Figure 7. The pH_o-conditioned Cl^– current is dependent on intracellular alkalinity.

(A) The pH_o-conditioned current is significantly reduced when changes in pH_i are buffered effectively with 40 mM HEPES (3.0 ± 0.6 pA/pF, n = 4 mice) compared with 10 mM HEPES (11.8 ± 2.0 pA/pF, n = 4 mice, **P < 0.01). However, the effect of hypoosmolarity is still preserved (23.7 ± 5.1, n = 2 mice in 40mM HEPES, vs. 31.0 ± 4.4 pA/pF, n = 4 mice in 10 mM HEPES, P > 0.05). (B) The pH-sensitive fluorescence of BCECF is changed from “blue” at pH_o 7.4 (first image) to purple during exposures to pH_o 6.0 (middle image) and to green, yellow, and red after the exposure (right image), reflecting variable increases in pH_i. Horizontal bar in the right panel represent 20 μm. (C) Tracings represent changes in fluorescence recorded from 5 different neurons simultaneously first during their transient exposures to pH_o 6.0 and then following the increases in pH to a variable degree in each neuron. Those increases are rapidly reduced when Zn²⁺ and amiloride are added (see arrow). (D) Normal pH_i of 7.2 ± 0.04 (n = 40 neurons from 3 mice at pH_o =7.4) drops to 7.11 ± 0.01 (n = 40 neurons from 3 mice, **P < 0.01) during the transient exposures to pH_o 6.0 and then increases to pH_i 7.54 ± 0.05 during the peak conditioned current (n = 40 neurons from 3 mice, **P < 0.001). (E) pH_i drops (ΔpH_i) by 0.13 ± 0.01 units (n = 40 neurons from 3mice, **P < 0.01) during exposures to pH_o 6.0, increases by 0.29 ± 0.05 units (n = 40 from 3 mice, 2 data points not shown, **P < 0.01) after exposure and then rapidly declines by 0.13 ± 0.02 units (n = 39 from 3 mice, one point not shown, **P < 0.01) with the addition of Zn²⁺ and amiloride. (F) The pH_o-conditioned current (black arrow) is inhibited from 23.4 ± 6.5 (n = 5 mice) to 3.6 ± 1.1 pA/pF (n = 4 mice, *P < 0.05) with amiloride and Zn²⁺, but the hypoosmolar-induced current remains intact (25.9 ± 6.3, n = 4 mice, vs. 28.6 ± 4.5 pA/pF, n = 4 mice, P > 0.05). (G) A drop in pH_i from 7.25 down to 7.0 and 6.0 does not induce a significant pH-conditioned current, with values of 1.4 ± 0.6 (n = 2 mice), 0.4 ± 0.1 (n = 2 mice), and 1.8 ± 0.8 (n = 2 mice) pA/pF, respectively. In contrast, increases in pH_i to 7.5, 7.6, and 7.8 cause progressively larger and prolonged inward currents, averaging 9.2 ± 1.8 (n = 3 mice), 16.3 ± 4.1 (n = 4 mice), and 27.5 ± 8.5 pA/pF (n = 3 mice), respectively (P = 0.021 by ANOVA). All panels include responses of individual neurons with means ± SE. Statistical comparisons are unpaired 2-tailed Student’s t test, except G.