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

Abstract

1. The effects of changes in extra- and intracellular pH (pHo and pHi, 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 pHo evoked changes in pHi. At room temperature, the ratio DeltapHi : DeltapHo (the slope of the regression line relating pHi to pHo) 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 pHi evoked by changes in pHo were employed in subsequent experiments to correct for the effects of changes in pHi on the Kd 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 pHo 7.3. Fifty percent inhibition of high-[K+]o-evoked rises in [Ca2+]i corresponded to pHo 7.23. In the presence of 10 microM nifedipine, 50 % inhibition of high-[K+]o-evoked responses corresponded to pHo 7.20, compared with a pHo of 7.31 for 50% inhibition of [Ca2+]i transients evoked by N-methyl-D-aspartate. 4. Changes in pHi at a constant pHo were evoked by exposing neurones to weak acids or bases and quantified in BCECF-loaded cells. Following pH-dependent corrections for the Kd 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 pHi. When changes in pHi similar to those observed during the application of weak acids or bases were elicited by changing pHo, 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 microM, 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 microM, 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 pHo. The effects of pHo cannot be accounted for by changes in pHi consequent upon changes in pHo. However, changes in pHi affect the unitary properties of a Ca2+-dependent K+ channel. The results support the notion that pHo and/or pHi transients may serve a modulatory role in neuronal function.

Figure 2. Changes in pH_o modulate NMDA-evoked increases in I₃₃₄/I₃₈₀ ratio values

A, under control conditions (pH_o 7.3), transient exposures to 50 mM [K⁺]_o (○) or 20 μM NMDA (□) evoked rises in the I₃₃₄/I₃₈₀ ratio value. Subsequent addition of 10 μM nifedipine to the Hepes-buffered perfusion medium reduced high-[K⁺]_o-evoked increases in the ratio value by 81 % (third, fourth and fifth responses) and NMDA-evoked increases in the ratio value by 54 % (sixth response). The pH of the perfusion medium was then changed sequentially from 6.7 to 7.9 in 0.2 pH unit increments, for the periods indicated by the bars above the trace, resulting in a gradual increase in NMDA-evoked rises in the I₃₃₄/I₃₈₀ ratio values. The final four responses shown are K⁺- and NMDA-evoked responses upon return to pH 7.3 medium. The record is a mean of data obtained from 10 neurones simultaneously, the experiment being performed at room temperature. B, pH-dependent corrections for the K_d of fura-2 for Ca²⁺ (see Methods) were applied to the data shown in A and other, similar experiments and a plot was made of pH_oversus changes in peak [Ca²⁺]_i responses evoked by NMDA in the presence of 10 μM nifedipine, normalized to the peak of the [Ca²⁺]_i response obtained at pH_o 7.3. Each point represents data obtained from a minimum of 3 neuronal populations. The 4-parameter logistic plot (r²= 0.99) had a pK of 7.31. The extrapolated maximum and minimum values were 160 and 47 %, respectively.

Figure 3. Changes in pH_o modulate high-[K⁺]_o-evoked increases in I₃₃₄/I₃₈₀ ratio values

A, under control conditions (pH_o 7.3) two consecutive applications of 50 mM [K⁺]_o evoked stable rises in the I₃₃₄/I₃₈₀ ratio. Subsequent responses to 50 mM K⁺ were obtained at the pH_o values indicated by the bars above the trace. The final 2 responses were obtained upon reperfusion with pH 7.3 medium. The record is a mean of data obtained from 16 neurones simultaneously. The experiment was performed at room temperature in HCO₃⁻/CO₂-buffered media. B, pH-dependent corrections for the K_d of fura-2 for Ca²⁺ (see Methods) were applied to the data shown in A and other, similar experiments and a plot was made of pH_oversus changes in peak high-[K⁺]_o-evoked [Ca²⁺]_i responses, normalized to the peak of the [Ca²⁺]_i response obtained at pH_o 7.3. Each point represents data obtained from a minimum of 4 neuronal populations. The 4-parameter logistic plot (r²= 0.99) had a pK of 7.23 and extrapolated maximum and minimum values of 158 and 31 %, respectively. C, under control conditions (pH_o 7.3) three consecutive applications of 50 mM [K⁺]_o evoked stable rises in the I₃₃₄/I₃₈₀ ratio. Addition of 10 μM nifedipine reduced the high-[K⁺]_o-evoked rises in the I₃₃₄/I₃₈₀ ratio by ≈80 % (fourth and fifth responses). Subsequent responses to 50 mM K⁺ were obtained at the pH_o values indicated by the bars above the trace. The final two responses were obtained upon reperfusion with pH 7.3 medium. The record is a mean of data obtained from 7 neurones simultaneously. The experiment was performed at room temperature in Hepes-buffered media containing 40 μM AP5 and 20 μM CNQX throughout. D, pH-dependent corrections for the K_d of fura-2 for Ca²⁺ were applied to the data shown in C and other, similar experiments and a plot was made of pH_oversus changes in peak high-[K⁺]_o-evoked [Ca²⁺]_i responses, normalized to the peak of the [Ca²⁺]_i response obtained at pH_o 7.3. Each point represents data obtained from a minimum of 4 neuronal populations. The 4-parameter logistic plot (r²= 0.99) had a pK of 7.20 and extrapolated maximum and minimum values of 131 and 54 %, respectively.

