Enzymatic And Regulatory Properties Of The Native Isozymes Of Retinal Guanylyl Cyclase, RetGC1 And RetGC2

Abstract

Purpose: : Retinal guanylyl cyclase (RetGC) in vertebrate photoreceptors is present as two isozymes, RetGC1 and RetGC2. Both RetGC1 and RetGC2 accelerate rod recovery from excitation when stimulated by Ca²⁺/Mg²⁺ -binding guanylyl cyclase activating proteins (GCAPs). However, the biochemical properties and the relative contribution of these isozymes to the overall cGMP synthesis in rod outer segments (ROS) are poorly understood. This study represents the first biochemical characterization of the native RetGC1 and RetGC2 isozymes in mouse ROS membranes.

Methods: : Gucy2F or Gucy2E gene knockout mice were bred into GCAPs1,2^-/- double knockout background in order to functionally isolate RetGC1 or RetGC2, respectively. Mouse ROS were then purified by density gradient centrifugation and assayed for their GCAPs- and Ca²⁺-sensitive cyclase regulation.

Results: : Basal RetGC activity measured at low free Ca²⁺ and physiological free Mg²⁺ in ROS isolated from GCAPs1,2^-/- mice was <3 nmol cGMP/min/mg rhodopsin, and GCAP1 and GCAP2 added at saturating concentrations increased the overall RetGC activity ~25 and 11-fold, respectively. Native RetGC1 was stimulated by GCAP1 and GCAP2 ~23 and 12-fold, whereas RetGC2 was stimulated at least 8 and 6-fold, respectively. Under the same conditions, the maximal activity of RetGC1 was ~ 5-times greater than RetGC2 activity. The native RetGC isozymes displayed similar apparent affinity for GCAP2 (1~2 µM), and RetGC1 displayed slightly higher (~1 µM) than RetGC2 (~4 µM) affinity for GCAP1. Calcium inhibited activation of each RetGC isozyme by GCAP1 and GCAP2 at EC₅₀~140 and 60 nM [Ca]_free, respectively, consistently with the GCAPs properties determined in vitro and in vivo. There was no detectable Ca²⁺-sensitive cyclase activity in the retinas lacking both RetGC1 and RetGC2.

Conclusions: : Both GCAP1 and GCAP2 can efficiently stimulate native RetGC1 and RetGC2 isozymes in vitro. The affinity of GCAP1 for RetGC1 is slightly higher than for RetGC2, but both GCAPs stimulate RetGC1 and RetGC2 within the estimated range of the free GCAP concentrations in ROS. Ca²⁺- sensitivity of RetGC is determined by GCAP affinity for Ca²⁺, and does not depend on the particular cyclase isozyme. RetGC1 contributes substantially more than RetGC2 to the total cGMP synthesis in rods, therefore, the reason why rods lacking RetGC1 are known to quickly recover from excitation remains to be determined.

Keywords: photoreceptors • signal transduction • enzymes/enzyme inhibitors