Electrochemical Gradient at different ATP Concentrations

The luminal pH of single SVs after acidification with different concentrations of ATP was measured by collecting the fluorescence of spH-SVs upon photolysis of 1-5 mM NPE-ATP in glycine buffer (Table ‎2-1). NPE-ATP uncaging led to fluorescence quenching of spH-SVs in a dose-dependent manner (Figure ‎3-4A). For each acidification measurement, a photobleaching image was acquired by measuring the fluorescence of spH-SVs with the same experimental settings used for pH measurements in the absence of NPE-ATP. In order to estimate the steady-state luminal pH at each ATP concentration, fluorescence traces were first corrected for photobleaching by dividing individual traces by the averaged fluorescence trace obtained from the corresponding photobleaching images. Corrected fluorescence traces were then converted to pH traces using the pH-fluorescence calibration curve (Figure ‎2-12). Luminal pH at each ATP concentration was then calculated from the individual pH traces as the averaged pH values between 15 to 20 seconds after NPE-ATP uncaging. The averaged pH values from 3-5 experimental replicates were then plotted against ATP concentrations, calculated from NPE-ATP with an uncaging efficiency of 60% (see section 2.7.1) (Figure ‎3-4B). These data show that at saturating concentrations of ATP (2-3mM) in a buffer which was free of membrane-permeable ions, the interior of SVs acidifies to a pH of ~ 6.57 ± 0.04 (SD).

Moreover, the acidification kinetics of single vesicles were obtained at each ATP concentration by a bi-exponential fit to individual pH traces as described in section 2.7.6.1. These data show that a faster rate of acidification is achieved when ATP is in abundant supply. The averaged acidification rate constants (1/sec) were plotted against the concentration of ATP, and fit with the Michaelis-Menten equation (Figure ‎3-4C). This resulted in a maximum acidification rate constant of 1.78 ± 0.06 1/sec (±SEM) and Km of 0.63 ± 0.12 mM ATP.

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Figure ‎3-4 Luminal pH of single spH-SVs after acidification at different ATP concentrations.

A) Averaged fluorescence traces of spH-SVs at different NPE-ATP (data from 3-5 independent experimental replicates are compiled for the averaged trace). UV flash indicates where ATP is released by photolysis. The trace in the absence of ATP shows photobleaching of the probe over the experimental timescale. Error bars represent SEM of n single SVs (n is indicated on the traces). B) The averaged luminal pH of spH-SVs at different free ATP (calculated based on 60%

uncaging efficiency). Error bars represent SD of 3-5 experimental replicates. C) Acidification rate constants of single spH SVs at different ATP concentrations; red line shows Michaelis-Menten fit to the data (R-Square = 0.99, Km = 0.63 ± 0.12 mM ATP, Vmax = 1.78 ± 0.06 1/sec, Error bars represent SEM of n single SVs; n = 112, 128, 43 and 69 for 0.6, 1.2, 2.4 and 3 mM ATP, respectively).

Next, the membrane potential generated across the membrane of single SVs upon acidification at different ATP concentrations was measured by labeling SVs with 100 nM of VF2.1.Cl in glycine buffer. In agreement with the pH measurements, addition of 0.6-3 mM ATP to labeled SVs induced an increase in VF2.1.Cl fluorescence in a dose dependent manner (Figure ‎3-5A). The averaged fluorescence values at 15-20 seconds after ATP

Results |67 application were converted to changes in membrane potential by Eq. 2.7. The results from 3-5 experimental replicates for each ATP concentration were averaged and plotted against ATP concentrations (Figure ‎3-5B). These results show that upon acidification of SVs with 3 mM ATP, a ∆ψ of 82.97 ± 16.9 mV (±SD) is formed across the membrane of vesicles.

Figure ‎3-5 Changes in membrane potential across the membrane upon acidification at different ATP.

A) Averaged fluorescence traces of VF2.1.Cl-labled SVs in response to addition of different Mg.ATP to the bath solution (data from 3-5 independent experimental replicates are compiled for the averaged trace). The trace in the absence of ATP shows photobleaching of the probe over experimental timescale. Error bars represent SEM of n single SVs (n is indicated on the traces).

B) Averaged extent of change in membrane potential upon ATP-induced acidification. Error bars represent SD of 3-5 experimental replicates.

Interestingly, when ∆pH and ∆ψ, measured at the same concentration of ATP, were plotted against each other, a non-linear relationship was observed (Figure ‎3-6).

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Figure ‎3-6 Relationship between ∆pH and ∆ψ.

Changes in the membrane potential upon acidification with different concentrations of ATP are plotted against the pH gradient induced by the corresponding concentration of ATP. It should be noted that the pH and potentiometric measurements were performed separately and the absolute values must be viewed cautiously.

3.2.1 ∆pH and ∆ψ in Glutamatergic and GABAergic SVs

After each pH or potentiometric measurement, on-stage antibody staining against VGAT was performed using the solution exchange system of the TIRF setup. This allowed for the detection of glutamatergic and GABAergic SVs in the same population of vesicles. Surprisingly, when the luminal pH of GABAergic and glutamatergic SVs were compared, a significantly lower luminal pH was observed in glutamatergic compared to GABAergic SVs. The same results were obtained at all tested ATP concentrations (0.6-3 mM) (Figure ‎3-7A). Consistent with these data, significantly larger ∆ψ was also measured in glutamatergic compared to GABAergic SVs in each potentiometric measurement (Figure ‎3-7B). However, no significant difference was observed between acidification rate constants of these SVs (Figure ‎3-7C), suggesting no difference in the force driving protons into the vesicular lumen. Thus, to unravel the underlying mechanism for the observed difference, two other factors regulating ∆µH+ were measured in these SVs: buffering capacity and proton permeability.

Results |69 Figure ‎3-7 Comparison between glutamatergic and GABAergic vesicles in their proton electrochemical gradient.

A) Luminal pH of glutamatergic and GABAergic SVs after acidification with different concentrations of ATP. Glutamatergic SVs reached 0.1 ± 0.03 (± SD) pH units lower luminal pH compared to GABAergic SVs. B) Changes in membrane potential across the membrane of glutamatergic and GABAergic SVs upon acidification with different ATP concentrations. The magnitude of the membrane potential was 11.99 ± 5.2 mV (± SD) larger in glutamatergic compared to GABAergic SVs. Two circles connected via a dashed line represent the average response of glutamatergic and GABAergic SVs from the same experiment. Error bars represent SEM of single SVs. Number of glutamatergic and GABAergic SVs per measurement was on average 444 ± 122 and 160 ± 78 (± SD), respectively. p-value = 2.7 x 10^-5 and 8.7 x 10^-4 in A and B, respectively. C) Acidification rate constant of glutamatergic and GABAergic SVs at 2.4 mM ATP. Averaged acidification rate constants (± SEM) were 1.24 ± 0.13 (1/sec) and 1.22 ± 0.14 (1/sec) for the glutamatergic and GABAergic SVs, respectively. Error bars represent SEM of n single SVs (n is indicated on the bars).