Extended Data Fig. 8: Non-equilibrium physical modelling of pumping, resting and leaky modes provides the single-molecule pumping and leaking rate.
From: Regulation of the mammalian-brain V-ATPase through ultraslow mode-switching
a, Schematic illustration of the main parameters used in the model. Note: this is illustration is also shown in Fig. 2a. b,c, Top: Representative single-molecule traces displaying mode-switching dynamics. Proton pumping was stochastically interrupted by inactive and proton-leaky modes. During active periods of the V-ATPase, a dynamic equilibrium between proton-pumping and passive leakage is established, therefore reaching a single acidification plateau. Leakage currents are a convolution of transprotein and passive membrane proton efflux. Transprotein leakage currents are temporally distinct from proton pumping and may activate directly after the enzyme switched off. Data was fitted with a non-equilibrium model as described in the supplementary information. Pumping rates and permeabilities (both membrane and transprotein) are calculated as free parameters by the model. Bottom: Pumping dynamics for the proton-pumping rate (red), the passive membrane efflux rate (grey) and the transprotein efflux rate (yellow). In b the transprotein leak was the primary efflux pathway while in c proton efflux manifested only passively through the membrane. d, Proton pumping rates and permeability estimates of the model for the data shown in this figure (b, c, and e). Pumping rates were found to be 7 ± 5 H+/s. Membrane and V-ATPase permeability were 3 ± 2 × 10−5 cm/s and 18 ± 9 × 10−5 cm/s. Transprotein permeability of the V-ATPase is nearly an order of magnitude larger than passive membrane permeability indicating the regulatory importance of the proton-leaky mode. Error bars correspond to one s.d. Number of independent model outputs are N = 8, 11 and 8 for membrane permeability, V-ATPase permeability and proton-pumping rates respectively. e, Additional single-molecule traces fitted the model. Arrows point out the mode-switching events during which dynamic (ΔpH = ΔpHmax) or static (baseline, ΔpH = 0) equilibrium was not reached
