Differential Sensitivity to Intracellular pH Among High- and Low-Threshold Ca2+ Currents in Isolated Rat CA1 Neurons

Geoffrey C. Tombaugh and George G. Somjen

Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Tombaugh, Geoffrey C. and George G. Somjen. Differential sensitivity to intracellular pH among high- and low-threshold Ca2+ currents in isolated rat CA1 neurons. J. Neurophysiol. 77: 639-653, 1997. The effects of intracellular pH (pHi) on high-threshold (HVA) and low-threshold (LVA) calcium currents were examined in acutely dissociated rat hippocampal CA1 neurons with the use of the whole cell patch-clamp technique (21-23°C). Internal pH was manipulated by external exposure to the weak base NH4Cl or in some cases to the weak acid Na-acetate (20 mM) at constant extracellular pH (7.4). Confocal fluorescence measurements using the pH-sensitive dye SNARF-1 in both dialyzed and intact cells confirmed that NH4Cl caused a reversible alkaline shift. However, the external TEA-Cl concentration used during ICa recording was sufficient to abolish cellular acidification upon NH4Cl wash out. With 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) in the pipette, NH4Cl exposure reversibly enhanced HVA currents by 29%, whereas exposure to Na-acetate markedly and reversibly depressed HVA Ca currents by 62%. The degree to which NH4Cl enhanced HVA currents was inversely related to the internal HEPES concentration but was unaffected when internal ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was replaced by equimolar bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). When depolarizing test pulses were applied shortly after break-in (Vh = -100 mV), NH4Cl caused a proportionally greater increase in the sustained current relative to the peak. The dihydropyridine Ca channel antagonist nifedipine (5 µM) blocked nearly all of this sustained current. A slowly inactivating nifedipine-sensitive (L-type) HVA current could be evoked from a depolarized holding potential of -50 mV; NH4Cl enhanced this current by 40 ± 3% (mean ± SE) and reversibly shifted the tail-current activation curve by +6-8 mV. L-type currents exhibited more rapid rundown than N-type currents; HVA currents remaining after prolonged cell dialysis, or in the presence of nifedipine, inactivated rapidly and were depressed by omega -conotoxin (GVIA). NH4Cl enhanced these N-type currents by 76 ± 9%. LVA Ca currents were observed in 32% of the cells and exhibited little if any rundown. These amiloride-sensitive currents activated at voltages negative to -50 mV, were enhanced by extracellular alkalosis and depressed by extracellular acidosis, but were unaffected by exposure to either NH4Cl or NaAC. These results demonstrate that HVA Ca currents in hippocampal CA1 neurons are bidirectionally modulated by internal pH shifts, and that N-type currents are more sensitive to alkaline shifts than are L- or T-type (N > L > T). Our findings strengthen the idea that distinct cellular processes governed by different Ca channels may be subject to selective modulation by uniform shifts in cytosolic pH.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The regulation of pH in the nervous system is critical for normal cellular function. In spite of this fact, modest extracellular pH shifts (0.1-1 pH unit) in the brain occur under both normal and pathological conditions and have been observed in many vertebrate species (Caspers and Speckmann 1972; Chesler and Kaila 1992; Siemkowicz and Hansen 1981). As these pHo shifts reflect the transmembrane flux of acid or base equivalents (H+, OH-, HCO-3), they almost certainly accompany changes in pHi, which may also influence cell function. However, little is known about the specific pathways by which pHi transients are capable of modulating neuronal physiology.

The generic ability of H+ to influence biological systems implies that H+ ions probably affect synaptic and excitable membranes in the nervous system by numerous mechanisms. However, several lines of evidence suggest that many membrane effects of H+ ions arise from their action on voltage-gated calcium channels. Ca channels are known to be very sensitive to internal pH in a variety of cell types including neurons (Dixon et al. 1993; Irisawa and Sato 1986; Umbach 1982). In the mammalian brain, Ca channels govern many diverse cellular functions, including neurotransmitter release, patterns of excitability, and second-messenger cascades believed to underlie certain forms of neuronal plasticity (Wyllie and Nicoll 1994). Even modest (e.g., 20%) shifts in high-threshold (HVA) Ca channel conductance can dramatically affect neurotransmitter release (Dodge and Rahamimoff 1967), as well as patterns and thresholds of cell firing (De Schutter and Bower 1994).

