Physiological Properties of Rod Photoreceptor Cells in Green-sensitive Cone Pigment Knock-in Mice

Abstract

Rod and cone photoreceptor cells that are responsible for scotopic and photopic vision, respectively, exhibit photoresponses different from each other and contain similar phototransduction proteins with distinctive molecular properties. To investigate the contribution of the different molecular properties of visual pigments to the responses of the photoreceptor cells, we have generated knock-in mice in which rod visual pigment (rhodopsin) was replaced with mouse green-sensitive cone visual pigment (mouse green). The mouse green was successfully transported to the rod outer segments, though the expression of mouse green in homozygous retina was ∼11% of rhodopsin in wild-type retina. Single-cell recordings of wild-type and homozygous rods suggested that the flash sensitivity and the single-photon responses from mouse green were three to fourfold lower than those from rhodopsin after correction for the differences in cell volume and levels of several signal transduction proteins. Subsequent measurements using heterozygous rods expressing both mouse green and rhodopsin E122Q mutant, where these pigments in the same rod cells can be selectively irradiated due to their distinctive absorption maxima, clearly showed that the photoresponse of mouse green was threefold lower than that of rhodopsin. Noise analysis indicated that the rate of thermal activations of mouse green was 1.7 × 10−7 s−1, about 860-fold higher than that of rhodopsin. The increase in thermal activation of mouse green relative to that of rhodopsin results in only 4% reduction of rod photosensitivity for bright lights, but would instead be expected to severely affect the visual threshold under dim-light conditions. Therefore, the abilities of rhodopsin to generate a large single photon response and to retain high thermal stability in darkness are factors that have been necessary for the evolution of scotopic vision.

Rod Visual Pigment Optimizes Active State to Achieve Efficient G Protein Activation as Compared with Cone Visual Pigments

“…At 37°C, V max /R* of mRh (38.8 G t */s) was about 2.5-fold greater than that of mG (15.4 G t */s). This difference in G t activation efficiency is consistent with our previous electrophysiological study (7), which showed that the amplification efficiency of mRh was about 3-fold greater than that of mG. Therefore, it is likely that the difference in G t activation efficiency between rhodopsin and cone visual pigments was the main contributor to the difference in the amplification efficiency of the single photon response in that previous study.…”

“…It has been reported previously that the G t activation efficiencies of rhodopsin and cone visual pigments were similar to each other based on experiments performed at about 0°C (7,9,10), although those of rhodopsin and cone visual pigments of a poikilothermic animal (carp) were recently reported to be different at 20°C (11,12). Thus, it is likely that the difference in amplification efficiency between wild-type rods and rods con-taining mG is caused by temperature-dependent intrinsic properties of visual pigments such as the thermal equilibrium between active state Meta-II and its precursor Meta-I and/or the lifetime of Meta-II that may compete with the deactivation process consisting of phosphorylation by rhodopsin kinase (7).…”

“…Electrophysiological assays showed that rods containing mG have amplification efficiency about one-third that of wild-type rods. Because the amplification efficiency of the transduction cascade per bleached pigment in mouse rods was about 6.8-fold higher than that in mouse cones (8), the electrophysiological assay results implied that about half of the difference in amplification efficiency between rods and cones is derived from the difference of visual pigments (7,8).…”

“…Thus, if a visual pigment efficiently absorbs a photon and efficiently activates G protein, the photosensitivity of the photoreceptor cell is high. To elucidate the contribution of rod and cone visual pigments to the photoresponse of photoreceptor cells, we have produced knock-in mice whose rods express mouse green-sensitive cone visual pigment (mG) 2 instead of rhodopsin (7). Electrophysiological assays showed that rods containing mG have amplification efficiency about one-third that of wild-type rods.…”

“…Thus, it is likely that the difference in amplification efficiency between wild-type rods and rods con-taining mG is caused by temperature-dependent intrinsic properties of visual pigments such as the thermal equilibrium between active state Meta-II and its precursor Meta-I and/or the lifetime of Meta-II that may compete with the deactivation process consisting of phosphorylation by rhodopsin kinase (7). Regarding the latter possibility, Shi et al (13) reported that the response profile of mouse rods containing mouse UV cone pigment differed depending on whether rod arrestin was present or absent, although the effect of arrestin was not very strong.…”