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¹ Department of Physiology, University of British Columbia, 2177 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3 ² Department of Anatomy & Cell Biology, University of British Columbia, 2177 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3

	Abstract

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We examined the effects of changes in pH_o and pH_i on currents contributing to the medium and slow afterhyperpolarizations (mI_AHP and sI_AHP, respectively) in rat CA1 neurones. Reducing pH_o from 7.4 to 6.7 inhibited mI_AHP and sI_AHP whereas increasing pH_o to 7.7 augmented mI_AHP and, to a greater extent, sI_AHP. The ability of changes in pH_o to modulate mI_AHP reflected changes in the Ca²⁺-activated K⁺ current, I_AHP, and a Co²⁺- and XE991-resistant component of mI_AHP, but not the muscarine-sensitive current, I_M. In the presence of 1 µM TTX and 5 mM TEA, low pH_o-evoked reductions in sI_AHP were associated with reductions in Ca²⁺-dependent depolarizing potentials; because neither effect was attenuated when internal H⁺ buffering power was raised by including 100 mM tricine in the patch pipette, the actions of reductions in pH_o to inhibit sI_AHP and, possibly, I_AHP in large part appear to reflect a low pH_o-dependent decrease in Ca²⁺ influx. In contrast, the effects of high pH_o to augment mI_AHP and sI_AHP were associated with relatively small increases in Ca²⁺ potentials but were significantly attenuated by 100 mM internal tricine, indicating that a rise in pH_i consequent upon the rise in pH_o was largely responsible. The possibility that changes in pH_i could act to modulate mI_AHP and sI_AHP, independently of changes in Ca²⁺ influx, was also suggested by experiments in which pH_i was lowered at a constant pH_o by the external application of propionate or by the withdrawal of HCO^-₃ from the perfusing medium. Lowering pH_i at a constant pH_o had little effect on Ca²⁺ potentials but inhibited mI_AHP and, to a greater extent, sI_AHP, effects that were attenuated by 100 mM internal tricine. Together, the results indicate that changes in pH_o and pH_i modulate mI_AHP and sI_AHP in rat CA1 neurones and suggest that, depending on the direction of the pH_o change, the sensitivities of the underlying currents to changes in Ca²⁺ influx and/or pH_i may contribute to the effects of changes in pH_o to modulate mI_AHP and sI_AHP.

(Received 18 June 2003; accepted after revision 3 November 2003; first published online 7 November 2003)
Corresponding author J. Church: Department of Anatomy & Cell Biology, University of British Columbia, 2177 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3.  Email: jchurch{at}interchange.ubc.ca

	Introduction

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In rat hippocampal CA1 pyramidal neurones, action potential(s) elicit temporally distinct afterhyperpolarizations (AHPs). Single spikes are followed by a fast AHP mediated by BK-type large-conductance Ca²⁺-activated K⁺ channels whereas spike trains elicit medium and slow AHPs that can be separated by kinetic and pharmacological criteria (reviewed by Storm, 1990; Vergara et al. 1998; Sah & Faber, 2002). The medium AHP (or current, mI_AHP) peaks rapidly and reflects, in part, the activation of both the apamin-sensitive Ca²⁺-activated K⁺ current, I_AHP, which is mediated by SK-type small-conductance Ca²⁺-activated K⁺ channels, and the muscarine-sensitive voltage-gated K⁺ current, I_M (Storm, 1989; Williamson & Alger, 1990; Köhler et al. 1996; Stocker et al. 1999; Sailer et al. 2002). In contrast, the slow AHP (or current, sI_AHP) follows a slower time course and has been ascribed to the activation of apamin-insensitive Ca²⁺-activated K⁺ channels, the molecular identity of which remains subject to debate (see Lancaster & Adams, 1986; Sah & Isaacson, 1995; Sah & Faber, 2002).

As befits their important roles in determining the excitability and firing patterns of central neurones, each of the above AHPs can be modulated by neurotransmitters acting through a variety of signal transduction pathways (Madison & Nicoll, 1986; Charpak et al. 1990; Holm et al. 1997; Haug & Storm, 2000; Melyan et al. 2002). They can also be modulated by changes in pH. Thus, reductions and increases in pH_o, respectively, attenuate and augment the fast AHP in CA1 neurones, effects that cannot be ascribed in their entirety to pH_o-induced changes in Ca²⁺ influx (Church & McLennan, 1989; Church, 1999). Rather, neuronal pH_i is steeply dependent on pH_o (e.g. Ou-Yang et al. 1993; Church et al. 1998) and, in agreement with findings in a variety of non-neuronal cell types (e.g. Cook et al. 1984; Kume et al. 1990; Copello et al. 1991; Laurido et al. 1991; Hayabuchi et al. 1998), changes in [H⁺]_i affect directly the unitary properties of the BK-type channel that underlies the fast AHP in CA1 neurones (Church et al. 1998; also see Liu et al. 1999). Changes in pH_o also modulate the medium and slow AHPs in CA1 neurones, effects that can be mimicked by changes in pH_i at a constant pH_o (Church & McLennan, 1989; Church, 1992, 1999). However, the pH sensitivities of the currents that contribute to the medium and slow AHPs have not been investigated and it remains unclear whether the effects of changes in pH_o and/or pH_i on these AHPs might be secondary to changes in Ca²⁺ influx.

In the present study we used the whole-cell patch-clamp technique to investigate the modulation of mI_AHP and sI_AHP by extra- and intracellular pH changes in CA1 pyramidal neurones in rat hippocampal slices. The results indicate that sI_AHP and, to a lesser extent, mI_AHP are sensitive to changes in pH_i and suggest that these effects may contribute to high pH_o-evoked increases in the currents. In contrast, a reduction in Ca²⁺ influx appears to be the major determinant of the inhibition of mI_AHP and sI_AHP observed at low pH_o.

	Methods

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Cell preparation

All procedures conformed to guidelines established by the Canadian Council on Animal Care and were approved by The University of British Columbia Animal Care Committee.

