PMC

In silico simulation of voltage-clamp traces of ClC-7. Starting from a resting potential of +20 mV, we simulated a pulse protocol from −140 mV to +100 mV in 20-mV steps for 6 s, followed by +100 mV for 1 s, after returning to the resting potential. Depicted are the turnover rates for (a) ClC-7^fast antiporter, and (b) ClC-7^WT antiporter with relatively slow (de) activation kinetics (τ_act = 1 s), (c) a ClC-7 antiporter with moderately accelerated (de) activation kinetics (τ_act = 0.25 s), and (d) a ClC-7 antiporter with an extremely accelerated (de) activation kinetics (τ_act = 10⁻¹⁰ s). The colour gradient varies from dark blue for negative voltages to red for positive voltages (cytosolic potential defined as zero) as indicated in the pulse protocol (inset in (a)).

Differences in ClC-7 kinetics do not affect lysosomal acidification. (a) Schematic representation of the model with ClC-7 antiporters, V-ATPases, potassium and sodium channels, and proton leak. The cartoon was created using Servier Medical Art templates (https://smart.servier.com), licensed under a Creative Commons License (https://creativecommons.org/licenses/by/3.0/). (b–h) Depicted for the different ClC-7 scenarios during lysosomal acidification (ClC-7^WT, dashed black line; ClC-7^fast, red; ClC-7^unc, blue; ClC-7^ko, green) are (b) luminal pH, (c) total membrane potential, (d) luminal concentrations of potassium, (e) sodium, and (f) chloride ions, as well as the turnover rates of (g) ClC-7^WT and ClC-7^fast, and (h) ClC-7^unc. Initial conditions provided in .

Schematic representation of all components included in the model of ion homeostasis. The V-ATPase pumps protons (H⁺) into the lysosomal lumen for acidification. ClC-7 antiporter transports chloride ions (Cl⁻) in exchange for protons. CAX transports calcium ions (Ca²⁺) in exchange for protons. Proton (H⁺), potassium (K⁺), sodium (Na⁺) and Ca²⁺ channels allow the passive movement of these ions. The Donnan particles (D) affect lysosomal acidification through the membrane potential, and buffering capacity (β) of the lumen affects the rate of pH changes. The names of the channels and transporters are indicated with labels. Variables and parameters are written in blue and black, respectively. The flux across the membrane irrespective of direction is represented by black arrows. The cartoon was created using Servier Medical Art templates (https://smart.servier.com), which are licensed under a Creative Commons License (https://creativecommons.org/licenses/by/3.0/).

Ca²⁺/H⁺ exchange mediates effective Ca²⁺ uptake in the presence of a Ca²⁺ leak. (a,b) Simulations of Ca²⁺ uptake via CAX with three different stoichiometries as depicted (1:1, 2:1, 3:1) in (a) absence and (b) presence of Ca²⁺ leak for wild-type ClC-7. The luminal free Ca²⁺ concentrations are shown for 1, 10, 20 and 30 CAXs. (b) For each case, the Ca²⁺ permeability was set to enable a steady-state luminal free Ca²⁺ concentration. (c) Simulations for the four ClC-7 scenarios considering different CAX configurations: 30 CAX, 1H⁺:1Ca²⁺; 20 CAX, 2H⁺:1Ca²⁺, and 10 CAX, 3H⁺:1Ca²⁺, using the same Ca²⁺ permeability as in (b) for all ClC-7 scenarios. The initial conditions were set to the steady-state values of (i.e., after Ca²⁺ release, ). The cartoons were created using Servier Medical Art templates (https://smart.servier.com), licensed under a Creative Commons License (https://creativecommons.org/licenses/by/3.0/).

