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. 2014 Sep 2;107(5):1042-1053.
doi: 10.1016/j.bpj.2014.05.050.

Explicit Spatiotemporal Simulation of receptor-G Protein Coupling in Rod Cell Disk Membranes

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Explicit Spatiotemporal Simulation of receptor-G Protein Coupling in Rod Cell Disk Membranes

Johannes Schöneberg et al. Biophys J. .
Free PMC article

Abstract

Dim-light vision is mediated by retinal rod cells. Rhodopsin (R), a G-protein-coupled receptor, switches to its active form (R(∗)) in response to absorbing a single photon and activates multiple copies of the G-protein transducin (G) that trigger further downstream reactions of the phototransduction cascade. The classical assumption is that R and G are uniformly distributed and freely diffusing on disk membranes. Recent experimental findings have challenged this view by showing specific R architectures, including RG precomplexes, nonuniform R density, specific R arrangements, and immobile fractions of R. Here, we derive a physical model that describes the first steps of the photoactivation cascade in spatiotemporal detail and single-molecule resolution. The model was implemented in the ReaDDy software for particle-based reaction-diffusion simulations. Detailed kinetic in vitro experiments are used to parametrize the reaction rates and diffusion constants of R and G. Particle diffusion and G activation are then studied under different conditions of R-R interaction. It is found that the classical free-diffusion model is consistent with the available kinetic data. The existence of precomplexes between inactive R and G is only consistent with the data if these precomplexes are weak, with much larger dissociation rates than suggested elsewhere. Microarchitectures of R, such as dimer racks, would effectively immobilize R but have little impact on the diffusivity of G and on the overall amplification of the cascade at the level of the G protein.

Figures

Figure 1
Figure 1
Reaction kinetics at two levels of modeling detail. (A) The first steps of the photoactivation cascade consist of the following activation reactions: M1/R equilibrium (1); G-activation reactions, comprising formation of RG complex, subsequent nucleotide exchange in G, and RG-complex dissociation (2); and dissociation of G from the membrane (3). A nonproductive RG-complex formation (4) is added for the precomplex case. These reactions are used for the ODE model of the cascade. (B) Using particle-based reaction-diffusion (ReaDDy), bimolecular association reactions have to be split into the explicitly simulated diffusional encounter and the first-order transition from encounter complex to stable complex. This affects the RG- and RG complex formation in (2.2) and (4.2) (red and blue). (C) Graphical representation of the microscopic diffusion and reaction components, illustrating space exclusions, molecular shape, and crowding. To see this figure in color, go online.
Figure 2
Figure 2
ODE fit and modeling. (A) ODE fits (solid lines) to representative dissociation signals (circles; measuring conditions and rate constants as in Tables 1, S1, and S2). (B) ODE modeling (solid lines) of representative dissociation signals (200 μM GTP, 0 μM GDP; values taken from Heck and Hofmann (38)), with rate constants as in Table 1. Note that the two sets of rate constants (sets A and B in Table S4) yield essentially identical traces. To see this figure in color, go online.
Figure 3
Figure 3
ReaDDy model. (A) Collision radii of R-type, G-type, and RG-type particles were chosen based on crystal structures (R and R, bovine 1U19 and 3PQR (40); G, bovine 1GOT (41); RG, 3SN6 (42)). Due to the different molecular shapes within and on the surface of the membrane, there are two types of collision radii: rc,mb for collisions within the membrane and rc,sol for collisions on the membrane surface (see Table 2 for radius parameters). (B) Disk-vesicle geometry with 4499 R (purple), 450 G (blue), and 1 R (yellow) on a 0.18 μm2 surface. The diffusion trajectory (black line; and see inset) of R is drawn for 2 ms. To see this figure in color, go online.
Figure 4
Figure 4
Microscopic ReaDDy simulation of the free-diffusion scenario. (A) Box-whisker plot of mean times required for individual reaction steps in the G activation: R hits the first free G (G hit), formation of the first RG complex (RG form), first GDP release (GDP rel), first GTP uptake (GTP up) and first G dissociation (RGdis). The cumulative time of all individual steps is depicted on the righthand side. Shown are data from 12 simulations that ran for 10 ms. (B) Microscopic model. (i) Model parameters. (ii) Snapshot of the simulated geometry, containing R (purple), R (yellow), and G (blue). Diffusion of R is depicted for 1 ms. Note that all particles are freely diffusing, similar to the trajectory of R. (iii) G activation over time. Compared are averages of 10 ReaDDy simulations (blue, standard error in light blue) with the ODE model (red) and the experimental data (black). To see this figure in color, go online.
Figure 5
Figure 5
Microscopic ReaDDy simulation of the pre-complex scenario. (A) Dependence of the G production rate on the pre-complex off rate, given that the on rate is in the diffusion limit (simulations ran for 10 ms, depicted are mean and standard error). The dashed blue line indicates the experimentally found G production rate. (B) Microscopic model: (i) model parameters, (bold) parameters different to the free diffusion scenario. (ii) Snapshot of the simulated geometry, containing R (purple), R (yellow), G (blue) and RG-precomplex (green) particles. The diffusion of R is depicted for 1ms. Note that all particles are freely diffusing similar to the trajectory of R. (iii) G activation over time. Averages of 10 ReaDDy simulations (blue, with standard error (light blue)) are compared with the ODE model (red) and the experimental data (black). To see this figure in color, go online.
Figure 6
Figure 6
Dependence of geometry on free lipid (D0) and apparent diffusion constant (D), encounter frequency, and G production rate. (A) In the free-diffusion case (free), R (purple), R (yellow), and G (blue) are all mobile. In the rack geometry, 80% of R is set as immobile in racks of dimers, whereas the other molecules diffuse freely. Here, R can be part of the mobile fraction (racksRfree) or be part of an immobile rack (racksRin rack). (B and C) Dependency of D0 (first row), D (second row), R-G encounter rate (third row), and G production, given the geometries in A. Note the smaller scale of the y axis between the plots of free lipid and apparent diffusion constant. In B, the D0 of the free-diffusion case is imposed for all geometries. Consequently, the crowding effects of the geometry can be seen in the apparent diffusion constant and the resulting encounter and G-production rates. In C, the crowding effects of the geometry are compensated by imposing higher free-lipid-diffusion constants on the particles (note that the diffusion of G is set to compensate the loss of diffusion contribution from the immobilized R in the R in rack case). As a consequence, apparent diffusion, encounter frequency, and G production are similar in all geometries. All data shown are averages and standard errors of six simulations per scenario. Simulation timescales are 100 μs for diffusion analysis, 1 ms for encounter analysis, and 10 ms for G catalysis. To see this figure in color, go online.
Figure 7
Figure 7
Microscopic ReaDDy simulation of the racks of rhodopsin dimers scenario. (i) Model parameters, highlighting (bold) parameters different from the free-diffusion scenario. (ii) Snapshot of the simulated geometry, containing R (purple), R (yellow), and G (blue). Note that only G, monomeric R, and R particles are freely diffusing. R oligomers are considered immobile. (iii) G activation over time. Averages of 10 ReaDDy simulations (blue, with standard error in light blue) are compared with the ODE model (red) and the experimental data (black). To see this figure in color, go online.

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