Transverse hippocampal slices (400 µm) were prepared from 17- to 21-day-old (patch pipette recordings) or 40-day-old (sharp microelectrode recordings) Wistar rats as previously described (Church & McLennan, 1989). In brief, animals were anaesthetized with 3% halothane in air, decapitated and the brains rapidly removed. Hippocampal slices were then cut and allowed to recover for at least 1 h in a holding chamber at room temperature (RT, 19–23°C). Individual slices were transferred as needed to the recording chamber and placed on a nylon mesh at the interface between a humidified atmosphere and a continuously (2 ml min^-1) superfusing medium at RT (patch pipette recordings) or 34°C (sharp microelectrode recordings).

Solutions and chemicals

The standard HCO^-₃/CO₂-buffered media employed at RT and 34°C (in parentheses) contained (mM): NaCl 123 (129), KCl 3, NaHCO₃ 26 (20), NaH₂PO₄ 1.5, MgSO₄ 1.5, D-glucose 10 and CaCl₂ 2 (pH 7.4 after equilibration with 5% CO₂–95% O₂). Low pH HCO^-₃/CO₂-buffered solutions contained either 5 mM or 4.5 mM NaHCO₃ (pH 6.7 at RT and 34°C, respectively) and high pH HCO^-₃/CO₂-buffered solutions contained either 55 mM or 47 mM NaHCO₃ (pH 7.7 at RT and 34°C, respectively); changes in [NaHCO₃] were balanced by equimolar changes in [NaCl]. For Ca²⁺-free medium, Ca²⁺ was omitted, [Mg²⁺] was increased to 3.5 mM and 200 µM EGTA was added. In solutions containing 2 mM CoCl₂, H₂PO^-₄ and SO^2-₄ were omitted. HCO^-₃/CO₂-buffered solutions containing Hepes (100 mM), the weak acid sodium propionate (40 mM) or the weak bases NH₄Cl or trimethylamine HCl (TMA) (40 mM in each case) were prepared by equimolar substitution for NaCl. In HCO^-₃ free media, 30 mM Hepes or 30 mMN-[tris(hydroxymethyl)methyl]glycine (tricine) isosmotically replaced NaHCO₃ and an appropriate concentration of NaCl; these media were saturated with O₂ and titrated to the appropriate temperature-corrected pH with 10 M NaOH. Hepes and tricine were employed at 30 mM to prevent the reduction in slice interstitial pH that would otherwise occur upon the transition from a HCO^-₃ containing to a HCO^-₃ free medium (see Cowan & Martin, 1995). During perfusion with HCO^-₃ containing media, the atmosphere in the recording chamber contained 5% CO₂–95% O₂; this was switched to 100% O₂ upon the introduction of HCO^-₃ free media. The pH of each solution was re-checked at the appropriate temperature following every experiment.

Salts and experimental compounds, which were applied by superfusion, were obtained from Sigma-Aldrich Canada Ltd (Oakville, ON, Canada), with the exceptions of potassium methylsulphate (ICN Pharmaceuticals Canada Ltd, Montréal, QC, Canada) and XE991 (a gift from Bristol-Myers Squibb Co., Wilmington, DE, USA).

Recording techniques

Whole-cell patch pipette current- and voltage-clamp recordings were obtained from CA1 neurones by the ‘blind’ technique. Patch pipettes were pulled from 1.2 mm o.d. x 0.9 mm i.d. borosilicate tubing (World Precision Instruments Inc., Sarasota, FL, USA) and heat-polished; open pipette resistance was 2–5 M when filled with one of the solutions detailed in Table 1. After a seal >1 G was achieved, recordings were made (Axoclamp 2, Axon Instruments Inc., Union City, CA, USA) when the application of light suction revealed a resting membrane potential (V_m) of at least –50 mV (after subtraction of the 5–7 mV junction and tip potentials; Neher, 1992), overshooting action potentials, an input resistance (R_in, measured at –50 mV) >100 M and a stable series resistance (8–30 M). The reference bath electrode was a 3 M KCl, 4% agar bridge. Voltage and current waveforms were low-pass filtered at 2.7 kHz and 1.2 kHz, respectively, and digitized at 5–10 kHz using a Digidata 1200, controlled by pCLAMP software (v.6, Axon Instruments). Leakage currents were subtracted off-line. In a small number of initial control experiments, conventional intracellular recordings were obtained with sharp microelectrodes (see Church & McLennan, 1989; Church, 1999). In brief, microelectrodes were pulled from thin-walled borosilicate tubing, filled with 4 M potassium acetate (final resistance 60–100 M) and connected to an active bridge circuit. Sharp microelectrode recordings were included in data analysis if, under control conditions, neurones exhibited a stable V_m of at least –55 mV with overshooting action potentials and R_in measured at V_m > 20 M.

During current-clamp recordings, the effects of changes in perfusate composition on the medium and slow AHPs which followed a 300 ms depolarizing current-evoked train of action potentials were quantified by comparing the amplitudes of the corresponding AHPs under control and test conditions as described (Church, 1999). The magnitude of the depolarizing current pulse employed to evoke the spike train was varied to elicit the same number of action potentials under each condition. Test measurements were conducted at the original control membrane potential by passing, when necessary, steady current through the recording electrode. In experiments conducted in the presence of 1 µM TTX and 5 mM TEA, a 40–100 ms depolarizing current pulse was employed to elicit a Ca²⁺-dependent depolarizing potential that was followed by a slow AHP; the half-amplitude duration of the Ca²⁺ potential and the peak amplitude of the subsequent slow AHP were measured (see Church, 1999).

Under voltage-clamp conditions, 80–200 ms depolarizing voltage steps from the holding potential (V_H, –50 mV) to 0–20 mV, applied approximately once per minute, elicited partially clamped inward currents that were followed by early (mI_AHP) and late (sI_AHP) outward K⁺ tail currents. Experimentally evoked changes in mI_AHP and sI_AHP were quantified by comparing the peak amplitudes of the currents obtained under control and test conditions; if a distinct sI_AHP peak was not evident, current amplitudes were compared isochronally at 400 ms after the end of the depolarizing voltage step. Larger depolarizing steps (from V_H to +20–40 mV) were employed to elicit Co²⁺ and Co²⁺- and XE991-resistant currents. The influence of time-dependent changes in current amplitude was minimized by comparing the computed means of responses obtained prior to and after recovery from the test condition with the test response. In experiments conducted in the presence of 1 µM TTX and 5 mM TEA, the depolarizing voltage step elicited a robust inward Ca²⁺ current (I_Ca) that was followed by a sI_AHP; the I_Cas evoked under control and test conditions were compared qualitatively whereas the peak amplitudes of the subsequent sI_AHPs were measured and compared statistically.