Figure 4. Modulation of high-[K⁺]_o-evoked increases in [Ca²⁺]_i by changes in pH_i

A, a 10 min application of the weak base trimethylamine (TMA, 10 mM) evoked a rise in pH_i. Following the withdrawal of TMA, pH_i fell to values below the initial resting level and then recovered gradually. The experiment was performed at room temperature in HCO₃⁻/CO₂-buffered media (pH 7.3). The record is a mean of data obtained from 17 neurones simultaneously. B, under control conditions (pH_o 7.3) an application of 50 mM [K⁺]_o evoked a rise in [Ca²⁺]_i (first response). Subsequent responses to 50 mM K⁺ were obtained at 6 min after the start of a 10 min period of perfusion with a pH 7.3 medium containing 10 mM TMA (second response), and at 5 and 25 min following the withdrawal of TMA (third and fourth responses, respectively). A single K_d value for fura-2 (169.13, corresponding to a resting pH_i of 6.93, measured in 5 experiments of the type illustrated in A) was employed to generate the continuous line. The open circles (○) represent the peak of the rise in [Ca²⁺]_i computed using a K_d 14 % lower than that employed for the continuous line, to reflect the rise in pH_i observed at 6 min after the start of application of TMA (see Results). The open squares (□) represent the peak of the rise in [Ca²⁺]_i computed using a K_d 19 % higher than that employed for the continuous line, to reflect the fall in pH_i observed at 5 min following the withdrawal of TMA (see Results). The record is a mean of data obtained from 14 neurones simultaneously. The experiment was performed in a sister culture to that employed in the experiment shown in A, under identical conditions.

Figure 5. Comparison of the effects of changes in pH_i and pH_o on high-[K⁺]_o-evoked increases in [Ca²⁺]_i

A, a 16 min application of the weak acid propionate (PROP, 40 mM) evoked a fall in pH_i. Following the recovery of pH_i to the initial resting level, the pH of the perfusate was reduced from 7.3 to 6.85 for 20 min. pH_i fell gradually and recovered to the initial resting level upon re-perfusion with pH 7.3 medium. The experiment was performed at room temperature in HCO₃⁻/CO₂-buffered media. The record is a mean of data obtained from 7 neurones simultaneously. B, under control conditions (pH_o 7.3) two consecutive applications of 50 mM [K⁺]_o evoked stable rises in [Ca²⁺]_i (first and second responses). The third response was obtained 8 min after the start of a 16 min period of perfusion with a pH 7.3 medium containing 40 mM propionate. The final three responses were obtained immediately prior to perfusion with pH 6.85 medium, at 16 min following the start of a 20 min exposure to pH 6.85 medium, and during a period of reperfusion with pH 7.3 medium. A single K_d value for fura-2 (corresponding to the resting pH_i measured in 3 experiments of the type illustrated in A) was employed to generate the continuous line. The open circles (○) represent the peak of the rise in [Ca²⁺]_i computed using a K_d 14 % higher than that employed for the continuous line, to reflect the fall in pH_i observed at 8 min after the start of application of propionate (see Results). The open squares (□) represent the peak of the rise in [Ca²⁺]_i computed using a K_d 16 % higher than that employed for the continuous line, to reflect the fall in pH_i observed at 16 min following the start of exposure to pH 6.85 medium (see Results). The record is a mean of data obtained from 6 neurones simultaneously. The experiment was performed in a sister culture to that employed in the experiment shown in A, under identical conditions.

Figure 1. Dependence of pH_i on pH_o

A, in an experiment conducted at 37 °C under HCO₃⁻/CO₂-buffered conditions, decreasing or increasing pH_o for the periods indicated by the bars above the trace and to the values shown above the bars, decreased and increased pH_i, respectively. The record is a mean of data obtained from 9 neurones simultaneously. B, linear regression analysis of the dependence of pH_i on pH_o at both room temperature (•) and at 37 °C (▪) under HCO₃⁻/CO₂-buffered conditions and under Hepes-buffered conditions (○ and □, respectively). Each point represents data obtained from a minimum of 4 neuronal populations; error bars are s.e.m. The equations relating pH_i to pH_o at room temperature and at 37 °C under HCO₃⁻/CO₂-buffered conditions were, respectively, pH_i= 4.27 + (0.37 × pH_o) (r²= 0.99) and pH_i= 1.92 + (0.71 × pH_o) (r²= 0.99). The equations relating pH_i to pH_o at room temperature and at 37 °C under Hepes-buffered conditions were, respectively, pH_i= 3.60 + (0.45 × pH_o) (r²= 0.98) and pH_i= 1.52 + (0.79 × pH_o) (r²= 0.99).

Figure 6. Unitary properties of a Ca²⁺-dependent K⁺ channel

A, single channel current recordings from an inside-out patch with internal (bath) [Ca²⁺] at 20 μM (upper trace) and lack of activity when internal [Ca²⁺] was decreased to 0.7 μM (lower trace). In both records, the internal pH was 7.3 and the patch potential was +20 mV. Channel activity was restored upon reperfusion with medium containing 20 μM Ca²⁺ (not shown). B, openings from a different inside-out patch with internal [Ca²⁺] at 20 μM and internal pH values of 7.3 (upper trace), 6.7 (middle trace) and following return to pH 7.3 (lower trace). The patch potential was +20 mV for all records. C, distribution of open times (at V=+20 mV) at pH 7.3 (collation of 462 events); the mean open time was 19.8 ms. D, distribution of open times (at V=+20 mV) at pH 6.7 (collation of 107 events); the mean open time was 5.4 ms.

Figure 7. Effects of changes in internal pH on transient activation of I_BK(Ca)

The protocol was to initially hold the potential of the inside-out patch at -80 mV for 10 s and then to step V to 0 mV. The records shown commence 4 ms following the steps to 0 mV. No further events were evident following the final closures shown in each of the traces, despite a maintained potential of 0 mV (see McLarnon, 1995). P_o values were determined from the analysis of 10 steps, for times of 70 ms following each step, for each of the pH values tested; internal [Ca²⁺] was 0.7 μM throughout.