On the basis of the marked pH sensitivity of many ligand-gated ion channels (Askalan and Richardson 1994; Pasternack et al. 1992; Tang et al. 1990), several investigators have suggested that pH transients associated with neuronal activity serve as local paracrine or feedback signals (Gottfried and Chesler 1994; Ransom 1992; Taira et al. 1993). Consistent with this hypothesis, our earlier results in hippocampal neurons revealed a striking sensitivity of HVA Ca channels to external pH (Tombaugh and Somjen 1996). In the present study, we explore the possibility that mammalian Ca channels also serve as targets for internal pH shifts. Our results confirm that HVA Ca currents are highly sensitive to internal H+ in CA1 neurons and also indicate that different classes of native Ca channels in these cells exhibit a range of pH sensitivities.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Cell isolation

Acutely dissociated pyramidal neurons were obtained from the CA1 region of rat hippocampal slices according to the method of Kay and Wong (1986). Adult male Sprague-Dawley rats (100-125 g) were decapitated under ether anesthesia; 500-µm hippocampal slices were prepared, and small tissue pieces (0.5 mm3) of the CA1 region were isolated and stirred gently for 90 min at room temperature in oxygenated N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline, which contained the following (in mM): 125 NaCl, 5 KCl, 1 CaCl2, 2 MgCl2, 25 dextrose, 10 HEPES, pH 7.0, containing 0.6 mg/ml trypsin (Sigma, St. Louis, MO). After digestion, tissue pieces were washed three times with trypsin-free buffer, pH 7.4, and incubated at room temperature in fresh perfusion buffer (see below) continually stirred under pure oxygen until needed. Cells were dissociated as needed in a small volume (200 µl) of control perfusion solution with a graded series of fire-polished Pasteur pipettes. The cell suspension was placed in an open perfusion chamber (volume approx 300-400 µl) mounted on the stage of an inverted microscope (Nikon Diaphot). The cells were allowed to settle (3 min), and the chamber was perfused at a rate of 0.5-1 ml/min. Pyramidal or spindle-shaped cells were chosen according to the following criteria: a smooth, nongranular appearance with a branched or unbranched apical dendrite of at least two somal lengths.

 
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TABLE 1. Enhancement of HVA currents by NH4Cl is sensitive to internal pH buffering


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FIG. 1. External tetraethylammonium (TEA+) modulates pHi shifts during NH4Cl exposure. Intensity plots depict changes in 570-nm fluorescence intensity (F570) recorded every 2 s from cells filled with the pHi-sensitive dye carboxy-SNARF1, excited with the 488-nm line of a krypton/argon laser. Reduced fluorescence reflects an alkaline shift. Schematics illustrate the experimental design. A: cell filled with 200 µM SNARF1 (free acid) via a patch pipette also containing 5 mM TEA-Cl; external TEA-Cl = 50 mM. B: cell loaded by incubation with SNARF1-AM (see METHODS); external TEA-Cl = 50 mM. C: cell treated as in B; external TEA-Cl = 0 mM.


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FIG. 2. High-threshold (HVA) Ca currents are modulated by weak acid or base exposure. Cells were exposed to 20 mM of either NaAC (n = 4) or NH4Cl (n = 8) at constant extracellular pH (7.4) for 50 s (heavy bar), followed by wash out. Currents were evoked by 120-ms depolarizing voltage ramps (inset). Peak current amplitudes (means ± SE) are expressed as a percent of baseline values (corrected for leak and rundown). Small shifts along the voltage axis are evident in the illustrated currents.

