C. Characterization of differences in cone PDE6 regulation

The dramatic differences in light sensitivity, response kinetics, and background adaptation of rods and cones have not been explained at the molecular level. Cone outer segments contain the same phototransduction cascade as rod photoreceptors, but in most instances, cone-specific isoforms of phototransduction proteins have been identified (including opsins, transducin, PDE6, cGMP-gated ion channel subunits, arrestin, recoverin, etc (Ebrey and Koutalos, 2001). In collaboration with Dr. Karen Carleton (Hubbard Genome Center, UNH), we have begun a systematic evolutionary analysis of rod and cone phototransduction proteins to test the hypothesis that one or more phototransduction proteins exhibit structural and functional differences between the rod and cone isoforms. We recently completed an evolutionary trace analysis of rod and cone opsins that demonstrates no significant differences between the rod and cone opsins in their regions of interaction with transducin or other proteins. Using existing sequence databases, along with selective sequencing of phototransduction proteins from phylogenetically diverse vertebrates, we are extending this functional genomic analysis to other phototransduction proteins likely to differ functionally in rod and cone visual transduction.

Since PDE6 is the central regulatory enzyme of rod phototransduction, we hypothesized that cone PDE6 would differ in its catalytic or regulatory properties, and therefore underlie some of the physiological differences between rods and cones. Having developed improved methods for extracting and purifying cone PDE6 from the retina, we are testing the hypothesis that differences in binding of the cone inhibitory ? subunit or in the cGMP binding GAF domain results in a shortened activated lifetime for cone PDE6 relative to the rod isoform.

References

Ebrey,T. and Koutalos,Y. (2001). Vertebrate photoreceptors. Prog. Retin. Eye Res. 20, 49-94.