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 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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