Experimental solutions

Membrane currents were recorded with the use of electrodes pulled from thin-walled 1.5-mm borosilicate glass capillaries (World Precision Instruments, Sarasota, FL) on a Brown-Flaming P-80 puller (Sutter). The internal solution contained 100 mM CsF, 20 mM tetraethylammonium chloride (TEA-Cl), 2 mM MgCl2, 0.5 mM CaCl2, 10 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 2 mM Na2-ATP, 0.3 mM Na2-GTP, 5 mM phospho-creatine, and 50 U/ml creatine kinase (Boehringer Mannheim, Indianapolis, IN), 10 mM HEPES, pH 7.2. In some cases, the internal HEPES was adjusted to 1 or 50 mM, and the CsF concentration was adjusted to maintain osmolarity; in others, EGTA was replaced by equim o l a r   b i s - ( o - a m i n o p h e n o x y ) - N , N , N ' , N ' - t e t r a a c e t i c   a c i d(BAPTA; Molecular Probes, Eugene, OR). These solutions yielded electrodes with an open-tip resistance of 2-3 MOmega . All internal solutions were adjusted to pH 7.2 with CsOH and stored at -20°C. In our initial trials (Table 1), the external control solution consisted of 125 mM NaCl, 5 mM TEA-Cl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 25 mM dextrose, and 0.5 µM tetrodotoxin (TTX; Calbiochem, La Jolla, CA). In our later experiments, which constitute the bulk of our study, we increased [TEA+] to 50 mM and omitted KCl (NaCl, 80 mM) to abolish residual K+ currents. Test solutions contained 20 mM of either NH4Cl or Na-acetate substituted for equimolar NaCl. At pH 7.4, a small amount of these compounds exists as the neutral conjugate base or acid (NH3, HAC), which can readily cross the cell membrane. Once in the cytosol, NH3 scavenges while HAC liberates protons (Thomas 1984). In some experiments designed to test the effect of external pH, these compounds were omitted, and the pH of the external solution was adjusted to 6.4 or 8.0 with HCl or NaOH; for the acidic solution, HEPES was replaced with equimolar piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES). In a few cases, CdCl2 (400 µM) was added to block all HVA calcium currents. Amiloride-HCl (RBI, Natick, MA) and nifedipine (Sigma, St. Louis, MO) stock solutions were prepared in dimethyl sulfoxide (DMSO) and diluted to a final concentration of 0.5 mM and 5 µM, respectively. The final concentration of DMSO never exceeded 0.05% and was included in the control perfusion solution when appropriate. omega -Conotoxin (GVIA, Sigma) was used at a final concentration of 0.5 µM. The osmolarity of all external (295-305 mosM) and internal (270-280 mosM) solutions was measured with a freezing-point osmometer.

Microfluorometry

Cells were filled with the pH-sensitive dye carboxy-SNARF1 (Molecular Probes); shifts in intracellular fluorescence were monitored by confocal microscopy. Cells were filled with the dye by one of two methods. In one protocol, cells were loaded directly via a patch pipette containing 200 µM SNARF1 (free acid). In the second protocol, cells were dissociated in standard perfusion buffer (see above) containing 5 µM of the methyl-ester from of the dye (SNARF1-AM, 0.1% DMSO) and were loaded for 8 min at room temperature. Cells were placed in an open perfusion chamber mounted on the stage of an upright confocal microscope (Biorad MRC600, Zeiss Axioskop) equipped with ×40 water-immersion objective (0.75 NA). The dye was excited with 488 nm light from a krypton/argon laser; emitted light was split with a dichroic mirror (605 nm long pass), filtered through 570 ± 20 nm and 640 ± 20 nm band-pass filters, and sent to two photomultiplier tubes. Image acquisition was controlled by COMOS software (Biorad); time series measurements were collected with TCSM software (Biorad). Fluorescent signals were collected at 2-s intervals from two rectangular regions traced 1) in a cell-free area ("background") and 2) within the cell soma. For the purposes of the present study, emission ratios were not calculated and actual pHi values were not determined; 570 nm fluorescence was monitored over time to verify whether expected pHi changes occurred. All data were background-corrected. Cell autofluorescence was below detection, and no correction for photobleaching was applied, because the rate of signal loss was small (1-2%/min).

