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pH modulation of Ca²⁺ responses and a Ca²⁺-dependent K⁺ channel in cultured rat hippocampal neurones

John Church, Keith A. Baxter and James G. McLarnon *

1. The effects of changes in extra- and intracellular pH (pH_o and pH_i, respectively) on depolarization-evoked rises in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and the activity of a Ca²⁺-dependent K⁺ channel were investigated in cultured fetal rat hippocampal neurones.

2. In neurones loaded with 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF), changes in pH_o evoked changes in pH_i. At room temperature, the ratio pH_i : pH_o (the slope of the regression line relating pH_i to pH_o) was 0·37 under HCO₃^-/CO₂-buffered conditions and 0·45 under Hepes-buffered conditions; corresponding values at 37 °C were 0·71 and 0·79, respectively. The measurements of changes in pH_i evoked by changes in pH_o were employed in subsequent experiments to correct for the effects of changes in pH_i on the K_d of fura-2 for Ca²⁺.

3. In fura-2-loaded neurones, rises in [Ca²⁺]_i evoked by transient exposure to 50 mM K⁺ were reduced and enhanced during perfusion with acidic and alkaline media, respectively, compared with control responses at pH_o 7·3. Fifty percent inhibition of high-[K⁺]_o-evoked rises in [Ca²⁺]_i corresponded to pH_o 7·23. In the presence of 10 µM nifedipine, 50 % inhibition of high-[K⁺]_o-evoked responses corresponded to pH_o 7·20, compared with a pH_o of 7·31 for 50 % inhibition of [Ca²⁺]_i transients evoked by N-methyl-D-aspartate.

4. Changes in pH_i at a constant pH_o were evoked by exposing neurones to weak acids or bases and quantified in BCECF-loaded cells. Following pH-dependent corrections for the K_d of fura-2 for Ca²⁺, rises in [Ca²⁺]_i evoked by high-[K⁺]_o in fura-2-loaded cells were found to be affected only marginally by changes in pH_i. When changes in pH_i similar to those observed during the application of weak acids or bases were elicited by changing pH_o, reductions in pH inhibited rises in [Ca²⁺]_i evoked by 50 mM K⁺ whereas increases in pH enhanced them.

5. The effects of changes in pH on the kinetic properties of a BK-type Ca²⁺-dependent K⁺ channel were investigated. In inside-out patches excised from neurones in sister cultures to those used in the microspectrofluorimetric studies, with internal [Ca²⁺] at 20 µM, channel openings at an internal pH of 6·7 were generally absent whereas at pH 7·3 (or 7·8) the open probability was high. In contrast, channel activity in outside-out patches was not affected by reducing the pH of the bath (external) solution from 7·3 to 6·7. In inside-out patches with internal [Ca²⁺] at 0·7 µM, a separate protocol was applied to generate transient activation of the channel at a potential of 0 mV following a step from a holding level of -80 mV. In this case open probabilities were 0·81 (at pH 7·8), 0·57 (pH 7·3), 0·19 (pH 7·0) and 0·04 (pH 6·7). Channel conductance was not affected by changes in internal pH.

6. The results indicate that, in fetal rat hippocampal neurones, depolarization-evoked rises in [Ca²⁺]_i mediated by the influx of Ca²⁺ ions through dihydropyridine-sensitive and -resistant voltage-activated Ca²⁺ channels are modulated by changes in pH_o. The effects of pH_o cannot be accounted for by changes in pH_i consequent upon changes in pH_o. However, changes in pH_i affect the unitary properties of a Ca²⁺-dependent K⁺ channel. The results support the notion that pH_o and/or pH_i transients may serve a modulatory role in neuronal function.

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