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. 2015 Feb 25;35(8):3701-10.
doi: 10.1523/JNEUROSCI.4160-14.2015.

Monitoring of vacuolar-type H+ ATPase-mediated proton influx into synaptic vesicles

Yoshihiro Egashira  1 , Miki Takase  1 , Shigeo Takamori  2
Affiliations

Monitoring of vacuolar-type H+ ATPase-mediated proton influx into synaptic vesicles

Yoshihiro Egashira et al. J Neurosci. .

Abstract

During synaptic vesicle (SV) recycling, the vacuolar-type H(+) ATPase creates a proton electrochemical gradient (ΔμH(+)) that drives neurotransmitter loading into SVs. Given the low estimates of free luminal protons, it has been envisioned that the influx of a limited number of protons suffices to establish ΔμH(+). Consistent with this, the time constant of SV re-acidification was reported to be <5 s, much faster than glutamate loading (τ of ∼ 15 s) and thus unlikely to be rate limiting for neurotransmitter loading. However, such estimates have relied on pHluorin-based probes that lack sensitivity in the lower luminal pH range. Here, we reexamined re-acidification kinetics using the mOrange2-based probe that should report the SV pH more accurately. In recordings from cultured mouse hippocampal neurons, we found that re-acidification took substantially longer (τ of ∼ 15 s) than estimated previously. In addition, we found that the SV lumen exhibited a large buffering capacity (∼ 57 mm/pH), corresponding to an accumulation of ∼ 1200 protons during re-acidification. Together, our results uncover hitherto unrecognized robust proton influx and storage in SVs that can restrict the rate of neurotransmitter refilling.

Keywords: V-ATPase; acidification; synaptic vesicle.