Whole cell recordings

Calcium currents were recorded under whole cell voltage clamp with a Dagan PC-1 amplifier, sampled at 4 kHz, filtered at 3 kHz with a 3-pole low pass Bessel filter and stored on a PC by the use of a TL-1 Labmaster A/D converter and "P-Clamp" software (v. 5) from Axon Instruments (Foster City, CA). After obtaining a stable recording, capacitive artifacts were canceled electronically, and series resistance was compensated 50-80%. Active currents were evoked by a series of depolarizing voltage steps or ramps from a holding potential of -100 mV, preceded by a 200-ms hyperpolarizing prepulse to -120 mV to remove residual channel inactivation. In some cases, the holding potential was changed to -50 mV to inactivate the N-type current. Whole cell currents were evoked by a series of +10-mV voltage steps from -100 mV to +40 mV at 10-s intervals. Voltage ramps designed to evoke Ca currents began 20 ms after hyperpolarization to -120 mV and increased to +60 mV over 120 ms. Ramps were useful for the frequent repetition of current-voltage (I-V) plots to follow changes over time and for comparisons between different current components in a given cell.

Experimental treatments

After obtaining the whole cell configuration, active currents were monitored by periodic voltage ramps (20- to 30-s intervals) until the currents had stabilized; recordings were typically begun 3-5 min after rupturing the membrane patch ("break-in"). Test solutions were applied through gravity-fed tubing; solution exchange in the chamber occurred within 1 min. All experiments were performed at room temperature (21-23°C).

Data analysis

All analyses of current records, including curve fitting, were done with the use of "P-Clamp" software. Statistical analyses were performed with the use of analysis of variance; between-group post hoc comparisons were made with the use of Sheffé's F-test. In experiments where 200-ms test pulses were applied, currents were measured at their peak, whereas the sustained current was measured as the mean current at the end of the voltage pulse (between 195 and 200 ms). The amplitude of ramp-evoked HVA currents was measured as the maximum current occurring between -15 mV and +15 mV. For summary I-V plots, current amplitudes were expressed as a percent of the maximum current recorded initially under control conditions and presented as means ± SE. In some experiments, currents were evoked by 16-ms test pulses from a holding potential of -50 mV, at which most of the N-type current is inactivated (Fox et al. 1987). Tail currents evoked by this protocol were normalized to the maximum amplitude and plotted against membrane voltage to examine shifts in voltage dependence. In a subset of cells, low-threshold (LVA) currents were visible during voltage ramps and could be evoked with voltage pulses negative to -50 mV from a holding potential of -100 mV. These currents usually contained a noninactivating component (Avery and Johnston 1995) that was subtracted before measurement of the peak current.


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FIG. 3. NH4Cl exposure enhances a sustained HVA current. A: representative HVA currents were evoked by 200-ms pulses before and 30 s after exposure to 20 mM NH4Cl (Vh = -100 mV); highlighted trace labeled "diff." (difference current) was derived by subtraction. Scale bars: 0.5 nA, 50 ms. B: summary current-voltage (I-V) curves derived from measurements of peak and sustained current amplitudes(n = 7); data (means ± SE) are expressed as a percent of the maximum current evoked under control conditions.

Series resistance (Rs) was estimated by fitting a single exponential to the cell capacitance current (filtered at 10 kHz) and then extrapolating to the point immediately before a -10-mV voltage step (Stuart et al. 1993). Input resistance (Rin) and steady-state leak currents were determined by hyperpolarizing prepulses; scaled leak currents derived from these prepulses were subtracted off-line from both ramp and step activated currents before analysis. During periods in which repeated voltage commands were applied, HVA calcium currents exhibited variable "rundown" (0.5-5%/min). Cells that exhibited current fluctuations due to unstable seal resistance were discarded. Illustrated I-V plots derived from voltage steps are spline-fitted. All summary data are presented asmeans ± SE.

