Investigating the Function of N74 in Phosphodiesterase 6 Subunit Gamma (PDE6)

Abstract

Purpose: : Phosphodiesterase 6 subunit gamma (PDE6γ) is a major component of the photoresponse and is required for rod integrity. In particular, PDE6γ is needed to for normal PDE6 activity. It has been previously shown in vitro, that by mutating Asparagine 74 (N74) in PDE6γ subunit, the regulatory control of catalytic core of PDE6, subunits α and β, is reduced. However, the function of this amino acid in PDE6γ is not known in vivo.

Methods: : To test this hypothesis, the murine opsin promoter was used to direct expression of wild-type PDE6γ (control) and N74A transgenes and the constructs were inject into normal oocytes. Founder mice were crossed with PDE6γ^tm1/PDE6γ^tm1, which lack the γ-subunit of c-GMP-PDE6, to generate mice only synthesizing either wild-type of N74A PDE6γ. Mice were kept on a 12 hour LD/DD cycle until 4-6 weeks of age. Mice were dark or light adapted and their retinae were harvested for Western blot analysis, Enzyme Immuno Assay (EIA) to measure cGMP-PDE activity, or PDE activity, measured indirectly via phosphate release via 5’ GMP through nucleotidease. Dark-adapted mice were used for photocurrent measurements of individual rods by suction electrode and electroretinograms (ERG).

Results: : The ERG, western and photocurrent measurements show no significant differences, when compared to control mice. EIA and PDE activity studies are ongoing.

Conclusions: : The mutation of N74 caused PDE6 activity to increase in comparison to the control in vitro. This suggests that this amino acid is necessary in regulating PDE6 activity. However, a phenotype consistent with a PDE6 defect was not detected in vivo, using ERG and photocurrent analysis. This suggests that PDE6γ interacts with other molecules or proteins in vivo to compensate for the N74A mutation.

Keywords: photoreceptors • protein structure/function • retina