The cation channels Na⁺ and K⁺ neutralize the influence of ClC-7 on Ca²⁺ release. (a) Schematic representation of the model with ClC-7 antiporters, V-ATPases, potassium and sodium channels, proton leak, and Ca²⁺ release channel. The cartoon was created using Servier Medical Art templates (https://smart.servier.com), licensed under a Creative Commons License (https://creativecommons.org/licenses/by/3.0/). (b–j) Depicted for the different ClC-7 scenarios during triggered Ca²⁺ release (ClC-7^WT, dashed black line; ClC-7^fast, red; ClC-7^unc, blue; ClC-7^ko, green) are (b) luminal free Ca²⁺ concentration, (c) Ca²⁺ flux, (d) luminal pH, (e) total membrane potential, (f) luminal concentrations of potassium, (g) sodium and (h) chloride ions, as well as the turnover rates of (i) ClC-7^WT and ClC-7^fast, and (j) ClC-7^unc. The initial conditions were set to the steady-state values of (). From t = 0 s, the lysosomal membrane was permeable to calcium ions (P_Ca²⁺ = 8.9 × 10⁻⁵ cm/s).

Cl-/H+ exchange supports channel-mediated lysosomal Ca²⁺ uptake independent of ClC-7 activation kinetics. (a) Schematic representation of the model with ClC-7 antiporters, V-ATPases, potassium, sodium, Ca²⁺ channels, and proton leak. The cartoon was created using Servier Medical Art templates (https://smart.servier.com), licensed under a Creative Commons License (https://creativecommons.org/licenses/by/3.0/). (b–i) The initial conditions were set to the steady-state values of () and from t = 0 s, the lysosomal membrane was permeable to calcium ions (P_Ca²⁺ = 5.7 × 10⁻⁴ cm/s) representing the opening of the uptake channel. (b) Luminal free Ca²⁺ concentration of ClC-7WT for five different values of cytosolic Ca²⁺ concentration ([Ca²⁺]_C). (c–k) Depicted for the different ClC-7 scenarios during triggered Ca²⁺ uptake with a cytosolic Ca²⁺ concentration ([Ca²⁺]_C) of 0.6 mM (ClC-7^WT, dashed black line; ClC-7^fast, red; ClC-7^unc, blue; ClC-7^ko, green) are (c) luminal free Ca²⁺ concentration, (d) Ca²⁺ flux, (e) luminal pH, (f) total membrane potential, luminal concentrations of (g) potassium, (h) sodium and (i) chloride ions, as well as the turnover rates of (j) ClC-7^WT and ClC-7^fast, and (k) ClC-7^unc. From t = 0 s, the lysosomal membrane was permeable to calcium ions (P_Ca²⁺ = 5.7 × 10⁻⁴ cm/s) representing the opening of the uptake channel. (l) Steady state value of luminal free Ca²⁺ concentration for 10 different [Ca²⁺]_c values for the different ClC-7 scenarios.

Ca²⁺ release accompanied exclusively by ClC-7 antiporter reveals differences between fast and WT scenarios. (a) Schematic representation of the model with ClC-7 antiporters, V-ATPases, potassium and sodium channels, proton leak, and Ca²⁺ release channel. The cartoon was created using Servier Medical Art templates (https://smart.servier.com), licensed under a Creative Commons License (https://creativecommons.org/licenses/by/3.0/). (b–j) Depicted for the different ClC-7 scenarios during subsequent Ca²⁺ uptake and release (ClC-7^WT, dashed black line; ClC-7^fast, red; ClC-7^unc, blue; ClC-7^ko, green) are (b) luminal free Ca²⁺ concentration, (c) Ca²⁺ flux with a zoom to the last simulated uptake (top) and release (bottom), (d) luminal pH, (e) total membrane potential, (f) luminal concentrations of potassium, (g) sodium, and (h) chloride ions, as well as the turnover rates of (i) ClC-7^WT and ClC-7^fast, and (j) ClC-7^unc. The initial conditions were set to the steady state values of (i.e., after Ca²⁺ release, ). Ca²⁺ uptake and release was induced every 2 s by increasing and decreasing the cytosolic Ca²⁺ concentration, respectively. Ca²⁺ uptake was simulated considering all the elements shown in (a). In order to induce a change in the activity of the ClC-7 antiporter, we simulated Ca²⁺ release in the presence of only Ca²⁺ channel and ClC-7 antiporters.