Depolarization-induced pH microdomains and their relationship to calcium transients in isolated snail neurones

Abstract

Neuronal electrical activity causes only modest changes in global intracellular pH (pH(i)). We have measured regional pH(i) differences in isolated patch-clamped neurones during depolarization, using confocal imaging of 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) fluorescence. The pH(i) shifts in the soma were as expected; however, substantially larger shifts occurred in other regions. These regional differences were still observed in the presence of CO(2)-HCO(3)(-), they decayed over many seconds and were associated with changes in calcium concentration. Lamellipodial HPTS fluorescence fell by 8.7 +/- 1.3 % (n = 9; approximately 0.1 pH unit acidification) following a 1 s depolarization to 0 mV; this was more than 4-fold greater than the relative shift seen in the soma. Depolarization to +40 mV for 1 s caused a 46.7 +/- 7.0 % increase (n = 11; approximately 0.4 pH unit alkalinization) in HPTS fluorescence in the lamellipodia, more than 6-fold that seen in the soma. Application of 5 % CO(2)-20 mM HCO(3)(-) did not significantly reduce the size of the +40 mV-evoked local pH shifts despite carbonic anhydrase activity. The pH(i) gradient between regions approximately 50 microm apart, resulting from acid shifts, took 10.3 +/- 3.1 s (n = 6) to decay by 50 %, whereas the pH(i) gradient resulting from alkaline shifts took only 3.7 +/- 1.4 s (n = 12) to decay by 50 %. The regional rates of acidification and calcium recovery were closely related, suggesting that the acidic pH microdomains resulted from Ca(2+)-H(+) pump activity. The alkaline pH microdomains were blocked by zinc and resulted from proton channel opening. It is likely that the microdomains result from transmembrane acid fluxes in areas with different surface area to volume ratios. Such neuronal pH microdomains are likely to have consequences for local receptor, channel and enzyme function in restricted regions.

Figure 1. Single wavelength (458 nm excitation) calibration of HPTS fluorescence

A, fluorescence intensities (F; open and filled circles), recorded as 12-bit numbers, of 14 calibration solutions plotted against their pH (pH range 3.8–8.9). The continuous line shows the least-squares best fit of the data to the standard equation: where pK_HPTS = 7.18, F_min = 0 and F_max = 3150. B, eqn (1) (see text) is shown plotted for 6 starting pH values, at which F = F₀. The y-axis shows the ΔpH corresponding to a given F/F₀. The circles show the open circle data from A replotted following normalization to the fluorescence at pH 7. The pH difference from pH 7, of each solution, has been calculated (ΔpH) and plotted against the fluorescence intensity normalized to that at pH 7 (F/F₀). See text for an explanation of the filled square.

Figure 2. Validation of HPTS F/F₀ plots as an indicator of pH_i shifts using long depolarization to induce steady-state homogeneous alterations in pH_i

Aa, transmitted image of neurone. Ab, z-series reconstruction of neurone showing adhesions to the coverslip (shown by the vertical yellow line) of the cell body and axon. Ac, greyscale confocal HPTS fluorescence image, just above the coverslip (confocal slice, 1 μm), showing two regions of interest (red and blue). In B, traces show membrane potential (E_m), clamp current (I_c), mean region intensity and F/F₀ during a long depolarization to +40 mV. The pH calibration was obtained from F/F₀ using eqn (1) (see text), assuming thermodynamic equilibrium of pH across the plasma membrane by the end of the trace.

Figure 8. Effect of 5 % CO₂-20 mm HCO₃⁻ on depolarization-induced pH shifts in lamellipodia

Aa, transmitted light image of neurone showing the region from which fluorescence was collected. Ab, greyscale image of confocal fluorescence (confocal slice, 2.5 μm thick). The yellow ‘ladder’ shows the regions of interest used for the linescans shown in the lower panels of C and D whilst the blue and red regions were used to produce the F/F₀ traces in B-D. B, membrane potential, clamp current and F/F₀ (for the red and blue regions) during depolarization in the absence and presence of 5 % CO₂-20 mm HCO₃⁻ (pH 7.25). C and D, F/F₀ for the two regions before, during and after depolarization to +40 mV in the absence (C) and presence (D) of CO₂-HCO₃⁻. The linescan images show the spatial distribution of the relative fluorescence shifts over time. The open rectangles, spanning the F/F₀ traces and the linescan images, indicate the period of depolarization. pH values were calculated from the fluorescence shift on removal of CO₂-HCO₃⁻ by assuming an intrinsic buffering power of 12 mm.

