Hydrogen ion dynamics in human red blood cells

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.Red blood cells are essential for the transport of oxygen and carbon dioxide around the body. Their ability to carry these gases depends strongly on intracellular pH (acid-base balance). Using a pH-sensitive dye, we were able to study, for the first time, dynamic changes of pH inside individual red blood cells. Using this approach, we have characterised the physiological mechanisms that regulate red blood cell pH in response to acid/base disturbances. Proteins, called anion exchangers, swap bicarbonate (HCO-3) for chloride anions across the cell membrane. These regulate the cell's pH. We show that the activity of these proteins underlies an important link between pH and cell volume, which has previously been predicted to occur in sickle-cell disease. We have extended our measurement system to sample pH from a large population of cells. These measurements will be useful for studying pH-related phenomena that occur in healthy or diseased red blood cells. © 2010 The Authors. Journal compilation © 2010 The Physiological Society.