Start the Simulator. Since we have no Simulation Description File yet click "Cancel" to continue.

Click "set model file".

Select "G_proteins.dbf". Click "Open".

Click "set geometry file" and select "GprotCell.geo". Click "Open". Click "Accept".

Set a concentration of 50 molecules per square micron for "Gabg_GDP" and "Rec inactive unbound". Click OK.

Deselect "Enable cellular potts model". Click OK.

Save the Simulation Description as "G_proteins.sdc". Click "Save" to proceed with the Simulation.

In the main "Simulation Window" click "Simulate For X Seconds", enter 40 and click OK.

Important! Shift-click the cell to select it for inspection. In the "Time Trace Window" select Gabg_GDP to see the initial equilibration of free vs. complexed (in Gabg_GDP) G proteins.

Deselect "Display in Mol/l" to view molecule complex numbers.

Click "Slice View" -> "Slice View of Entire Region".

Click "Align Cut XZ" in the "Slice Selection Window". Using the Translation slider, move the cut plane until it cuts through the middle of the cell. Moving the mouse in the display region and zooming out (by pressing the 'x' key on the keyboard, move the view until you see the slice plane.

Select "Rec inactive unbound" and click "Update Display". Then Click "View Slice".

In the "Slice View Window" click "Add a Concentration Profile", select a Linear Gradient of "Ligand" and set the concentrations at Point 1 and 2.

Click "Set First Anchor Point" and then click at the position shown as a black point.

Similarly for the second anchor point. Click "Apply Profile".

In the "Slice Selection Window" select "Volume Concentration" and from the "Molecules" select "Ligand". Click "Update Display". Then Click "View Slice".

The "Slice View Window" now shows the Ligand gradient. Clicking on any extracellular point will provide information about the local concentration of the selected chemical species (here: Ligand).

In the main "Simulation Window" click "Simulate For X Seconds", enter 50 and click OK.

In the "Time Trace Window" select "Now showing: Patterns" and "Galpha_GTP_pattern" to see the kinetics of the production of GTP Galpha.

In the "Slice Selection Window" select "Surface Concentration" and from the "Complexes with Molecule" select "Ligand". Click "Update Display". The cellular gradient of ligated receptors is displayed.

Now, select "Surface Concentration" and from the "Complexes" list select "Gbetagamma_1" (The default name of the Gbetagamma complex that was automatically created when we defined the association of Gbetagamma with Galpha). Click "Update Display" to see how this receptor gradient is translated into free Gbetagamma.

Click "View Slice" and in the "Slice View Window" click "Draw New Straight Line Profile". Then draw a line along the adge of the cell (parallel to the gradient). Click OK.

A window opens that displays the concentration of Gbetagamma along the line. Note that the steep (almost 10:1) extracellular Ligand gradient has been translated into a relatively shallow intracellular gradient of free Gbetagamma. This is due to several factors, the most important among them diffusion of the G proteins in the membrane (interfering with the formation of a strong gradient within the membrane) and the saturation of receptors with Ligand that reduces the differences in receptor occupancy between front and back of the cell. We will now explore the contribution of the second factor by switching to a stimulus with the same gradient but lower absolute concentration.

Set a zero concentration field for the Ligand - don't forget to click "Apply Profile".

In the "Slice Selection Window" select "Volume Concentration" and from the "Molecules" select "Ligand". Click "Update Display". Then Click "View Slice". The "Slice View Window" now shows zero concentration for the Ligand. Click on an extracellular volume element to see its concentration value for the Ligand. Note that the volume elements containing membrane will display non-zero concentrations and that their values will depend on how much membrane area they contain (since we are querying per volume element): corner elements will have a high concentration relative to side elements.

Simulate for 80 seconds. The simulation will enforce a zero extracellular concentration thereby inducing a dissociation of the Ligand from the receptor without rebinding (and thus determined entirely by the dissociation rate). In the Time Trace Window" select "Gabg_GDP" to see the recovery of heterotrimeric G proteins to pre-stimulus level.

Apply a gradient with 10-fold lower absolute concentration of Ligand.

Simulate for 40 seconds. The Time Trace Window" shows the dissociation of the heterotrimeric G proteins

In the "Slice Selection Window" select "Surface Concentration" and from the "Molecules" select "Ligand". Click "Update Display". The cellular gradient of ligated receptors is displayed.

Now, select "Surface Concentration" and from the "Complexes" list select "Gbetagamma_1". Click "Update Display" and "View Slice" to see how this receptor gradient is translated into free Gbetagamma.

This time, the intracellular gradient of free Gbetagamma is considerably steeper, reflecting the fact that the absolute (average) concentration of Ligand is in a region of the Ligand-Receptor binding curve that is more sensible to Ligand concentration differences.