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

摘要

Neuronal electrical activity causes only modest changes in global intracellular pH (pHi). We have measured regional pHi differences in isolated patch-clamped neurones during depolarization, using confocal imaging of 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) fluorescence. The pHi 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 CO2-HCO3⁻, they decayed over many seconds and were associated with changes in calcium concentration. Lamellipodial HPTS fluorescence fell by 8.7 ± 1.3 % (n = 9; ∼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; ∼0.4 pH unit alkalinization) in HPTS fluorescence in the lamellipodia, more than 6-fold that seen in the soma. Application of 5 % CO2-20 mm HCO3⁻ did not significantly reduce the size of the +40 mV-evoked local pH shifts despite carbonic anhydrase activity. The pHi gradient between regions ∼50 μm apart, resulting from acid shifts, took 10.3 ± 3.1 s (n = 6) to decay by 50 %, whereas the pHi 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 Ca²⁺-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.