To examine the effects of experimental manoeuvres on I_AHP in relative isolation, the current was evoked by a previously described protocol (Stocker et al. 1999) in the presence of 1 µM TTX and 1 mM TEA to block voltage-gated Na⁺ channels and Ca²⁺ and voltage-dependent K⁺ channels, respectively. Given previous findings that cAMP, acting via protein kinase A, modulates the activities of the Na⁺–H⁺, Na⁺-dependent Cl^--HCO^-₃ and Na⁺-independent Cl^--HCO^-₃ exchangers found in rat CA1 neurones and thereby changes pH_i (see Brett et al. 2002), we avoided the use of cAMP analogues in the patch pipette to suppress sI_AHP in these experiments (cf. Stocker et al. 1999). Rather, the decay phase of I_AHP and the rising phase of sI_AHP were fitted by a double exponential and sI_AHP was subtracted off-line (see Fig. 3A). The double exponential had the form

(1)

where y₀ is the current offset at time zero (i.e. the time immediately after the depolarizing voltage step); A₁ is the amplitude of current 1 at time zero; A₂ is the steady-state amplitude of current 2; and ₁ and ₂ are the time constants of current 1 and current 2, respectively. Parameters were seeded with estimates obtained from the experimental record and allowed to run free in a Levenberg-Marquardt algorithm; r² values were typically > 0.90.

View larger version (28K): [in this window] [in a new window]   Figure 3.  Effects of pHo changes on currents that contribute to mIAHP All recordings were obtained under HCO-3/CO2-buffered conditions. A, whole-cell recording of Ca2+-activated K+ currents evoked at pHo 7.4 in the presence of 1 µM TTX and 1 mM TEA. The decay phase of IAHP and the rising phase of sIAHP were fitted by a double exponential (grey lines) and sIAHP was subtracted off-line; the addition of 100 nM apamin markedly reduced the early outward current remaining after subtraction of sIAHP. B, the overall effects of decreasing and increasing pHo to 6.7 and 7.7, respectively, on the peak amplitude of the early outward current (IAHP) remaining after subtraction of sIAHP, expressed as percentage changes from the current measured at pHo 7.4 in the same cells. C, mIAHP and sIAHP were evoked under control pHo 7.4 conditions. The application of 2 mM Co2+ attenuated mIAHP and blocked sIAHP; mIAHP was then further reduced by the addition of 10 µM XE991. D, the overall effects of decreasing and increasing pHo to 6.7 and 7.7, respectively, on the early outward current remaining either in the presence of 2 mM Co2+ (hatched bars) or in the presence of 2 mM Co2+ and 10 µM XE991 (Co2++ XE; cross-hatched bars). The experimental traces show the effects of changes in pHo on the Co2+- and XE991-resistant current. Scale bars in B also apply to A and C. *P < 0.05 and **P < 0.01 compared to the appropriate controls obtained at pHo 7.4; n.s., not statistically significant.

	Results

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Characterization of AHPs and underlying currents

The medium and slow AHPs (and their underlying currents) recorded under whole-cell conditions may differ from those obtained with sharp microelectrodes (see Zhang et al. 1994). Initially, therefore, we performed a limited series of experiments to assess the characteristics of the AHPs and currents under our experimental conditions.

In 21 CA1 neurones impaled with sharp microelectrodes under control pH_o 7.4 HCO^-₃/CO₂-buffered conditions, V_m was -65 ± 1 mV and R_in was 27 ± 2 M, values similar to those reported previously by this laboratory (e.g. Church & McLennan, 1989; Church, 1999) and others (e.g. Azouz et al. 1996) under similar experimental conditions. Corresponding values for V_m and R_in obtained in whole-cell patch pipette recordings (n= 48) were -55 ± 1 mV and 160 ± 4 M, respectively. Under both recording conditions, 300 ms depolarizing current pulses elicited trains of action potentials that were followed by medium and slow AHPs (Fig. 1A and B). Despite the use of the methylsulphate anion in the pipette solution (see Zhang et al. 1994), the peak amplitudes of the medium and slow AHPs in the whole-cell patch configuration (5.2 ± 0.4 mV and 4.0 ± 0.3 mV, respectively) were significantly (P < 0.05 in each case) smaller than those obtained in sharp microelectrode recordings (medium AHP, 6.7 ± 0.9 mV; slow AHP, 5.5 ± 0.8 mV). Because of these differences, all subsequent recordings were obtained with patch pipettes in the whole-cell configuration.

View larger version (25K): [in this window] [in a new window]   Figure 1.  Medium and slow AHPs and their underlying currents All recordings were made under pHo 7.4 HCO-3/CO2-buffered conditions. A and B, depolarizing current-evoked trains of action potentials evoked medium and slow AHPs recorded in different neurones with a sharp microelectrode (A) or with a patch pipette under whole-cell conditions (B). The magnitudes of the depolarizing current pulses employed to elicit the spike trains are shown on the figure; the dashed lines indicate Vm (–60 mV in both cases). Action potentials are truncated at this voltage gain. Scale bars in A also apply to B. C and D, patch pipette recordings under whole-cell voltage clamp illustrate the outward tail currents mIAHP (C) and sIAHP (D) elicited by a 140 ms depolarizing voltage step from VH (–50 mV) to +20 mV in a CA1 neurone. In C and D, mIAHP is identified by an arrow and the dashed lines indicate the zero current level. The histograms show the percentage reductions of mIAHP (C) and sIAHP (D) observed under the conditions shown on the figure (Nifed, nifedipine; Iso, isoprenaline). In this and subsequent figures, error bars are S.E.M. and n values are shown in the columns. *P < 0.05 and **P < 0.01 compared to the respective untreated controls.

