Determination of basal phosphodiesterase activity in mouse rod photoreceptors with cGMP clamp

Teemu T Turunen,Ari Koskelainen

doi:10.1038/s41598-018-37661-w

Teemu T Turunen, Ari Koskelainen

Open Access

https://doi.org/10.1038/s41598-018-37661-w

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Journal: Scientific Reports	Publication Date: Feb 4, 2019
Citations: 3	License type: open-access

Affiliation: Aalto University

Abstract

Light regulates cGMP concentration in the photoreceptor cytoplasm by activating phosphodiesterase (PDE) molecules through a G-protein signalling cascade. Spontaneous PDE activity is present in rod outer segments even in darkness. This basal PDE activity (βdark) has not been determined in wild type mammalian photoreceptor cells although it plays a key role in setting the sensitivity and recovery kinetics of rod responses. We present a novel method for determination of βdark using local electroretinography (LERG) from isolated mouse retinas. The method is based on the ability of PDE inhibitors to decrease βdark, which can be counterbalanced by increasing PDE activity with light. This procedure clamps cytoplasmic cGMP to its dark value. βdark can be calculated based on the amount of light needed for the “cGMP clamp” and information extracted from the registered rod photoresponses. Here we apply this method to determine βdark values for the first time in the mammalian rods and obtain the following estimates for different mouse models: 3.9 s−1 for wild type, 4.5 s−1 for guanylate cyclase activating proteins (GCAPs) knockout, and 4.4 s−1 for GCAPs and recoverin double knockout mice. Our results suggest that depletion of GCAPs or recoverin do not affect βdark.

Highlights

Photoreceptor cells convert light information to sensory signals in a process called phototransduction
In addition to wild type (WT) mice, the performance of the method was investigated with guanylate cyclase activating proteins (GCAPs)−/− and GCAPs−/− recoverin−/− double knock out mice (DKO)
In the cyclic guanosine monophosphate (cGMP) clamp procedure, a phosphodiesterase-6 molecules (PDE) inhibitor is introduced to the retina while monitoring changes in the extracellular voltage with local ERG recording across the outer segment layer (LERG-OS) or across the whole photoreceptors (LERG-PR)

Summary

Introduction

Photoreceptor cells convert light information to sensory signals in a process called phototransduction. The amount of active PDE in darkness determines the rate constant for spontaneous cGMP hydrolysis, i.e. the basal PDE activity (βdark), which sets the steady state level and the turnover rate of cGMP It is one of the main factors in setting the kinetics of photoresponse deactivation and spatial propagation of cGMP concentration drop during photoresponses[5]. Gross et al (2012) demonstrated that when the calcium mediated feedback to guanylate cyclase is abolished by knocking out the guanylate cyclase activating proteins (GCAPs) and the lifetime of activated PDE is decreased by overexpressing RGS9, the basal PDE activity becomes the dominant factor determining the light response deactivation kinetics[5] In these circumstances, the late recovery of a single-photon response allows the determination of βdark. The utilization of the new method is not limited to mice but it is applicable in quantitative determination of steady state phosphodiesterase activity regardless of the species or the genetic background of the model animal

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Conclusion