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Figures

Figure 1.
Figure 1.
SV re-acidification monitored by mOrange2. A, Titrations of sypHy (green) and syp–mOr (red) expressed in cultured hippocampal neurons. pHluorin and mOrange2 were positioned at the luminal part of synaptophysin. Fluorescence intensities at each pH were normalized to those at pH 8.6 and were expressed as a function of pH. Both data were well fitted by a single-site titration model (see Materials and Methods). The pKa and nH were determined as 6.54 ± 0.05 and 0.99 ± 0.02 for syp–mOr and 7.09 ± 0.02 and 1.35 ± 0.02 for sypHy (n = 6 experiments with 38 boutons for sypHy and n = 5 experiments with 64 boutons for syp–mOr). B, Comparison of fluorescence between sypHy and syp–mOr during the acid-quench experiment. Acidic solution (pH 5.5) was applied before (Q0) and after (Q1) field electrical stimulation (50 Hz, 2 s). The decay in fluorescence during poststimulus acid quench (Q1) represented re-acidification of the endocytosed SVs retrieved within an 8 s interval (n = 14 experiments with 280 boutons for sypHy and n = 14 experiments with 214 boutons for syp–mOr). C, Fluorescence images of presynaptic boutons expressing sypHy (top) and syp–mOr (bottom), during Q0, 50 Hz stimulus and at the onset of Q1. T indicates a time point on the horizontal axis of the plot shown in B. Scale bar, 5 μm. D, The fluorescence from sypHy and syp–mOr during poststimulus acid quench (Q1) shown in B was normalized to the fluorescence during Q0. Inset, The time constant obtained from a single-exponential fit to the syp–mOr fluorescence was significantly larger than that to sypHy fluorescence (**p < 0.01, Student's t test). E, Temperature-dependent effect of acid exposure on syp–mOr fluorescence. Fluorescence was monitored at either 24°C (room temperature; black open circles) or 34°C (physiological temperature; red filled circles) and normalized to the fluorescence obtained by 50 mm NH4Cl application (n = 7 experiments with 104 boutons for 24°C and n = 7 experiments with 140 boutons for 34°C). The fluorescence apparently declined at 34°C, which was linearly fitted with a slope of −0.0016 ± 0.0004 s−1. This artificial decline of baseline fluorescence also potentially contributed to the fluorescence decay during poststimulus acid quench when measured at 34°C. F, Normalized fluorescence decay during re-acidification measured at 34°C (n = 10 experiments with 154 boutons). To obtain this, acid-quench experiments were performed similar to B and combined with 50 mm NH4Cl application. F/FNH4Cl during acid treatment was corrected by subtracting the re-acidification-independent decline seen in E. Dashed line indicates the fluorescence during Q0. Error bars indicate SEM.
Figure 2.
Figure 2.
Estimate of pHv from syp–mOr fluorescence. A, Average trace of syp–mOr fluorescence during acid-quench experiments with 30 s Q1 (n = 27 experiments with 263 boutons) and a schematic illustration of the experiment. We divided the time course of fluorescence measurement into seven steps, indicated by I–VII (trace), and modeled the contributions of individual probe fractions to the total fluorescence at each step (images depicting the presynaptic terminal). The three different probe fractions consisted of the surface, resting, and endocytosed “re-acidifying” SVs, which were indicated by gray, red, and green lines, respectively. From step I to step VI, each fraction was described by a combination of the initial surface fraction (S), released fraction (R) and the endocytosed fraction retrieved by the onset of poststimulus quench (E). The endocytosed fraction retrieved during acid quench (brown SV in step VI) was indistinguishable from the quenched surface fraction and was thus not considered in the analysis. The resting pHv was indicated as pHi. The pHv of endocytosed SVs at the onset of (T = 0 s) and during (T = t s) the poststimulus quench are shown as pHo and pHt, respectively. Fluorescence during the application of 50 mm NH4Cl (step VII) was used for signal normalization. B, Average changes in syp–mOr fluorescence (n = 10 experiments with 72 boutons) in control (black) and in the presence of 120 nm folimycin (blue) measured sequentially in the same boutons. Acid quenching (Q0, Q1) was performed only in the presence of folimycin (Foli). The peak amplitude of ΔF was not significantly changed in the presence of folimycin (p = 0.84, Student's t test), indicating that substantial endocytosis did not occur during electrical stimulation (50 Hz, 2 s). C, Normalized fluorescence of the endocytosed re-acidifying fraction calculated according to the above model. Results in the absence (red) and presence (blue) of folimycin were obtained from the acid-quench experiment shown in A and B, respectively. The dashed line refers to normalized fluorescence from resting SVs, calculated from the trace shown in A. D, Average pHv of endocytosed fraction. Normalized fluorescence from C (red trace) was segmented into bins with a width of 1 s and converted to pH. The binning process was performed to reduce uncertainty in the pH calculation, especially at low signal level. However, in a few cases, fluorescence values in the last few seconds could not be converted to pH and were thus excluded. A single-exponential fitting yielded a time constant for re-acidification of 14.9 ± 1.7 s. The dashed line indicates the resting pHv, which corresponded to 5.64 ± 0.03. Inset, [H+]v in the lumen of endocytosed SVs calculated from the average plot of the pHv. Dashed line indicates [H+] in the resting state. Error bars indicate SEM.
Figure 3.
Figure 3.
Theoretical evaluation of the mOrange probe for re-acidification measurement. A, The sensitivity of the re-acidification measurement was compared for the sypHy (green) and syp–mOr (red) probes. An index of the SNR was plotted as a function of pHt, the pHv of endocytosed re-acidifying SVs. Equations VII and VIII and the values used to calculate this parameter are described in Results. The dashed lines indicate values of the same index calculated for the exocytosis of single vesicles. B, Theoretical trace of sypHy fluorescence during SV re-acidification. The left shows an exponential decay of pH with τ of 15 s, as measured from the syp–mOr fluorescence imaging. Resting pHv of 5.64 (Fig. 2) was used as a baseline (dashed line). Time course of normalized fluorescence of sypHy was calculated according to the Henderson–Hasselbalch equation and was shown in the right (black trace). Single-exponential fitting of the trace yielded a τ of 6.3 s (green trace). Note that this value was almost identical to that of decay kinetics of sypHy fluorescence measured in our experimental condition (Fig. 1D).
Figure 4.
Figure 4.
Re-acidification kinetics of different SV populations. A, Luminal pH of SVs endocytosed during varying stimulus-to-quench intervals (Δt). After stimulus, a 15 s acid quench was applied after Δt of 4 s (n = 9 experiments with 112 boutons), 8 s (n = 9 experiments with 129 boutons), 12 s (n = 10 experiments with 145 boutons), and 16 s (n = 10 experiments with 131 boutons). pHs of the endocytosed vesicle fraction were calculated as described in Figure 2. B, Comparison of the re-acidification kinetics measured with different Δt. The time constants were ∼15 s regardless of Δt (p = 0.96, ANOVA). C, pHvs of endocytosed fraction at the onset of poststimulus acid quench (pHo) plotted against Δt. A single-exponential fitting yielded a time constant of 17.8 s. Error bars indicate SEM.
Figure 5.
Figure 5.
Luminal BC of SVs. A, Average trace of syp–mOr fluorescence in response to stepwise NH4+ treatments (n = 14 experiments with 215 boutons). For pH calibration, a solution of pH 5.5 was applied for 2 s before the NH4+ applications, and a solution of pH 7.4 containing a mixture of ionophores was applied at the end of imaging. Fluorescence was normalized to the value at pH 7.4. B, Resulting pHv as a function of NH4+ concentration. C, Relationship between quenched [H+] and resulting pHv. The slope of a linear fit revealed that the BC of SVs was 57.4 ± 4.8 mm/pH regardless of luminal pH. D, Estimated H+ influx during acid quench. Net H+ influx (in millimolar at the left axis) and the number of accumulated protons (right axis) were calculated based on Figure 2D and the measured BC.
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