Changes in pH_o modulate mI_AHP and sI_AHP

Lowering pH_o from 7.4 to 6.7 (HCO^-₃/CO₂-buffered conditions) significantly decreased the peak amplitudes of mI_AHP and sI_AHP (Fig. 2A and C). Conversely, increasing pH_o from 7.4 to 7.7 significantly increased mI_AHP and, to a greater extent, sI_AHP (Fig. 2B and D). Corresponding changes were observed in the medium and slow AHPs recorded from the same neurones under whole-cell current-clamp conditions (data not shown; see Church, 1999). The augmented sI_AHP observed at pH_o 7.7 remained sensitive to manoeuvres that reduced the current at pH_o 7.4. Thus, sI_AHP at pH_o 7.7 was reduced from 109 ± 14 pA (n= 12) to 10 ± 4 pA (n= 4) under [Ca²⁺]_o-free conditions (Fig. 2B; the corresponding current measured in the absence of external Ca²⁺ at pH_o 7.4 was 10 ± 3 pA; n= 2); to 10 ± 2 pA (n= 8) in the presence of 2 mM Co²⁺ (8 ± 2 pA at pH_o 7.4; n= 14); and to 42 ± 7 pA (n= 4) by the application of 2–4 µM isoprenaline (at pH_o 7.4, sI_AHP was reduced to 30 ± 9 pA by 8 µM isoprenaline; n= 3). The results indicate that decreases and increases in pH_o reduce and enhance sI_AHP, respectively. However, because sI_AHP overlaps temporally with mI_AHP(see Storm, 1990), the potential effect of changes in pH_o to modulate mI_AHP is less clear. This difficulty is compounded by the fact that a number of currents, including I_AHP and I_M, contribute to mI_AHP. Therefore, to further explore the effects of changes in pH_o on mI_AHP, I_AHP and I_M were studied in relative isolation.

View larger version (25K): [in this window] [in a new window]   Figure 2.  Changes in pHo modulate mIAHPand sIAHP All recordings were obtained under HCO-3/CO2-buffered conditions. A, reducing pHo from 7.4 to 6.7 reduced the peak amplitudes of mIAHP (inset) and sIAHP. B, in a different neurone, increasing pHo from 7.4 to 7.7 produced a small increase in mIAHP (inset) and a large increase in sIAHP. The augmented currents were subsequently inhibited by exposure to pH 7.7 Ca2+-free medium. C and D summarize the effects of decreasing (C) and increasing (D) pHo on the peak amplitudes of mIAHP (open bars) and sIAHP (hatched bars), expressed as percentage changes from the currents measured at pHo 7.4. *P < 0.05 and **P < 0.01 for the difference to the respective currents measured at pHo 7.4.

To study the effects of changes in pH on mI_AHP without contributions from Ca²⁺-activated currents, 2 mM Co²⁺ was applied (Fig. 3C). The residual current observed in the presence of Co²⁺ at pH_o 7.4 was insensitive to 100 nM apamin (n= 2; data not shown) but was reduced by 10 µM XE991 from 69 ± 10 pA to 40 ± 5 pA (a 40 ± 5% reduction; n= 4; P < 0.05; Fig. 3C), consistent with the involvement of not only I_M but also other early outward currents. Decreasing pH_o to 6.7 decreased the Co²⁺-resistant current whereas increasing pH_o to 7.7 increased it (Fig. 3D). However, when pH_o was reduced to 6.7 in the presence of 2 mM Co²⁺ and 10 µM XE991, the decline in the residual current was not significantly different (P > 0.17) to that observed in the presence of 2 mM Co²⁺ alone (Fig. 3D). Similarly, there was no significant difference between the increases in the Co²⁺-resistant current observed at pH_o 7.7 in the absence and presence of 10 µM XE991 (Fig. 3D).

Taken together, the results indicate that mI_AHP and sI_AHP are sensitive to changes in pH_o and further suggest that the effect of pH_o to modulate mI_AHP reflects changes in I_AHP and current(s) other than I_M that also contribute to the medium AHP.

Changes in pH_i modulate mI_AHP and s_AHP

In rat CA1 neurones, changes in pH_o lead to changes in pH_i in the same direction (see Introduction). Therefore, we examined whether changes in pH_i affect mI_AHP and sI_AHP and, if so, whether these effects might contribute to pH_o-induced changes in the currents.

Decreasing pH_i at a constant pH_o.  Initially, slices were exposed to a pH 7.4 HCO^-₃/CO₂-buffered medium containing 40 mM sodium propionate, a manoeuvre that leads to a transient fall in pH_i in hippocampal neurones (Church et al. 1998; Bonnet et al. 2000). As illustrated in Fig. 4A, this manoeuvre reduced mI_AHP by a maximum of 39 ± 2% and sI_AHP by a maximum of 56 ± 9% (n= 3 in each case), thereby resembling the effects of reducing pH_o on both currents. In contrast to the transient effects of externally applied weak acids, the transition from a HCO^-₃/CO₂-buffered medium to a HCO^-₃ free medium buffered with either 30 mM Hepes or 30 mM tricine (pH_o constant at 7.4) produces a transient increase in pH_i (reflecting CO₂ washout) followed by a sustained reduction in pH_i consequent upon the blockade of HCO^-₃ dependent acid extrusion mechanism(s) (Church, 1992; Cowan & Martin, 1995; Bonnet et al. 1998; Brett et al. 2002). Therefore, in the next series of experiments we examined the effects of this manoeuvre on mI_AHP and sI_AHP; test measurements in the absence of HCO^-₃ were obtained 20 min after the start of perfusion with HCO^-₃ free medium to allow pH_i to stabilize at the new lower resting level. The effects of switching to HCO^-₃ free Hepes- (n= 11) or tricine- (n= 2) buffered media were indistinguishable and the results were pooled.

