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Voltage-gated proton channels help regulate pH_i in rat alveolar epithelium

Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois

Submitted 9 August 2004 ; accepted in final form 25 October 2004

Voltage-gated proton channels are expressed highly in rat alveolar epithelial cells. Here we investigated whether these channels contribute to pH regulation. The intracellular pH (pH_i) was monitored using BCECF in cultured alveolar epithelial cell monolayers and found to be 7.13 in nominally HCO₃^–-free solutions [at external pH (pH_o) 7.4]. Cells were acid-loaded by the NH₄⁺ prepulse technique, and the recovery was observed. Under conditions designed to eliminate the contribution of other transporters that alter pH, addition of 10 µM ZnCl₂, a proton channel inhibitor, slowed recovery about twofold. In addition, the pH_i minimum was lower, and the time to nadir was increased. Slowing of recovery by ZnCl₂ was observed at pH_o 7.4 and pH_o 8.0 and in normal and high-K⁺ Ringer solutions. The observed rate of Zn²⁺-sensitive pH_i recovery required activation of a small fraction of the available proton conductance. We conclude that proton channels contribute to pH_i recovery after an acid load in rat alveolar epithelial cells. Addition of ZnCl₂ had no effect on pH_i in unchallenged cells, consistent with the expectation that proton channels are not open in resting cells. After inhibition of all known pH regulators, slow pH_i recovery persisted, suggesting the existence of a yet-undefined acid extrusion mechanism in these cells.

proton conductance; pH regulation; hydrogen ion; acid load; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; intracellular pH

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