Intracellular pH regulation of CA1 neurons in Na+/H+ isoform 1 mutant mice
Hang Yao, Enbo Ma, Xiang-Qun Gu, Gabriel G. Haddad
Hang Yao, Enbo Ma, Xiang-Qun Gu, Gabriel G. Haddad
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Intracellular pH regulation of CA1 neurons in Na+/H+ isoform 1 mutant mice

Abstract

To understand the role of Na+/H+ exchanger 1 (NHE1) in intracellular pH (pHi) regulation and neuronal function, we took advantage of natural knockout mice lacking NHE1, the most ubiquitously and densely expressed NHE isoform in the central nervous system (CNS). CA1 neurons from both wild-type (WT) and NHE1 mutant mice were studied by continuous monitoring of pHi, using the fluorescent indicator carboxy-seminaphthorhodafluor-1 (SNARF-1) and confocal microscopy. In the nominal absence of CO2/HCO3–, steady-state pHi was higher in WT neurons than in mutant neurons. Using the NH4Cl prepulse technique, we also show that H+ flux in WT neurons was much greater than in mutant neurons. The recovery from acid load was blocked in WT neurons, but not in mutant neurons, by removal of Na+ from the extracellular solution or by using 100 μM 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate (HOE 694) in HEPES buffer. Surprisingly, in the presence of CO2/HCO3–, the difference in H+ flux between WT and mutant mice was even more exaggerated, with a difference of more than 250 μM/s between them at pH 6.6. H+ flux in CO2/HCO3– was responsive to diisothiocyanato-stilbene-2,2′-disulfonate (DIDS) in the WT but not in the mutant. We conclude that (a) the absence of NHE1 in the mutant neurons tended to cause lower steady-state pHi and, perhaps more importantly, markedly reduced the rate of recovery from an acid load; and (b) this difference in the rate of recovery between mutant and WT neurons was surprisingly larger in the presence, rather than in the absence, of HCO3–, indicating that the presence of NHE1 is essential for the regulation and/or functional expression of both HCO3–-dependent and -independent transporters in neurons.

Authors

Hang Yao, Enbo Ma, Xiang-Qun Gu, Gabriel G. Haddad

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Examination of recovery from acid loading in both WT and NHE1 mutant CA1...
Examination of recovery from acid loading in both WT and NHE1 mutant CA1 neurons in the presence and absence of CO2/HCO3–. (a) CO2/HCO3– enhances the recovery from an acid load in a WT CA1 neuron. A single CA1 neuron was acid-loaded twice with HEPES solution containing 20 mM NH4Cl. The cell was allowed to recover first in the absence, and then in the presence, of CO2/HCO3–. This WT cell promptly recovered from the acid load in both solutions, and with a faster recovery rate when the CO2/HCO3– was present. (b) CA1 neurons isolated from NHE1 mutant mice exhibit different patterns of pHi recovery from acid load. Although recovery from acid loads was present in this mutant neuron, the rates in the presence or absence of CO2/HCO3– were lower than in the WT. (c) Another mutant neuron was studied. In this experiment, the mutant neuron does not recover in HEPES buffer, even 10 minutes after acid loading. (d) With CO2/HCO3–, pHi of this kind of mutant cell recovers from acid loading to the starting level, albeit at a much reduced rate. At the end of each experiment, the cell was exposed to nigericin calibration solution (pH = 7.0) for calibration.