higher expression levels of GC in cones (GC-C) than in rods (GC-R). With identification and quantification of GC-activating protein (GCAP) subtypes expressed in rods and cones together with determination of kinetic parameters of GC activation in the presence and absence of GCAP, we estimated the in situ GC activity in rods and cones at low and high Ca2+ concentrations. It was revealed that the GC activity would be >10 times higher in cones than in rods in both the dark-adapted and the light-adapted states. Electrophysiological estimation of the GC activity measured in the truncated preparations of rod and cone outer segments gave consistent results. Our estimation of the in situ GC activity reasonably explained the rapid recovery and nonsaturating behavior of cone light responses.