VOLTAGE-DEPENDENT CLAMP OF INTRACELLULAR PH OF IDENTIFIED LEECH GLIAL-CELLS

1. The intracellular pH (pH(i)) was measured in voltage-clamped, giant neuropile glial cells in isolated segmental ganglia of the leech Hirudo medicinalis, using double-barrelled, pH-sensitive microelectrodes and a slow, two-electrode voltage-clamp system. The potential sensitivity of the pH(i) regulation in these glial cells was found to be due to an electrogenic Na+-HCO3- cotransporter (Deitmer & Szatkowski, 1990). 2. In the presence of 5% CO2 and 24 mM HCO3- (pH 7.4), pH(i) shifted by 1 pH unit per 110 mV, corresponding to a stoichiometry of 2 HCO3- : 1 Na+ of the cotransporter, while in Hepes-buffered CO2-HCO3--free saline (pH 7.4), pH(i) changed by 1 pH unit per 274 mV. The potential sensitivity of pH(i) decreased at lower pH(o), being 1 pH unit per 216 mV at external pH (pH(o)) 7.0. 3. Changing pH(o) between 7.8 and 6.6 induced pH(i) shifts with a slope of 0.72 pH(i) units per pH(o) unit in non-clamped, and of 0.80 pH(i) units per pH(o) unit in voltage-clamped cells, indicating that pH(i) largely followed pH(o). The electrochemical gradient of H+-HCO3- across the glial membrane was around 56 mV, and remained almost constant over this pH(o) range. 4. The membrane potential-dependent and pH(o)-sensitive shifts of pH(i) were unaffected by amiloride, an inhibitor of Na+-H+ exchange. 5. The intracellular acidification upon lowering pH(o) could be reversed by depolarizing the membrane as predicted from a cotransporter, whose equilibrium follows the membrane potential by resetting pH(i). 6. The results indicate that the pH(i) of leech glial cells is dominated by the electrogenic Na+-HCO3- cotransporter, and is hence a function of the membrane potential, and the Na+ and H+-HCO3- gradients, across the cell membrane.