The methods we used to separate L- and N-type HVA Ca currents, like all techniques currently available, yield only semiquantitative estimates of a cell's channel composition. For example, it is possible that N-type channel inactivation at -50 mV was incomplete and that currents evoked from this holding potential (referred to herein as "L-type") were contaminated by N-type currents. However, this contamination is considerably smaller at Vh = -50 mV than at Vh = -100 mV. Similarly, the use of nifedipine above 1 µM has been reported to partially block N-type channels (Jones and Jacobs 1990). However, Ca currents remaining in the presence of 5 µM nifedipine probably contained little if any L-type currents.


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FIG. 4. Nifedipine blocks a slowly inactivating current while fast-inactivating currents are blocked by omega -conotoxin. A: nifedipine blocks a slowly inactivating current that accounts for most of the sustained current. Trace marked "diff." (difference current) was derived by subtraction (Vh = -100 mV). Scale bars: 0.5 nA, 50 ms. B: HVA currents were evoked 1st from -120 mV and then again from -50 mV (top panel). Subsequently, HVA currents were repeatedly evoked by a -10-mV test pulse from -120 mV before and during exposure to 0.5 µM omega -conotoxin (GVIA; bottom panel). Both protocols blocked a rapidly inactivating N-type current (diff.). Scale bars: 0.2 nA, 50 ms.

The use of F- as the internal anion during whole cell recording does not affect peak HVA currents, but it has been reported to accelerate whole cell HVA Ca current inactivation in hippocampal neurons by depressing the sustained component of the current (Kay et al. 1986). We cannot rule out the possibility that sustained currents in our cells were depressed by the presence of F- ions, but there is no reason to believe a priori that F- interferes with effects of H+ on Ca channels.

The superior space clamp of acutely isolated neurons relative to other preparations has been attributed to the absence of extensive axonal and dendritic processes. Numann et al. (1987) reported nearly isopotential current-clamp responses in isolated CA1 neurons and estimated that the steady-state voltage attenuation at the ends of dendritic stumps (80-150 µm) in CA1 neurons would not exceed 1% and would still remain less than 10% during active current flow. Because this physical length represents the upper limit for dendrites in our cells, errors associated with inadequate space clamp in the present study would have been even smaller in the majority of our cells and were therefore considered negligible.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Electrotonic parameters

Under control conditions, cell input resistance (Rin) ranged from 400 to 5,000 MOmega . Rin reversibly declined during NH4Cl treatment (in mOmega : control, 1,189 ± 154; NH4Cl,653 ± 56; wash, 1,095 ± 124, n = 22), and reversibly increased during NaAC exposure (in mOmega : control, 954 ± 129; NaAC, 1,250 ± 205; wash, 1,002 ± 126, n = 12). Series resistance (Rs) ranged from 4.6 to 7.5 MOmega and was typically twice the open pipette resistance, corresponding to a voltage clamp error of a few millivolts for a typical 1-nA current when using Rs compensation. In a subset of cells examined, Rs did not change significantly either during or after exposure to NH4Cl (in mOmega : control, 5.2 ± 0.8; NH4Cl, 5.3 ± 0.8; wash, 5.2 ± 0.8, n = 6).


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FIG. 5. Sustained depolarization isolates a slowly inactivating L-type current. Currents were evoked in a single cell 1st from -100 mV and then from -50 mV (see voltage protocols); in the latter case, voltage steps evoked a slowly inactivating current. Scale bars: 0.5 nA; 50 ms (Vh = -100 mV), 4 ms (Vh = -50 mV). I-V plots from currents illustrate that the I-V relationship for the sustained current (Vh = -100 mV) was nearly identical to that for maximum currents evoked from -50 mV.