Figure 9. Effect of 5 % CO₂-20 mm HCO₃⁻, in the presence of the carbonic anhydrase inhibitor acetazolamide (25 μm), on depolarization-induced alkalinizations

Aa, transmitted light image of the neurone and patch electrode. Ab, confocal image of lamellipodia below the soma and axon (confocal slice, 1 μm thick). The yellow ‘ladder’ shows the regions used for the linescans shown in C and D. The red and blue regions were used to produce the traces shown in B-D. B, membrane potential, clamp current and mean region intensity before, during and after addition of CO₂-HCO₃⁻ and acetazolamide. C and D, F/F₀ following depolarization to +40 mV in the absence (C) and presence (D) of CO₂-HCO₃⁻ with acetazolamide. The linescan images show the spatial distribution of the relative fluorescence shifts over time. Calibration of F/F₀, using a buffering power of 12 mm, gives an approximate peak shift of 0.35 pH units (F/F₀ of 1.5) for the alkalinization evoked by depolarization to +40 mV.

Figure 3. pH-sensitive fluorescence shifts resulting from membrane depolarization for 1 s to 0, +20 and +40 mV

Aa, transmitted image showing the axonal lamellipodia and soma with attached patch electrode. Ab, background-subtracted greyscale confocal image (optical slice, 2.4 μm thick) of HPTS fluorescence in the lamellipodia. Ac, overlay of images a and b. Ad, the three regions of interest from which fluorescence data plotted in B were obtained. B, membrane potential and relative HPTS fluorescence shift (F/F₀) plotted against time for the three regions. The red region (lamellipodia) has an absolute fluorescence intensity (arbitrary greyscale units) of 13 compared with 85 in the blue region (axon foot) and 142 in the green region (soma). There is a 3 min break in the traces, during which 50 μm zinc was applied, before the final depolarization to +40 mV.

Figure 4. Mean data for the HPTS fluorescence shifts in the soma, axon and lamellipodia on depolarization for 1 s to 0 and +40 mV

Open bars, percentage change in fluorescence on depolarization to +40 mV (n = 6, 11 and 11, respectively, for the three regions); shaded bars, percentage change in fluorescence on depolarization to 0 mV (n = 6, 9 and 9, respectively). The areas of the three regions were 180 ± 24, 64 ± 8 and 182 ± 42 μm² (n = 10, 18 and 19), respectively. *P < 0.05, **P < 0.01, significant differences between data sets.

Figure 5. Depolarization-induced calcium microdomains

Aa, transmitted light image of neurone showing the cell body, axon, lamellipodia and patch pipette. Ab, greyscale image of confocal Oregon Green fluorescence (confocal slice, 1 μm thick) showing the three regions of interest from which data shown in B were obtained. B, membrane potential and relative Oregon Green fluorescence shift (F/F₀) plotted against time for the three regions. The neurone was sequentially depolarized for 1 s to three potentials, 0, +20 and +40 mV.

Figure 6. Mean data for the Oregon Green fluorescence shifts in the soma, axon and lamellipodia on depolarization for 1 s to 0 and +40 mV

Mean data (percentage change in fluorescence) on depolarization to 0 and +40 mV were pooled (n = 6, 9 and 6, respectively, for the three regions). *P < 0.05, **P < 0.01, significant difference between data sets.

Figure 7. Comparison of the calcium recovery and acidification rates in the three regions

A, example traces of Oregon Green fluorescence and HPTS fluorescence from the lamellipodia of two different neurones on depolarization to 0 mV for 1 s. The thick lines show the exponential fits used to calculate the rates plotted in B. B, plot of the mean acidification rate against the calcium recovery rate following depolarization to 0 mV for 1 s. Rates were calculated from the least-squares fit of a single exponential over the regions illustrated in A. The acidification rate data for the three regions (soma, axon and lamellipodia) were derived from 5, 11 and 7 cells, respectively, whilst the calcium recovery rate data were derived from 6, 9 and 6 cells, respectively.