Highly effective phosphorylation by G protein-coupled receptor kinase 7 of light-activated visual pigment in cones

Abstract

Cone photoreceptors show briefer photoresponses than rod photoreceptors. Our previous study showed that visual pigment phosphorylation, a quenching mechanism of light-activated visual pigment, is much more rapid in cones than in rods. Here, we measured the early time course of this rapid phosphorylation with good time resolution and directly compared it with the photoresponse time course in cones. At the time of photoresponse recovery, almost two phosphates were incorporated into a bleached cone pigment molecule, which indicated that the visual pigment phosphorylation coincides with the photoresponse recovery. The rapid phosphorylation in cones is attributed to very high activity of visual pigment kinase [G protein-coupled receptor kinase (GRK) 7] in cones. Because of this high activity, cone pigment is readily phosphorylated at very high bleach levels, which probably explains why cone photoresponses recover quickly even after a very bright light and do not saturate under intense background light. The high GRK7 activity is brought about by high content of a highly potent enzyme. The expression level of GRK7 was 10 times higher than that of rod kinase (GRK1), and the specific activity of a single GRK7 molecule was Ϸ10 times higher than that of GRK1. The specific activity of GRK7 is the highest among the GRKs so far known. Our result seems to explain the response characteristics of cone photoreceptors in many aspects, including the nonsaturation of the cone responses during daylight vision.rod ͉ photoreceptors ͉ retina ͉ phototransduction O ur visual system consists of two components: rods and cones (1, 2). These photoreceptors differ in their light sensitivity so that rods mediate twilight vision, and cones mediate daylight vision. Rods and cones are distinguished not only in their light sensitivity, but also in other response characteristics. The photoresponse time course is much briefer in cones, which improves the time resolution of our daylight vision greatly. Rods are saturated with bright background light and do not respond to more intense light (3). In contrast, cones are not saturated and respond to very bright light (4, 5). The molecular mechanisms underlying in the differences of these response characteristics are not yet known. In previous biochemical studies on the cone phototransduction mechanism, only slight quantitative differences were known in the transduction components between rods and cones (6-8).In our previous study, we obtained a large quantity of isolated cones (enough to perform biochemistry) and showed that transducin activation and cGMP phosphodiesterase activation, the reactions involved in the generation of a photoresponse, are less efficient in cones (9). These findings reasonably explained the lower light sensitivity in cones. Another remarkable difference was found in the phosphorylation of light-activated visual pigment. When light-activated, visual pigment is phosphorylated by a class of kinase known as rhodopsin kinase (rod kinase or G protein-coupled receptor kinase (GRK) 1) in...

Dephosphorylation during Bleach and Regeneration of Visual Pigment in Carp Rod and Cone Membranes

“…It took ϳ2.5 h to prepare Opsin-P and VP-P in cone membranes, and the phosphorylation levels were similar in Opsin-P and VP-P: 2.5 Ϯ 0.1 phosphates were present per Opsin-P (n ϭ 3) and 2.5 Ϯ 0.1 phosphates per VP-P (n ϭ 12). Because the maximum phosphorylation level was three phosphates per R* in native cone membranes (17), most of the phosphates were retained in Opsin-P and VP-P also in cone membranes.…”

“…It took ϳ3.5 h to prepare Opsin-P and VP-P in rod membranes, and the phosphorylation levels were similar in these preparations at the beginning of the measurement of phosphorylation levels or of dephosphorylation: 2.8 Ϯ 0.2 phosphates were present per Opsin-P (n ϭ 3) and 2.2 Ϯ 0.1 phosphates per VP-P (n ϭ 12). The maximum level of the phosphorylation in our native rod membranes was three per R* (17), so that most of the phosphates incorporated into the pigment were retained in Opsin-P and VP-P in rod membranes.…”

“…and Cones-Carp (Cyprinus carpio) rods and cones were purified as described previously (17). Animal care was conducted according to the institutional guidelines (approval number FBS-14-005).…”

RDH13L, an Enzyme Responsible for the Aldehyde-Alcohol Redox Coupling Reaction (AL-OL Coupling Reaction) to Supply 11-cis Retinal in the Carp Cone Retinoid Cycle

“…Preparation of Cone Membranes-Cones from carp (Cyprinus carpio, ϳ30 cm in length) were purified as described (18). Experimental animals were cared for in accordance with our institutional guidelines (approval number: FBS-14-005).…”

Phototransduction in mouse rods and cones

“…In all other species studied, another pigment kinase (the so-called "cone pigment kinase"), GRK7, is present in cone photoreceptors [119][120][121][122]. Indeed, many animal species, including human, have both GRK1 and GRK7 in cones.…”