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

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 Delta pH(i): Delta 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 degrees 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 mnr 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 mu M 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 bq 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+ 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 mu M, channel openings at an internal pH of 6.7 mere 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 mu M, 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 mas serve a modulatory role in neuronal function.