Abstract
Purpose :
Retinal membrane guanylyl cyclase 1 (RetGC1) plays important role in rod and cone physiology. Two types of regulatory proteins control enzymatic activity of RetGC1: Ca2+-sensitive guanylyl cyclase activating proteins (GCAPs) and retinal degeneration 3 (RD3) protein, which inhibits RetGC1 activity. Recently, bicarbonate was proposed as a novel phototransduction-linked RetGC regulator [1,2 ], contradicting other studies on the same subject [3,4]. The goal of this study was to independently verify the effects of bicarbonate on the basal and GCAP-regulated RetGC activity
Methods :
The enzymatic activity of a recombinant RetGC1 expressed in HEK293 cells and the native RetGC in wild type and transgenic mouse retinas was assayed in the presence of sodium bicarbonate and GCAPs using a direct method monitoring conversion of 32P-GTP into 32P-cGMP by thin-layer chromatography
Results :
No measurable effect of bicarbonate on basal activity of recombinant RetGC1 was detected in the presence of 0-100 mM bicarbonate. While GCAP1 activated recombinant RetGC1 expressed in HEK293 cells >100-fold, there was no increase in GCAP1-stimulated RetGC1 activity in the presence of bicarbonate. We also assayed the effects of bicarbonate on the activity of the native guanylyl cyclase in the retinas present in double-gene knockout mouse retinas lacking GCAP1 and GCAP2 (GCAPs-/-) and of the RetGC1 isozyme present in RetGC2-/-GCAPs-/- triple-knockout retinas. Bicarbonate did not stimulate basal guanylyl cyclase activity, while GCAP1 and GCAP2 produced 18- and 11-fold stimulation, respectively. There was no additional stimulation of the cyclase by bicarbonate in the presence of GCAPs. Instead, there was a small decrease in GCAP-stimulated guanylyl cyclase activity in the presence of 50 mM bicarbonate, which could be attributed to a non-specific effect of high salt concentrations in the assay
Conclusions :
Our results, together with the earlier studies [3,4], do not confirm with the reports that bicarbonate can directly activate photoreceptor guanylyl cyclase [1,2]. Our data also argue that the effects of bicarbonate on rod physiology reported in [2] cannot be explained by its direct regulation of guanylyl cyclase activity.
References: [1] Duda et al. (2016) Front. Mol. Neurosci. 9:5; [2] Duda et al. (2015) J. Biol. Chem. 290, 11052-60; [3] Guo et al. (2009) Biochemistry 48, 4417-22; [4] Sun et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 2041-46
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.