Phototransduction and Signaling in Photoreceptors

PROJECT SUMMARY There have been great advances over the past 40 years in understanding how light initiates vision in rod and cone photoreceptors of the eye. Not only is the sequence of events in this process known, but the genes coding for the key phototransduction proteins have also been cloned. Mutations of many of these genes are found to be associated with various vision-impairing diseases. Phototransduction starts from the visual pigment. A little over ten years ago, we made major advances by showing that the dark spontaneous activity of rod and cone pigments indeed comes from canonical isomerization of the pigment albeit being driven by thermal instead of light energy. We developed a physicochemical theory that is able to predict quantitatively the 107-fold range in thermal activity across canonical (i.e., native) rod and cone pigments in the visible spectrum as well as that of a man-made, hybrid pigment with both rod-pigment-like and cone-pigment-like properties. Considering visual pigments’ fundamental importance in both basic and clinical science, we propose in Aim 1 to understand native pigments further. In particular, we would like to examine the effect of protonation (found in the great majority of L, M and S- pigments, which detect visible light) versus non-protonation (found in mouse S(UV)-sensitive pigment and some others) at the Schiff-base linkage between apo-opsin and 11-cis-retinal on the spontaneous isomerization of a pigment. There is suggestion from chemistry by others that unprotonated pigments are much noisier in darkness. Additionally, an unusual property of S-pigments is that they belong to two distinct evolutionary sub-groups: SWS1 (minor subgroup) and SWS2 (major subgroup). In preliminary experiments during this grant period, we have found some evidence to suggest that SWS1 pigments are much noisier in darkness. We propose in Aim 2 to address this question closely and quantitatively by examining several pigments belonging to one or the other subgroup. Finally, unlike rod pigments, cone pigments have some tendency to re-dissociate spontaneously in darkness into apo-opsin and 11-cis-retinal (i.e., without isomerization). Importantly, because apo-opsin has weak constitutive activity, it triggers transduction to produce electrical noise even in darkness. Most recently, we found surprisingly that, in fully dark-adapted goldfish L(red)-cones, the fractional apo-opsin content is as high as ~30%, although only ~3% in green (M) cones and negligible in blue (S) cones. In Aim 3, we would like to know whether mammalian cones when fully dark-adapted also have a significant amount of apo-opsin. We shall examine several species, including marmoset (a primate) and, if available, macaque. Techniques involved primarily suction- pipette recording and microspectrophotometry from native cones.