View larger version (36K): [in this window] [in a new window]   Figure 4.  Reductions in pHi at a constant pHo inhibit mIAHPand sIAHP In all experiments, pHo was 7.4 throughout. A, representative examples of mIAHP and sIAHP recorded prior to and during propionate application. Above the records are summary histograms of the maximum reductions in mean peak amplitudes of mIAHP and sIAHP observed upon exposure to medium containing 40 mM propionate (Prop), compared to controls. B, representative examples of mIAHP and sIAHP recorded prior to and during perfusion with HCO-3 free medium. Above the records are summary histograms of the effects on mIAHP and sIAHP of the transition from a HCO-3 containing medium to a HCO-3 free medium buffered with either 30 mM Hepes or 30 mM tricine. Scale bars in A also apply to B. C, to the left are shown representative records of the early outward current (IAHP) remaining after subtraction of sIAHP, obtained prior to and during perfusion with HCO-3 free medium; 1 µM TTX and 1 mM TEA were present throughout. Results from 5 similar experiments are summarized on the right. D, to the left are shown representative records of the early outward current (Iresidual) remaining in the presence of 2 mM Co2+ and 10 µM XE991, obtained prior to and during perfusion with HCO-3 free medium. To the right are summaries of the effects of the transition from a pH 7.4 HCO-3 containing medium to a pH 7.4 HCO-3 free medium on the Co2+-resistant (hatched bars) and Co2+- and XE991-resistant (Co2++ XE; cross-hatched bars) early outward currents. Scale bars in C also apply to D. *P < 0.05 and **P < 0.01 compared to the respective control responses obtained in standard pH 7.4 HCO-3 containing medium; n.s., not statistically significant.

Given the variable effects of exposure to HCO^-₃ free medium on mI_AHP, I_AHP was studied in isolation using the protocol described earlier. Examined in the presence of 1 µM TTX and 1 mM TEA, decreasing pH_i at a constant pH_o significantly reduced I_AHP in 5 out of 5 neurones tested (Fig. 4C). Under the same conditions, sI_AHP remained sensitive to reductions in pH_i, being reduced from 85 ± 15 pA to 36 ± 9 pA (a 58 ± 1% reduction) upon exposure to HCO^-₃ free medium (n= 5; P < 0.05). To study the effects of changes in pH_i on mI_AHP without contributions from Ca²⁺-activated currents, 2 mM Co²⁺ was again employed. When the transition to HCO₃^- free medium was conducted in the presence of 2 mM Co²⁺, the Co²⁺-resistant current was reduced in 4 out of 4 neurones tested (Fig. 4D). Four additional neurones were treated with 2 mM Co²⁺ and 10 µM XE991. In three of these cells, reducing pH_i at a constant pH_o caused a decline in the residual current that was not significantly different (P > 0.5) to the decrease observed in the presence of 2 mM Co²⁺ alone (Fig. 4D). Interestingly, in the remaining neurone, reducing pH_i at a constant pH_o caused a 15% increase in the Co²⁺- and XE991-resistant early outward current, an effect that may contribute to the increased mI_AHP observed in 2 out of 13 cells when pH_i was reduced at a constant pH_o in the absence of either Co²⁺ or XE991 (see above).

Taken together, these observations indicate that reductions in pH_i at a constant pH_o inhibit sI_AHP and, less consistently, mI_AHP. The variable effects of low pH_i on mI_AHP may reflect the net effect of the pH_i change on I_AHP (which consistently declined, albeit to a lesser extent than sI_AHP) and the Co²⁺- and XE991-resistant current(s) that also contribute to mI_AHP (which sometimes exhibited an increase).

Increasing pH_i at a constant pH_o.  In rat hippocampal neurones, the external application of a weak base transiently increases pH_i at a constant pH_o (see Church et al. 1998; Bonnet & Wiemann, 1999). Therefore, in an attempt to examine the effects of increasing pH_i at a constant pH_o on mI_AHP and sI_AHP, slices were exposed to a pH 7.4 HCO^-₃/CO₂-buffered medium containing 40 mM TMA (n= 6). Unexpectedly, TMA reduced mI_AHP and sI_AHP in a manner that was independent of the increase in pH_i, most likely reflecting a direct effect of TMA to block the currents (T. Kelly & J. Church, unpublished observations; also see Bruening-Wright et al. 2002). Similar results, which are reminiscent of the effects of the weak base 4-aminopyridine (Andreasen, 2002), were obtained with 40 mM NH₄Cl (n= 2; also see Díaz et al. 1996). In light of these difficulties, we employed an alternative approach to assess the potential contribution of increases in pH_i to the effects of increasing pH_o on mI_AHP and sI_AHP, i.e. to prevent the change in pH_i by including a high concentration of H⁺ buffer in the patch pipette solution.

Buffering pH_i changes.  High internal H⁺ buffer concentrations ( 50 mM) must be employed in the whole-cell configuration to effectively control pH_i in the face of a change in pH_o (e.g. Byerly & Moody, 1986; Korn & Horn, 1991; Kapus et al. 1993). Initially, therefore, we examined the effects of changing pH_o on mI_AHP and sI_AHP recorded with 100 mM Hepes in the patch pipette; under these conditions, increasing pH_o from 7.4 to 7.7 failed to significantly increase either mI_AHP or sI_AHP (P > 0.1 and P > 0.2, respectively; Fig. 5A). Although these results are consistent with the possibility that a rise in pH_i consequent upon a rise in pH_o contributes to the effects of increasing pH_o to augment mI_AHP and sI_AHP, it is also apparent that mI_AHP and sI_AHP recorded at pH_o 7.4 with 100 mM internal Hepes were significantly reduced compared to the amplitudes of the currents measured with the standard pipette solution containing 10 mM Hepes, also at pH_o 7.4 (Fig. 5A and B). In turn, this raised the possibility that the lack of effect of increasing pH_o on mI_AHP and sI_AHP recorded with 100 mM internal Hepes might simply reflect an effect of high internal [Hepes] to modify the currents (also see Wanke et al. 1979). This possibility was supported by findings that the amplitudes of mI_AHP(not shown) and sI_AHP (Fig. 5B) recorded at pH_o 7.4 with 50 mM internal Hepes or 50 mM internal Tes (a structurally related buffer; not shown) were also significantly reduced, compared to the currents measured with 10 mM internal Hepes.

