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

• Published in: Scientific reports Vol. 9; no. 1; p. 1183
• Main Authors: Turunen, Teemu T., Koskelainen, Ari
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 04.02.2019; Nature Publishing Group
• Subjects: 13/95; 631/378/2613/1786; 631/378/340; 64/60; 9/30; Animal models; Cyclic GMP; Cytoplasm; Guanylate cyclase; Humanities and Social Sciences; Mammals; multidisciplinary; Phosphodiesterase; Photoreceptors; Photoresponse; Recoverin; Rodents; Science; Science (multidisciplinary)
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-018-37661-w

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 (β dark ) 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 β dark using local electroretinography (LERG) from isolated mouse retinas. The method is based on the ability of PDE inhibitors to decrease β dark , which can be counterbalanced by increasing PDE activity with light. This procedure clamps cytoplasmic cGMP to its dark value. β dark 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 β dark 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 β dark .