Under-expression of GAP proteins: Effect on Mouse Rod Response Decay

Abstract

 

Purpose

 

Our previous results (Chen et al., J. Neurosci, 32:15998-16006, 2012) indicate that rhodopsin kinase (GRK1) and background light accelerate rod response decay by modulating decay of phosphodiesterase (PDE). To test this hypothesis, we under-expressed mouse rod GAP proteins to slow decay of PDE, in order to increase the difference between the time constants of decay of PDE and light-excited rhodopsin (Rh*).

 

Methods

 

R9AP-/- rods were generously provided by Vadim Arshavsky. Suction-electrode recordings were made from single mouse rods as previously described (e.g. Chen et al., 2012).

 

Results

 

We mated R9AP-/- and RGS9-/- mice to obtain R9AP+/-; RGS9+/- animals, which we call GAPux. Quantitative Western blotting showed that expression of RGS9 and Gbeta5 was about 30% of wild-type (WT) levels. Rod responses of dark-adapted GAPux mice decayed more slowly than WT (see Figs. 1A and B): τ_REC increased from 197 ± 10 ms (SE, n=44) in WT to 281 ± 25 ms (8) in GAPux, and the limiting time constant τ_D grew from 173 ± 10 ms (15) in WT to 284 ± 23 ms (8) in GAPux (p<0.0001). These are increases of 60-70% which we attribute to decreased binding probability of GAP to TαGTP and PDEγ, resulting in slower TαGTP hydrolysis. When we over-expressed GRK1 by 12 times in GAPux mice (GAPux;RKS561L, see Fig. 1C), τ_REC decreased to 191 ± 28 ms and τ_D to 179 ± 17 ms (n=13), which are close to WT values and significantly different from GAPux (p<0.002). GAPux rod responses decline more quickly in background light, confirming our view that backgrounds modulate PDE decay.

 

Conclusions

 

The effect of GRK1 over-expression is absolutely greater (though proportionately nearly the same) for GAPux rods than for WT rods, even though GAP under-expression significantly increased the disparity between the time constants of PDE and Rh* decay. This result greatly increases the likelihood that GRK1 speeds rod decay by acting on the PDE rather than on rhodopsin, in opposition to the recently published results of Gross et al (Neuron 76: 370-382, 2012).

 

 

Figure 1. Mean responses of 11 WT, 8 GAPux, and 13 GAPux/RKS561L rods to 10 ms flashes of intensities from 2.4 to 2000 photons μm^-2.

 

Keywords: 648 photoreceptors • 646 phosphorylation • 689 retina: distal (photoreceptors, horizontal cells, bipolar cells)