View larger version (20K): [in this window] [in a new window]   Figure 5.  Effects of high internal concentrations of H+ buffers on mIAHPand sIAHP All recordings were obtained under HCO-3/CO2-buffered conditions. A, whole-cell recordings obtained from a CA1 neurone held at –50 mV with 100 mM Hepes in the patch pipette. Under pHo 7.4 conditions, an 80 ms depolarizing step to 0 mV was followed by a mIAHP and a small sIAHP. In contrast to the results obtained with 10 mM internal Hepes (see Fig. 2B and D), increasing pHo to 7.7 failed to significantly increase mIAHP or sIAHP. Inset, histograms summarizing the results obtained in 9 similar experiments. B, plot of the mean peak amplitudes (±S.E.M.) of sIAHP recorded at pHo 7.4 with 10, 50 or 100 mM Hepes, or 100 mM tricine, in the patch pipette. *P < 0.05 and **P < 0.01 compared to control (10 mM internal Hepes).

View larger version (33K): [in this window] [in a new window]   Figure 6.  Whole-cell recordings of mIAHPand sIAHP with 100 mM internal tricine In all experiments, the patch pipette solution contained 100 mM tricine. A–D are whole-cell recordings of mIAHP and sIAHP made under control pHo 7.4 HCO-3/CO2-buffered conditions (black traces) and during subsequent exposure to pH 7.7 HCO-3 containing medium (A), pH 7.4 HCO-3 containing medium with 40 mM propionate (B), pH 7.4 HCO-3 free medium (C) and pH 6.7 HCO-3 containing medium (D) (grey traces). Below the experimental traces in A–D are histograms summarizing the percentage changes of mIAHP and sIAHP observed under each of the test conditions (grey bars), compared to the percentage changes evoked by the same experimental manoeuvres with 10 mM internal Hepes (open bars). Scale bars in A also apply to B–D. *P < 0.05 and **P < 0.01 for the difference to the respective change measured with 10 mM internal Hepes.

pH effects on Ca²⁺ influx

Changes in pH_o and pH_i modulate the activities of neuronal high-voltage-activated Ca²⁺ channels (e.g. Tombaugh & Somjen, 1996, 1997; Church et al. 1998; Kiss & Korn, 1999). Because the modulation of sI_AHP(and, to a lesser extent, mI_AHP) by changes in pH_o and/or pH_i might simply reflect changes in Ca²⁺ influx through voltage-activated Ca²⁺ channels, we examined the effects of changes in pH on Ca²⁺-dependent depolarizing potentials (‘Ca²⁺ spikes’) and inward Ca²⁺ currents recorded in the presence of 1 µM TTX and 5 mM TEA.

Current-clamp recordings.  In agreement with previous reports (e.g. Higashi et al. 1990; Church, 1999), a 40–100 ms depolarizing current pulse applied under control pH_o 7.4 HCO^-₃/CO₂-buffered conditions (10 mM internal Hepes) elicited a Ca²⁺-dependent, Co²⁺- and nifedipine-sensitive depolarizing potential followed by a slow AHP (see Fig. 7A–C). A reduction in pH_o to 6.7 significantly inhibited both the half-amplitude width of the Ca²⁺ spike and the peak amplitude of the subsequent slow AHP, which declined from 4.0 ± 0.4 mV to 0.3 ± 0.2 mV (n= 5; Fig. 7A and D). Conversely, as illustrated in Fig. 7B and D, at pH_o 7.7 both the half-amplitude width of the Ca²⁺ spike and the peak amplitude of the subsequent slow AHP increased significantly (the latter from 4.0 ± 1.0 mV to 6.8 ± 1.0 mV; n= 7). Finally, slices were exposed to HCO^-₃ free medium to reduce pH_i at a constant pH_o; this manoeuvre significantly reduced the peak amplitude of the slow AHP (from 7.5 ± 0.8 mV to 3.9 ± 1.3 mV; n= 3) but, in contrast to the effect of a reduction in pH_o, the half-amplitude width of the preceding Ca²⁺ spike was not significantly affected (Fig. 7C and D).

View larger version (24K): [in this window] [in a new window]   Figure 7.  Effects of changing pHo and/or pHi on Ca2+-dependent depolarizing potentials and slow AHPs Records in A–C were obtained from three different neurones in the presence of 1 µM TTX and 5 mM TEA; in each case, the patch pipette contained 10 mM Hepes and Ca2+ spikes were evoked by 60–100 ms depolarizing current pulses from Vm (–60 mV in all cases). A, lowering pHo from 7.4 to 6.7 reduced both the half-amplitude width of the Ca2+ spike (inset) and the peak amplitude of the subsequent slow AHP. B, raising pHo from 7.4 to 7.7 increased both the half-amplitude width of the Ca2+ spike and the peak amplitude of the subsequent slow AHP. C, removing HCO-3 from the perfusate to lower pHi (pHo constant at pH 7.4) attenuated the slow AHP with little effect on the half-amplitude width of the Ca2+ spike. Scale bars in A also apply to the corresponding traces in B and C. D, summaries of the effects of the experimental manoeuvres illustrated in A–C on the half-amplitude widths of Ca2+ spikes () and the peak amplitudes of the subsequent slow AHPs (•), expressed as percentage changes from control values established under pHo 7.4 HCO-3 containing conditions. Also shown are the effects of the same experimental manoeuvres on the peak amplitudes of sIAHP (), as well as their effects on the half-amplitude widths of Ca2+ spikes () and the peak amplitudes of the slow AHPs () and sIAHPs () recorded with 100 mM internal tricine. Error bars are S.E.M. and n values are provided in parentheses. *P < 0.05 and **P < 0.01 for the difference between the corresponding measurements obtained with 10 mM internal Hepes and 100 mM internal tricine.

Voltage-clamp recordings.  Depolarizing from V_H to 0–20 mV in the presence of 1 µM TTX and 5 mM TEA revealed a robust partially clamped inward current that could be blocked by 2 mM Co²⁺(n= 3) and which was followed by a sI_AHP (Fig. 8A). Consistent with results obtained under current-clamp conditions, decreasing pH_o to 6.7 decreased the magnitude of the Ca²⁺ current (I_Ca) in 9 out of 9 neurones examined and significantly reduced sI_AHP by 76% (Figs 7D and 8A). Conversely, increasing pH_o to 7.7 increased I_Ca in 8 out of 8 neurones and significantly enhanced sI_AHP by 76% (Figs 7D and 8B). Also consistent with the current-clamp results, reducing pH_i at a constant pH_o by exposing slices to HCO^-₃ free medium significantly reduced sI_AHP by 51% in 9 out of 9 neurones with little apparent change in I_Ca (Figs 7D and 8C).

