Regulation of AE2-mediated Cl⁻ Transport by Intracellular or by Extracellular pH Requires Highly Conserved Amino Acid Residues of the AE2 NH₂-terminal Cytoplasmic Domain

A.K. Stewart's present address is University Laboratory of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, UK.

Abbreviation used in this paper: aa, amino acids.

Lowering bath pH from 7.4 to 5.0 in the presence of 5 μM butyrate lowered pH_i by 0.13 ± 0.017 pH units (n = 6) within 15 min (a single case is shown in Fig. 1 B), but only 0.07 ± 0.01 pH units (n = 3) within the same period in the absence of extracellular butyrate (Fig. 1 A presents a representative single trace). Thus, an acidification of ∼0.06 pH units can be attributed to butyric acid entry over this time period in this condition. With mean resting pH_i = 7.34 ± 0.02 (n = 11) at pH_o 7.40 (7.21 in example of Fig. 1 C), addition of 5 μM butyrate at pH_o 5.0 is predicted to lead to an equilibrium intracellular butyrate concentration of 0.66 mM. (This is a minimum estimate that assumes free diffusion of butyric acid and no permeability of butyrate anion.) Proton flux represented by this acidification requires knowledge of oocyte buffer capacity. The intrinsic buffer capacity of the oocyte has been estimated by the fall in pH_i after response to a CO₂ pulse of a single concentration (5% CO₂/95% air). In eight oocytes with mean resting pH_i of 7.24 ± 0.06 under HEPES-buffered (i.e., nominally CO₂/HCO₃⁻ free) conditions, intrinsic buffer capacity was 18.9 ± 1.8 mM/pH unit (n = 8) measured at the midpoint of the 5% CO₂-induced pH_i change, 7.00 ± 0.04. This value for oocyte intrinsic buffer capacity agrees with that of 19.8 mM/pH unit (Cooper and Boron, 1998), and with the value for Xenopus early embryo of 18 mM/pH unit (Turin and Warner, 1980). The magnitude of butyric acid entry required to acidify oocytes with intrinsic buffer capacity of 18.9 mM/pH unit is predicted to be 1.13 mM. This value is not much greater than the minimum estimate of 0.66 mM butyrate calculated to have entered the average oocyte in our experiments, especially considering the variation in recorded pH_i with depth of microelectrode penetration, and the strong dependence of intrinsic buffer capacity upon oocyte pH_i prior to CO₂ pulse (buffer capacity values estimated from multiple published pH_i traces of individual oocytes range between 9 and 21 mM/pH unit).

AE2 (A)₆342-347 is the only hexa-Ala substitution mutant among those tested that failed to approach zero activity at pH_o 5.0. Therefore, the curve fit to the data of the AE2 mutant (A)₆342-347 (inverted triangles) differed from all others computed for this work, such that an extra parameter was used in the sigmoid fit, in order not to force the fit through a zero point (see materials and methods). The pH_o(50) value of 6.11 ± 0.11 arising from this fit for AE2 (A)₆342-347 and shown in Fig. 2 D can be considered a maximum estimate. The curve fit using the standard sigmoid equation yields a predicted pH_o(50) value of 5.62 ± 0.20, with lower confidence limits.

Muller-Berger et al. (1995) found that reduction of bath pH to 6.1 either from 7.2 or from 8.3 did not change oocyte pH_i when measured after 90 min preincubation (in 110 mM KCl Barth's solution). Thus, 90 min preincubation at the desired pH_o achieved conditions of nominal pH_i-clamp which were suitable for their experiments in which each oocyte was exposed first to control pH and then to a single altered bath pH. Our experimental protocol exposed each oocyte to the complete range of bath pH values in the course of a single efflux experiment (Stewart et al., 2001 and the current work). In this setting, pH_i measured either by pH-sensitive microelectrode (Fig. 1) or by BCECF ratio fluorimetry (Zhang et al., 1996; unpublished data) did change acutely in parallel with changing pH_o. Thus, our experimental conditions required coincident changes in pH_o and butyrate concentration to achieve oocyte pH_i clamp. These and other experimental differences might contribute to the difference of ≥1 pH unit between the AE1 pH_o(50) values reported by Muller-Berger et al. (1995) and Zhang et al. (1996). Muller-Berger et al. (1995) lowered pH_i at constant pH_o through use of 40 or 60 mM NH₄Cl. In our experiments, 20 mM NH₄Cl did not inhibit AE1. The use of NH₄Cl for intracellular acidification at constant pH_o could not be applied to experiments with AE2, since NH₄Cl activates AE2 despite its accompanying acidification of the oocyte (Humphreys et al., 1997).