Intracellular SNARF1 fluorometry

Under conditions identical to those used for Ca current recording, NH4Cl exposure caused a marked and reversible reduction in SNARF1 570-nm fluorescence, consistent with an intracellular alkalinization (Fig. 1A). Identical changes occurred in intact cells loaded with the esterified form of the dye (Fig. 1B). In the absence of external TEA+, this fall in fluorescence was followed by an abrupt increase in signal intensity upon wash out, indicating rebound acidification. In all cases, fluorescence intensity gradually recovered to baseline values (Fig. 1C).

HVA currents

HVA Ca currents could be recorded in all cells examined, whereas a subset of cells also exhibited LVA Ca currents (discussed later). As we reported previously, HVA Ca currents in CA1 neurons activate at -40 mV, peak near 0 mV, can be carried by barium, and are blocked completely by cadmium (Tombaugh and Somjen 1996). HVA Ca currents stabilized within a few minutes of break-in and could be recorded for up to 1 h.

When NH4Cl was applied 5-7 min after break-in (internal HEPES, 10 mM), ramp-evoked HVA currents increased by an average of 29 ± 6% (Fig. 2). NaAC exposure depressed HVA currents by 62 ± 4%. Recovery was always incomplete after NaAC wash out, even after correcting for rundown. Apparent shifts in voltage dependence (on the order of a few millivolts) associated with NH4Cl exposure or Na-acetate were often seen during voltage ramps (Fig. 2).


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FIG. 6. Internal alkalinization causes modest shifts in L-type current amplitude and voltage dependence. A: currents evoked by voltage pulses (+10 mV) before and during exposure to NH4Cl. Scale bars: 0.4 nA, 4 ms. B: summary I-V curves for L-type currents (means ± SE, n = 6). C: normalized tail currents from a representative cell recorded upon return to -50 mV from a depolarizing test pulse (abscissa) delivered before, during, and after NH4Cl exposure.


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FIG. 7. L-type currents exhibit rapid rundown. A: examples of ramp-evoked HVA currents evoked from 2 different cells at 4 and 18 min after break-in (for voltage protocol, see Fig. 2). The size of the initial nifedipine-sensitive component (diff, highlighted) varied from cell to cell, but nifedipine (5 µM) consistently blocked a proportionally smaller fraction of the total current after prolonged periods of dialysis. B: examples of L-type and N-type current rundown in a single cell. L-type currents(Vh = -50 mV) were evoked with repeated test pulses (0 mV, 10-s intervals) beginning at 15 min after break-in; N-type currents were similarly evoked (Vh -100 mV) beginning at 45 min after break-in. Currents were leak corrected, and peak amplitudes are expressed as a fraction of the initial current. Scale bars: N-type: 0.2 nA, 50 ms; L-type: 0.5 nA, 4 ms.

In a separate series of experiments, we addressed whether solution exchange affected HVA Ca currents via changes in pHi. We varied the internal pH buffering and examined whether this influenced the change in current amplitude during NH4Cl exposure. Lowering the internal HEPES to 1 mM significantly amplified the increase in HVA current amplitude, whereas raising HEPES to 50 mM had the opposite effect (Table 1).

Because EGTA's affinity for Ca2+ is strongly pH sensitive (Tsien 1980), we also examined whether the effects of NH4Cl on HVA Ca currents could have been due in part to shifts in intracellular Ca2+ buffering. When we replaced EGTA with the pH-insensitive Ca2+ buffer BAPTA (using the same HEPES concentration, 10 mM), NH4Cl enhanced HVA currents to the same degree (Table 1).

During NH4Cl exposure, the sustained (195-200 ms) portion of step-evoked HVA currents (Vh = -100 mV) was proportionally more enhanced than the peak current (Fig. 3). Current inactivation during 200-ms depolarizing pulses could be fitted with the sum of two exponentials (Vreugdenhil and Wadman 1992), neither of which was significantly affected by NH4Cl exposure when examined with test pulses between -20 and +40 mV [at 0 mV: tau 1 = 15 ± 2 ms (control), 16 ± 2 ms (NH4Cl); tau 2 = 74 ± 5 ms (control), 74 ± 5 ms (NH4Cl); n = 10].