View larger version (39K): [in this window] [in a new window]   Figure 8.  Changing pHo and/or pHi modulates ICa and the subsequent sIAHP Traces in A–C were obtained from three different neurones, each held at –50 mV in the presence of 1 µM TTX and 5 mM TEA; in all cases, the patch pipette solution contained 10 mM Hepes. A, under control pHo 7.4 conditions, a depolarizing step elicited a partially clamped ICa (upper panel) that was followed by a sIAHP (middle panel). Decreasing pHo to 6.7 attenuated ICa and reduced the peak amplitude of sIAHP; both effects were reversed upon reperfusion with pH 7.4 medium. Subsequently, ICa and sIAHP were blocked by the addition of 2 mM Co2+. The time course of the effect of pHo 6.7 to reduce sIAHP is shown in the lower panel; asterisks denote the time points prior to, during and after exposure to pHo 6.7 medium at which the records shown in the upper and middle panels were obtained. B, increasing pHo reversibly increased ICa and sIAHP. C, lowering pHi at a constant pHo by removing HCO-3 from the perfusate (pHo constant at 7.4) attenuated sIAHP with little apparent effect on ICa. The gap in the record in the lower panel in C represents a 4 min break in data acquisition. Scale bars in A apply to the appropriate panels in B and C.

View larger version (30K): [in this window] [in a new window]   Figure 9.  Effects of changes in pHo and/or pHi on ICaand sIAHPrecorded with 100 mM internal tricine Traces in A–C were obtained from three different neurones, each held at –50 mV in the presence of 1 µM TTX and 5 mM TEA; in all cases, the patch pipette solution contained 100 mM tricine. A, under control pHo 7.4 HCO-3/CO2-buffered conditions, a depolarizing step elicited an ICa (upper panel) that was followed by a sIAHP (middle panel). Decreasing pHo to 6.7 reversibly reduced both ICa and sIAHP (the ICa record obtained after the return to pHo 7.4 has been omitted for clarity). The time course of the effect of pHo 6.7 to reduce sIAHP is shown in the lower panel; asterisks denote the time points prior to, during and after exposure to pHo 6.7 medium at which the records shown in the upper and middle panels were obtained. B, increasing pHo to 7.7 increased ICa but sIAHP was not enhanced to the same extent as observed with 10 mM internal Hepes (compare with Fig. 8B). C, exposure to a pH 7.4 HCO-3 free medium failed to reduce ICa or the subsequent sIAHP; in contrast, the same manoeuvre significantly reduced sIAHP recorded with 10 mM internal Hepes (see Fig. 8C). The gap in the record in the lower panel in C represents a 7 min break in data acquisition. Scale bars in A apply to the appropriate panels in B and C.

	Discussion

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Effects of changes in pH_o

sI_AHP.  Decreases and increases in pH_o reduce and augment, respectively, the Ca²⁺ influx through high-voltage-activated Ca²⁺ channels that contributes to the activation of sI_AHP (Lancaster & Adams, 1986; Shah & Haylett, 2000); in rat hippocampal neurones, the pH_o for 50% Ca²⁺ channel inhibition (pK) is 7.1–7.2 (Tombaugh & Somjen, 1996; Church et al. 1998). In the present study, the inhibition of sI_AHP observed at pH_o 6.7 was associated with a reduction in Ca²⁺ potentials and was not significantly affected by the inclusion of 100 mM tricine in the patch pipette. These findings indicate that when pH_o is reduced, decreased Ca²⁺ influx is primarily responsible for the attenuation of sI_AHP and reductions in pH_i consequent upon reductions in pH_o play little or no role. In contrast, pH_o 7.7-induced increases in sI_AHP appeared to reflect an action of increases in pH_i to modulate sI_AHP downstream from any high pH_o and/or high pH_i-induced changes in the priming Ca²⁺ signal. Thus, although increasing pH_o increased the magnitudes of Ca²⁺ potentials and sI_AHP, buffering high pH_o-induced pH_i changes with 100 mM internal tricine significantly attenuated only the augmented sI_AHP. Although it remains possible that changes in pH_o might also act to modulate sI_AHP directly, in no case did pH_o-induced changes in sI_AHP occur in the absence of parallel changes in Ca²⁺ potentials. In this regard we have shown previously, in outside-out patches, that changes in pH_o fail to alter the unitary properties of the BK-type Ca²⁺-dependent K⁺ channel that underlies the fast AHP in rat hippocampal neurones (cf. changes in pH_i; Church et al. 1998; also see Hayabuchi et al. 1998; Pedersen et al. 2000).

The differences between the ways in which low and high pH_o modulate sI_AHP may be due to a number of factors. Most notably, decreasing pH_o to 6.7 reduced the half-width of Ca²⁺ potentials by 57%, compared to the 19% increase observed at pH_o 7.7. These findings are consistent with the possibility that a marked decrease in Ca²⁺ entry at pH_o 6.7 inhibits sI_AHP to such an extent that any effect of a low pH_o-induced reduction in pH_i to further directly reduce the current is prevented. In contrast, Ca²⁺ entry at pH_o 7.7, although increased, may remain insufficient to maximally activate sI_AHP and high pH_o-induced decreases in [H⁺]_i may act to further modulate the current. The latter possibility receives support from previous findings that high pH_i-induced increases in the open probability of BK-type channels in hippocampal neurones are observed only when [Ca²⁺]_i is below saturation (Church et al. 1998; also see Copello et al. 1991; Liu et al. 1999; Pedersen et al. 2000).

mI_AHP.  Like the sI_AHP, mI_AHP was inhibited at pH_o 6.7 and increased at pH_o 7.7; these effects reflected changes in I_AHP and the Co²⁺- and XE991-resistant component of mI_AHP. Although reducing pH_o to 6.7 inhibited the Co²⁺-resistant component of mI_AHP more than the Co²⁺- and XE991-resistant component, the difference (which represents the contribution of I_M) did not reach statistical significance; similarly, increasing pH_o to 7.7 augmented the Co²⁺-resistant and Co²⁺- and XE991-resistant components of mI_AHP to a similar extent. The apparent insensitivity of I_M to the relatively small changes in pH_o (and pH_i) employed in the present study is consistent with the reported pK for the effect of changes in pH to modulate the KCNQ 2/3 heteromultimers that are believed to underlie I_M(pK6.1; Prole & Marrion, 2002; also see Peretz et al. 2002).

