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

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.

Functional modulation of phosphodiesterase-6 by calcium in mouse rod photoreceptors

“…A recording chamber used in the experiments allowed light stimulation and perfusion of the retina and had a clear passage for microelectrodes 19 . The retina was placed on a filter paper photoreceptor side upwards.…”

“…The inhibition constants against light-activated and basally activated PDE6, and the basal PDE6 activity with cGMP clamp were determined in normal Ca 2+ (n = 9 retinas) and in low Ca 2+ solution (n = 7 retinas) as described in 19 . The cGMP clamp experiments in normal and low Ca 2+ were conducted with separate retinas.…”

“…The phototransduction models used in this study and the theoretical background of cGMP clamp have been described in detail in 19 , while the methods for determining the phosphodiesterase inhibitor inhibition constants are published in 26 . Here we give a summary of the utilized phototransduction models and the core ideas behind the methods.…”

“… can be calculated based on certain phototransduction parameter values and on the amount of light needed to clamp the cGMP to its dark-value, expressed as rhodopsin isomerizations per rod per second (R*rod −1 s −1 ) 19 : where and are the average lifetimes of activated rhodopsin and PDE6, respectively, and is Hill’s coefficient for CNG channels. is the rod amplification constant that describes the molecular amplification of phototransduction, where denotes the rate constant by which the activation of rhodopsin leads to the activation of PDE6 subunits, and is the catalytic efficacy of a single light-activated PDE6 subunit.…”

“…We demonstrate that lowering of the extracellular calcium concentration reduces the light-sensitivity of rods by accelerating the photoresponse recovery in GCAPs −/− recoverin −/− background. Using the cGMP clamp paradigm 19 and the modeling of dim flash responses, we found that a drop in calcium concentration can cause ~ 30% increase in the basal PDE6 activity. In addition, we found that the decrease in the extracellular calcium concentration modulates the inhibition efficacy of the non-selective competitive inhibitor, 3-isobutyl-1-methylxanthine (IBMX), for PDE6, and surprisingly, this modulation shifts the inhibition constants against the light-activated and basally activated forms of PDE6 to opposite directions.…”