Determination of basal phosphodiesterase activity in mouse rod photoreceptors with cGMP clamp
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...
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Published in | Scientific reports Vol. 9; no. 1; p. 1183 |
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Main Authors | , |
Format | Journal Article |
Language | English |
Published |
London
Nature Publishing Group UK
04.02.2019
Nature Publishing Group |
Subjects | |
Online Access | Get full text |
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Summary: | 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
. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 2045-2322 2045-2322 |
DOI: | 10.1038/s41598-018-37661-w |