Also like sI_AHP, changes in pH_i did not contribute to low pH_o-induced reductions in mI_AHP (although reductions in pH_i at a constant pH_o usually inhibited the current; see below), whereas high-pH_o-induced increases in mI_AHP were attenuated by including 100 mM tricine in the patch pipette. The greater effect of pH_o 6.7 to reduce mI_AHP (a 59% decrease) than of pH_o 7.7 to increase it (by 12%) may reflect the previously mentioned larger change in Ca²⁺ entry in the acidic compared to the alkaline direction (with consequent effects on I_AHP, which was reduced by 58% at pH_o 6.7 and increased by 16% at pH_o 7.7) as well as the greater sensitivity of the Co²⁺- and XE991-resistant component of mI_AHP to acidic versus alkaline pH_o shifts. Although the current(s) that underlie the Co²⁺- and XE991-resistant component of mI_AHP remain unknown, the effects of changes in pH_o (and pH_i) on this current are consistent with the pH sensitivities of, for example, a variety of voltage-gated K⁺ channels (e.g. delayed rectifier channels) that are expressed in hippocampal neurones and may contribute to this component (see Maletic-Savatic et al. 1995; Steidl & Yool, 1999).

Effects of changes in pH_i

Although reductions in pH_i did not app_ilie low pH_o-induced reductions in mI_AHP and sI_AHP, changes in pH_i were able to modulate both currents. Thus, high pH_o-induced increases and low pH_i-induced decreases in mI_AHP and sI_AHP were significantly attenuated when internal H⁺ buffering power was raised by the inclusion of 100 mM tricine in the patch pipette. The latter effects were not accompanied by significant changes in Ca²⁺ potentials (or I_Ca), indicating that they are not dependent on marked alterations in Ca²⁺ influx (cf. the effect of reductions in pH_o). Rather, the results are consistent with the possibility that, under these conditions, pH_i becomes an important factor in the control of I_AHP and sI_AHP.

The modulation by pH_i of I_AHP and sI_AHP is reminiscent of previous findings that protons acting on the cytoplasmic side of the membrane are potent modulators of BK-type channels in a variety of cell types (e.g. Cook et al. 1984; Kume et al. 1990; Copello et al. 1991; Laurido et al. 1991; Hayabuchi et al. 1998; Liu et al. 1999), including rat hippocampal neurones (Church et al. 1998). Internal protons have been proposed to affect BK channels either by competing with Ca²⁺ at regulatory binding sites (Cook et al. 1984; Kume et al. 1990; Copello et al. 1991) and/or via an allosteric site on the channel complex (Laurido et al. 1991). Although kinetic studies at the single channel level are required to determine how protons modulate I_AHP and sI_AHP, it is of interest that SK-type Ca²⁺-activated K⁺ channels use calmodulin (CaM) constitutively associated with the C-terminus of the SK -subunit as their high affinity Ca²⁺ sensor (Xia et al. 1998). Calcium binding to CaM is pH sensitive (e.g. increasing pH from 6.5 to 7.5 increases the affinity of CaM for Ca²⁺ > 4-fold; Tkachuk & Men'shikov, 1981), an effect that indirectly confers a pH dependency on the many Ca²⁺-dependent processes that are normally regulated by CaM. This raises the possibility that protons may affect I_AHP and, possibly, sI_AHP by modulating Ca²⁺ binding to CaM, although this could not explain the pH_i sensitivity of BK-type channels (which are not gated by the interaction of Ca²⁺ ions and CaM; see Sah & Faber, 2002). Single channel studies might also reveal the basis for the differential sensitivity of I_AHP and sI_AHP to changes in pH_i.

Functional implications

Given that medium and slow AHPs are important determinants of neuronal excitability, the pH-dependent modulation of mI_AHP and sI_AHP observed in the present study would be expected to profoundly affect neuronal function. By inhibiting mI_AHP and sI_AHP, decreases in pH_o and/or pH_i would be expected to slightly depolarize neurones, reduce spike frequency adaptation and increase the number of action potentials elicited by a depolarizing current pulse, whereas increases in pH_o and/or pH_i would exert opposite effects. Indeed, these are precisely the consequences of changes in pH_o and pH_i on neuronal firing patterns reported previously in CA1 pyramids (Church & McLennan, 1989; Church, 1992, 1999) and other mammalian neurones (e.g. CO₂-sensitive medullary neurones; Wiemann et al. 1998). In addition, changes in pH may modulate the link between membrane excitability and cellular metabolism that is provided by Ca²⁺-activated K⁺ channels which, when activated, act as a negative feedback mechanism that limits Ca²⁺ influx into neurones (see Chono et al. 2003). It has been suggested, for example, that the inhibition of voltage-activated Ca²⁺ channels by reductions in pH_i might help to prevent excessive rises in [Ca²⁺]_i under conditions, such as ischaemia, which are associated with an internal acidosis (e.g. Tombaugh & Somjen, 1997). The present findings suggest, however, that reductions in pH_i may uncouple [Ca²⁺]_i increases from the activation of I_AHP and sI_AHP (as well as I_C; see Church et al. 1998) and may thereby act to offset any effect of a reduction in pH_i to reduce Ca²⁺ flux through voltage-activated Ca²⁺ channels. This possibility is supported by our previous findings (Church et al. 1998) that, in contrast to changes in pH_o, changes in pH_i at a constant pH_o fail to significantly affect the magnitudes of depolarization-evoked [Ca²⁺]_i transients in fura-2-loaded hippocampal neurones. Conversely, augmented Ca²⁺-activated K⁺ currents may help to limit the increases in Ca²⁺ entry and epileptiform burst-firing that occur in the CA1 region under high pH_o conditions (Church & McLennan, 1989; Church et al. 1998).

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