Determination of basal PDE activity in WT, GCAPs^-/- and GCAPs^-/- rec^-/- mice rods

Abstract

 

Purpose

 

Absorption of a photon activates rhodopsin (R^*) which starts a G-protein signaling cascade leading to an activation of phosphodiesterase (PDE^*) hydrolyzing cGMP. Due to thermal energy, there is always some PDE activity present even in darkness. The amount of PDE^* in darkness determines the rate of spontaneous cGMP hydrolysis, i.e. the basal PDE activity (β_dark), which is one of the main factors setting the kinetics of photoresponse recovery. Here we introduce and test a novel approach to determine β_dark.

 

Methods

 

PDE inhibitors decrease the catalytic activity of PDE. A decrease in the basal PDE activity can be compensated by increasing [PDE^*] with light. We assume that the amount of light-induced PDE activity needed to keep the [cGMP]_in, and thus the photoreceptor signal, constant after introducing PDE inhibitor to the retina matches with the decrease in β_dark. This was accomplished with a PID controlled background light feedback. β_dark was calculated based on the activation factor of the cascade (determined), the lifetimes of R^* (from literature) and PDE^* (determined), the inhibition constant of the inhibitor (determined) and the amount of light needed for the compensation. Local ERG was used to determine β_dark from isolated WT, GCAPs^-/- and GCAPs^-/- rec^-/- mouse retinas in HEPES buffered solution. The PDE inhibitor IBMX was used at concentrations of 10, 20 and 40 µM.

 

Results

 

The β_dark values obtained from a linear fit to data (value ± SER) were 1.7 ± 0.01 s^-1 for WT, 2.2 ± 0.2 s^-1 for GCAPs^-/- and 1.8 ± 0.1 s^-1 for GCAPs^-/- rec^-/- mice. The time constant of an exponential fit to the late recovery phase of dim flash responses of GCAPs^-/- believed to be set mainly by basal PDE activity was 440 ms, corresponding to β_dark of 2.3 s^-1.

 

Conclusions

 

β_dark was determined for the first time for WT mice and the β_dark values were comparable between the mouse strains used. β_dark obtained from light compensation (≈ 2 s^-1) and from dim flash recovery (2.3 s^-1) are somewhat smaller than previously obtained for GCAPs^-/- mice (4.1 s^-1, Gross et al., Biophys. J., vol. 102, 2012).  

 

Determination of β_dark (β_{light compensation} = β_dark[IBMX]/K_I) for WT, GCAPs^-/- and GCAPs^-/- rec^-/- mice. Error bars indicate SEMs.

 

 

Exponential fits to the late recovery of GCAP^-/- dim flash response (local ERG across outer segment layer, average from 6 retinas).