Regulation of Photoreceptor Signal Termination

Abstract

Abstract: : Purpose:. The gamma subunit of rod phosphodiesterase (PDE6γ) inhibits the catalytic α and ß subunits in darkness; binding of activated transducin alpha (Tα–GTP) removes this inhibition, stimulating PDE activity. PDE6γ has also been shown to act as a GTPase Accelerating Protein (GAP) for Tα by potentiating the action of another GAP, RGS9. Previous experiments have suggested that excess PDE6γ could turn off transduction and modulate the decay of the light response. We have tested this idea by over–expressing wild–type PDE6γ in a transgenic mouse. Methods: The murine opsin promoter was used to over–express the wild–type PDEγ transgene. These constructs were injected into normal oocytes. The founders were then crossed with Pde6g^tm1/Pde6g^tm1mice that lack the γ–subunit of PDE6 to generate mice that could only synthesize transgenic PDE6γ. Electrical responses were recorded conventionally with suction electrodes and analyzed as previously described. Results:While Pde6g^tm1/Pde6g^tm1mice mice have severe photoreceptor degeneration as in human retinitis pigmentosa, over–expression of the wild type transgene in the knockout Pde6g^tm1/Pde6g^tm1mice background prevents this degeneration. Rods with over expressed PDE6γ were desensitized ∼4–fold compared to wild–type controls; the intensity of light (20ms flashes, 500 nm) needed for 50% of saturation was about 6 photons µm^–2 for controls, but about 25 photons µm^–2 for PDE6γ over–expressers. Single photon analysis revealed that the light response wave form of PDE6γ over–expressers was similar to wild–type during the rising phase of the response, but recovery of current following the flash occurred more rapidly. The single photon time–to–peak for controls was about 200 ms, with a full return of the current to the dark level at about 1 s; whereas the PDE6γ over expressers had a time–to–peak of 150 ms, and a return of current to the dark level at about 0.5 s. Conclusions:Excess PDE6γ accelerates the falling phase of the photoresponse. It is possible that PDE6γ normally attached to the PDE complex dissociates from PDE6 α and ß far enough to allow excess PDE6γ to penetrate to the inhibitory binding site. Acceleration of the turn–off of PDE then speeds the waveform of decay of the light response. It remains unclear whether excess PDE6γ does this entirely by itself or by means of some interaction with RGS9. Our results may provide new evidence that will help us separate the roles of the various components responsible for turn–off and may also indicate a role for free PDE6γ in normal phototransduction.

Keywords: transgenics/knock–outs • photoreceptors • genetics