Hydrogen ion dynamics in human red blood cells

Authors

  • Pawel Swietach,

    1. Department of Physiology, Anatomy and Genetics, Burdon Sanderson Cardiac Science Centre, Parks Road, Oxford OX1 3PT, UK
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  • Teresa Tiffert,

    1. Physiological Laboratory, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK
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  • Jakob M. A. Mauritz,

    1. Physiological Laboratory, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK
    2. Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge CB2 3RA, UK
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  • Rachel Seear,

    1. Physiological Laboratory, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK
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  • Alessandro Esposito,

    1. Physiological Laboratory, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK
    2. Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge CB2 3RA, UK
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  • Clemens F. Kaminski,

    1. Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge CB2 3RA, UK
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  • Virgilio L. Lew,

    1. Physiological Laboratory, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK
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  • Richard D. Vaughan-Jones

    1. Department of Physiology, Anatomy and Genetics, Burdon Sanderson Cardiac Science Centre, Parks Road, Oxford OX1 3PT, UK
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Corresponding author P. Swietach: Department of Physiology, Anatomy and Genetics, Burdon Sanderson Cardiac Science Centre, Parks Road, Oxford OX1 3PT, UK.  Email: pawel.swietach@dpag.ox.ac.uk

Abstract

Our understanding of pH regulation within red blood cells (RBCs) has been inferred mainly from indirect experiments rather than from in situ measurements of intracellular pH (pHi). The present work shows that carboxy-SNARF-1, a pH fluorophore, when used with confocal imaging or flow cytometry, reliably reports pHi in individual, human RBCs, provided intracellular fluorescence is calibrated using a ‘null-point’ procedure. Mean pHi was 7.25 in CO2/HCO3-buffered medium and 7.15 in Hepes-buffered medium, and varied linearly with extracellular pH (slope of 0.77). Intrinsic (non-CO2/HCO3-dependent) buffering power, estimated in the intact cell (85 mmol (l cell)−1 (pH unit)−1 at resting pHi), was somewhat higher than previous estimates from cell lysates (50–70 mmol (l cell)−1 (pH unit)−1). Acute displacement of pHi (superfusion of weak acids/bases) triggered rapid pHi recovery. This was mediated via membrane Cl/HCO3 exchange (the AE1 gene product), irrespective of whether recovery was from an intracellular acid or base load, and with no evident contribution from other transporters such as Na+/H+ exchange. H+-equivalent flux through AE1 was a linear function of [H+]i and reversed at resting pHi, indicating that its activity is not allosterically regulated by pHi, in contrast to other AE isoforms. By simultaneously monitoring pHi and markers of cell volume, a functional link between membrane ion transport, volume and pHi was demonstrated. RBC pHi is therefore tightly regulated via AE1 activity, but modulated during changes of cell volume. A comparable volume–pHi link may also be important in other cell types expressing anion exchangers. Direct measurement of pHi should be useful in future investigations of RBC physiology and pathology.

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