Fig 1.
Modeling the effects of drugs on lysosomal ion homeostasis.
We utilized an established physiological framework to model lysosomal ion transport (A). Accordingly, the V-ATPase actively pumps protons into the lysosome (1) while the proton-chloride antiporter CLC7 dissipates the ensuing increase in membrane potential by coupling the efflux of protons with the influx of chloride (2). Protons escape the lysosomes through diffusion across the lysosomal membrane (3), and protons in the lysosome are sequestered through the buffering capacity of resident lysosomal components (4). Free protons that accumulate in the lysosomal lumen contribute to the decrease in lysosomal pH. Based on this mechanism, the effect of drugs was captured by modeling different lysosomal shapes and volumes (B). Concomitantly, the drug-dependent inhibition of V-ATPase function was modeled by varying the proton pumping activity (C); the drug-dependent change in membrane proton permeability was modeled by varying the proton leak from the lysosome (D); and, the drug-dependent perturbation of membrane potential regulation was modeled by inhibiting CLC7 (E) or decreasing cytoplasmic chloride (F).
Table 1.
Model parameters.
Fig 2.
The effects of individual lysosomal ion stressors on spherical versus tubular lysosomal physiology.
(A) The effect of inhibiting the number of V-ATPase molecule per lysosome showed significant changes in lysosomal pH and Cl-, with minimal change in membrane potential in both spherical and tubular lysosomes. (B) The effect of increasing proton-specific membrane permeability per lysosome showed significant changes in lysosomal pH and Cl-, with minimal change in membrane potential in both spherical and tubular lysosomes. In both (A) and (B), only a slight difference is observed in the effect of the lysosomal ion stressors on spherical versus tubular lysosomal physiology.
Fig 3.
The effects of lysosomal surface expansion and associated stress tolerance on lysosomal physiology.
(A) Simultaneous increment in lysosomal radius and surface area, with a constant volume of 1.65x10-16 L induced progressive perturbation in lysosomal pH, Cl-, and membrane potential. (B) Individual effects of varying V-ATPase number and membrane proton permeability on the lysosomal physiology of surface area expansion-mediated lysosomal stress. Either increasing V-ATPase number or reducing membrane proton permeability reduces transient perturbations in lysosomal pH, Cl-, and membrane potential following the simultaneous increment of lysosomal radius and surface area shown in (A).
Fig 4.
The effect of a combination of lysosomal ion stressors in spherical versus tubular lysosomes.
(A) Dimensions of tubular and spherical lysosomes. (B) The simultaneous inhibitions of V-ATPase and CLC7 numbers induced changes in both spherical and tubular lysosomes with minimal difference between the two lysosomal morphologies. The increment in V-ATPase inhibition induced the most significant change while only the complete depletion of CLC7 induced physiological perturbation which mainly arose from the significant change in membrane potential (> 250 mV, as indicated by the black arrow sign). (C) The simultaneous inhibition of V-ATPase and membrane proton permeabilization induced very similar and significant changes in the overall physiology of both spherical and tubular lysosomes.
Fig 5.
The effect of a simultaneous inhibition of the transport of chloride and proton ions in spherical versus tubular lysosomes.
(A) Dimensions of tubular and spherical lysosomes. (B) The simultaneous inhibitions of the cytoplasmic chloride and V-ATPase number per lysosome induced significant changes in lysosomal pH, Cl-, and membrane potential. The effect was magnified in the tubular lysosome where the > 4 pH unit increment in lysosomal pH, > 150 mM reduction in lysosomal Cl- accumulation, and > 250 mV increment in membrane potential were observed (as indicated by the black arrow signs).
Fig 6.
The effect of a simultaneous inhibition of proton and chloride transport in spherical versus various sized disc-shaped lysosomes.
The simultaneous inhibitions of the cytoplasmic chloride and V-ATPase number per lysosome induced significant changes in lysosomal pH, Cl-, and membrane potential. The effect was more magnified in the disc-shaped lysosomes with lysosomal radius (> 2.2 fold) and height (< 3.7 fold) than in the spherical lysosome where the > 4 pH unit increment in lysosomal pH, > 150 mM reduction in lysosomal Cl- accumulation, and > 250 mV increment in membrane potential were observed (as indicated by the black arrow signs).
Fig 7.
The effect of a simultaneous V-ATPase inhibition and membrane permeabilization in spherical versus various sized disc-shaped lysosomes.
The alterations of the V-ATPase number and membrane proton permeability per lysosome induced very similar changes to lysosomal pH, Cl-, and membrane potential. For all of the disc-shaped lysosomes, as the lysosomal radius and height increased and decreased, respectively, more increment in the lysosomal pH, more reduction in the lysosomal Cl- accumulation, and a slight increment in the lysosomal membrane potential were observed when there were no or very minimal alterations in both the V-ATPase number and membrane proton permeability, thus indicating the effect that the change in morphology alone has on lysosomal physiology.