Deletion of Guanylate Cyclase 1 (GC1/GC–E) and Guanylate Cyclase 2 (GC2/GC–F) Eliminates Photoreceptor Function

Abstract

Purpose: : To determine the consequence of deletion of the two guanylate cyclases (GC1 and GC2) present in mouse photoreceptors.

Methods: : Generation of GC2 knockout mice by targeted deletion; generation of GC1/GC2 double knockout mice; characterization of each phenotype by single– and double–flash ERGs, rod single–cell recording, immunocytochemistry, western blotting.

Results: : Restoration of dark cGMP levels in photoreceptors after light stimuli is mediated by activation of GCs by calcium–sensitive GC activating proteins, termed GCAPs. We show by single cell recording and electroretinography that deletion of both GC1 and GC2 (GC double knockout, or GCdko) rendered rod and cone photoreceptors nonfunctional. In single–cell recordings of GC1–/– rods, time–to–peak (t_p) in response to a dim flash was prolonged while in GC2–/– rods, recovery of the dark current (t_i) was delayed. In GCdko rods, GCAP1 and GCAP2 were nearly completely absent. In GC1–/– and GCdko cones, S– and M/L–cone pigments mislocalized in the outer segment layer. Moreover, cone transducin α subunit and cone PDE α’ subunit were undetectable in these mutant cones suggesting that outer segments did not form correctly and/or are devoid of phototransduction components.

Conclusions: : These results exclude additional cyclases from participating in photoreceptor recovery. Absence of PDEα’ and Tα in GC1–/– cones establish a role for GC1 in transport/stability of membrane–associated proteins to cone outer segments. Downregulation of GCAPs suggests that functional expression of GCs is essential for GCAP stability. Single–cell recording in mouse rods indicate that GC1 is responsible for early feedback to Ca²⁺ while GC2 may restore the final levels of cGMP.

Keywords: photoreceptors • signal transduction • degenerations/dystrophies