Different knockout models

To further assess the suitability of the model for simulating single photon responses, I simulated single photon responses in different knockout models. These results can be compared to various experimentally recorded single photon responses, which are shown in figure 4.6 on the left.

Before doing any of the simulations, I improved the model by introducing re-coverin, using a quasi-steady state assumption that the free, the calcium-bound and the rhodopsin kinase-calcium-bound states of recoverin are in their respective steady-state dark concentrations throughout the entire simulation. As previously discussed, this simplification is only valid for brief flashes of light without any sustained background - this is the case for the single photon responses simulated here.

Next, I created models for the different knockout conditions. As explained earlier, we cannot use the experiment description with IQMmergemodexpwith the stochas-tic simulations and have to create separate models. To create the knockout models, I only removed the knocked-out molecular species, and none of the kinetic param-eters of the model were changed for the simulations. For the implementation of the GCAPs knockout, see section3.1.1: the activity of the GC is fixed at its dark activity.

I implemented models with a knockout of the rhodopsin kinase, a knockout of the GCAPs, a knockout of arrestin, and a completely substituted mutant of rhodopsin (CSM) where all the phosphorylation sites are substituted, i.e. knocked out. The results of a first deterministic investigation can be found in figure 4.6.

0 0.2 0.4 0.6 0.8 1 1.2

t/s 0

0.5 1 1.5 2 2.5

J/pA

Old model

WT Arr GCAPs -/-CSM RK

-/-Figure 4.6: Single photon responses in different knockout models. Left: experimen-tal data from mouse rods. Arrestin knockout (Arr -/-, red) from (Xu et al.,1997), completely substituted mutant (CSM, green) from (Mendez et al.,2000), GCAPs knockout (GCAPs -/-, yellow) from (Burns et al.,2002), rhodopsin kinase knock-out (RK -/-, blue) from (Chen et al., 1999). The respective wild type responses are plotted in matching colors with a thinner line, and all responses have been normalized to a maximum wild type amplitude of 1 pA. The arrestin knockout response has been scaled to the same amplitude as the wild type response. Right:

deterministically simulated single photon responses in models with the respective knockouts: arrestin (Arr -/-, red), GCAPs (GCAPs -/-, yellow), rhodopsin kinase (RK -/-, blue) and the completely substituted mutant (CSM, green) as well as the wild type (WT, black).

We can see that the model is generally able to reproduce the changed kinetics of the single photon response in the different knockout conditions.

In the GCAPs knockout (yellow line in the left and right panel), the amplitude of the single photon response is increased about four-fold and the shut-off is much slower. This is reproduced well in the simulated responses. The reason for the changed kinetics is that the GC is not regulated in a calcium-dependent manner anymore when the GCAPs are missing. As a consequence of the light response, the cGMP and calcium concentrations drop. In the wild type, the GCAPs then activate the GC to produce more cGMP than the dark rate because of the drop in calcium concentration. This restores the cGMP concentration, which opens the

cyclic nucleotide gated channels, in turn restoring the calcium concentration.

In the knockout, this is not the case: the GC stays at its dark production rate of cGMP. The upstream part of the signalling cascade (rhodopsin, transducin, PDE) is normally shut off, but the low GC activity in the absence of the GCAPs can-not counterbalance the hydrolytic rate of cGMP. Thus, ∆J reaches a much higher amplitude and takes a longer time to return to the dark state.

Knocking out the rhodopsin kinase (blue lines) and removing all of rhodopsin’s phosphorylation sites (green lines) has the same effect on the light response. The consequence of the modifications is the same: rhodopsin can no longer be phos-phorylated. In the experimental and simulated data, we observe that the light response rises to about the double amplitude of the wild type single photon re-sponse and then stays at this amplitude, with no shut-off taking place. This is because rhodopsin does not get phosphorylated and the activation of the photo-transduction cascade is thus not terminated. The phosphorylation of rhodopsin is an essential first step of the shut-off: not only does it decrease rhodopsin’s activ-ity by reducing the affinactiv-ity for transducin, but it also enables binding to arrestin, which completely terminated rhodopsin’s activity.

When no phosphorylation can take place, because the rhodopsin kinase is missing or because rhodopsin has no phosphorylation sites, rhodopsin cannot be shut off and it cannot bind to arrestin. The only possibility for the shut-off is the thermal decay of activated rhodopsin, which has a low rate and thus takes a long time to spontaneously occur. Thus, the light response increases to a higher amplitude and stays there, because the activity of rhodopsin is not terminated.

Finally, the arrestin knockout (red lines) also impacts the shut-off of the response.

Unfortunately, the experimental data from (Xu et al., 1997) only extend for 0.5 s.

Still, we can see that the response amplitude decreases after the maximum, but less strongly than in the wild type. In the simulations, this looks slightly different.

We cannot compare the response amplitudes, since the experimental trace for the knockout is normalized to the same amplitude as the wild type response. However, the shut-off in the simulated data is weaker than in the experimental data: the decrease in amplitude after the maximum is slower.

In summary, the model reproduces the experimental data well, except for the shut-off for the arrestin knockout. This is further discussed in chapter 5.