Hypothesized diprotomeric enzyme complex supported by stochastic modelling of palytoxin-induced Na/K pump channels

Figures

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  Figure 1.. Statistical model of the PTX–Na+/K+ pump complex. (a) Diagram of the reactions and substates for the PTX–pump complex model. (b) General principle of the statistical model. (c) One of the four enzyme units in the substate PTXEc transitioning to the substate PTXEo, according to one of the directions of reaction p8. (d) Network of stochastic hybrid automata used to model the six possible transitions between the substates indicated in (b).

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  Figure 2.. Activation of the PTX-induced current. (a) Current simulated with the SM. Grey curve (simulation) is compared with the normalized experimental data (circles—extracted from Artigas & Gadsby [6]) and with the simulation performed with the model of differential equations (MDE; black curve—extracted from Rodrigues et al. [9]). (b) Simulation performed with the MDE. The concentrations of the substates responsible for the induced current are shown. (c) Number of pumps occupying the different substates of the PTX–pump complex (as shown in (b), the substates are responsible for the induced current).

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  Figure 3.. Single PTX-induced channel activity. (a) Perfusion with solution containing 2 nM of PTX caused the channel to open, showing epochs with intense open/close transitions and epochs where the induced channel remains in the closed state. (b) Simulation of the activity of the PTX-induced channel. Representing the experimental procedure, the [PTX]o was maintained equal to 2 nM up to the channel opening until the pump changed to the substate PTXEo. (Left) Simulation of the most intense period of activity, associated with transitions between the substates PTXEc and PTXEo. (Right) Simulation of period where the pump remains in the closed substate PTXEc most of the time.

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  Figure 4.. Reaction and substate diagram for the PTX–pump complex assuming the pump functions as a diprotomer.

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  Figure 5.. Single-channel activity and activity of a few channels induced by the presence of PTX. (a) The perfusion with solution containing PTX (2 nM) caused the channel to open, showing several open/close transitions and epochs where the channel remains in the closed state most of the time. After a few minutes of channel activity, the perfusion with PTX (2 nM) continues, causing the appearance of more channel activity. (b) Simulation of the induced channel activity. Reproducing the experimental manoeuvre, the [PTX]o was maintained at 2 nM, until the channel opened, therefore, until the pump occupies the substate PTXEo. After the channel remains active for a few minutes, the [PTX]o is changed from 0 to 2 nM, inducing more channels. (Left) The period of most intense activity is characterized by transitions between the substates E1:PTXEc and E1:PTXEo (p6 reaction). In the period where the activity is less intense, the pump occupies the substates (Na+)2E2:PTXEo and (Na+)2E2:PTX(Na+)2Ec. (Right) The continuous perfusion with PTX induces channels occupying all substates E1:PTXEc and E1:PTXEo.

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  Figure 6.. Each column shows a simulation with a different number of pumps (top traces). The corresponding PTX-induced current (below). The number of pumps occupying the different substates is considered in the model.