Currents evoked at positive potentials were contaminated by residual outward currents that, theoretically, could have been pHi sensitive (Byerly and Moody 1986). However, when HVA Ca currents were abolished with 400 µM Cd2+, we failed to detect any change in the amplitude of these residual currents during NH4Cl exposure (n = 3).

At negative holding potentials (-100 mV), a nifedipine-sensitive current accounted for most or all of the sustained current (Fig. 4A). omega -Conotoxin (GVIA, 0.5 µM) blocked a rapidly inactivating component of the HVA current (Fig. 4B) that was comparable with the current inactivated at a holding potential of -50 mV. When the holding potential was raised to -50 mV to inactivate N-type currents (Fox et al. 1987), a slowly inactivating current could be evoked whose activation and amplitude appeared to correspond to the sustained current evoked from -100 mV (Fig. 5). This current was also nifedipine sensitive (not shown), identifying it as an L-type current. When corrected for rundown, these L-type currents increased by 40 ± 3% during NH4Cl exposure (Fig. 6, A and B). Normalized tail currents, evoked upon return to -50 mV, were shifted by 6-8 mV during NH4Cl exposure (Fig. 6C).


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FIG. 8. HVA current sensitivity to NH4Cl is enhanced by nifedipine. A: time course of NH4Cl effect on ramp-evoked HVA currents under control conditions(n = 8) and in the presence of 5 µM nifedipine (n = 7). Currents (means ± SE) are expressed as a percent of baseline values following correction for leak and rundown. B: representative ramp-evoked currents (different cells) for each experimental group before, during and after NH4Cl exposure (Vh = -100 mV; for voltage protocol, see Fig. 2).

Nifedipine blocked a large fraction of the whole cell ramp-evoked current within 5 min after break-in but became less effective after prolonged periods of dialysis (Fig. 7A). L-type currents were observed in a majority of cells shortly after break-in but exhibited a more rapid rate of rundown compared with N-type currents (Fig. 7B). In the presence of nifedipine, the effect of NH4Cl was more pronounced, increasing whole cell HVA currents by 76 ± 9% (Fig. 8). This result was confirmed when N-type currents were evoked by voltage steps in the presence of nifedipine (Fig. 9). In addition, the ability of NH4Cl to enhance HVA currents was always amplified in a given cell during a second NH4Cl exposure (Fig. 10). This increase in efficacy was not affected when nifedipine was present during the second treatment, but was significantly blunted when nifedipine was present during both the first and second NH4Cl application (Fig. 10).


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FIG. 9. N-type currents are highly sensitive to internal alkalosis. A: N-type currents evoked in the presence of nifedipine (5 µM) before and during exposure to NH4Cl. Scale bars: 0.2 nA, 50 ms. B: summary I-V relation (n = 3; mean ± SE) before, during, and after (wash) exposure to NH4Cl. Data are presented as a percent of the maximum current recorded under control conditions (compare with L-type currents in Fig. 7).


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FIG. 10. Enhancement of HVA currents by NH4Cl is time dependent and nifedipine sensitive. Summary of changes in the efficacy of NH4Cl on ramp-evoked whole cell HVA currents (Vh = -100 mV). Data are presented as the percent of baseline value for the peak current (corrected for leak and rundown). The increase in the efficacy of NH4Cl occurred during a 2nd NH4Cl exposure. This increase was unaffected when nifedipine was present during the 2nd trial but was significantly blunted when nifedipine was present during both trials. All experiments were begun 5-10 min after break-in (n = 5) time between trials ranged from 3 to 10 min. *Significantly different from control, P < 0.02. **Significantly different from control,P < 0.01.

LVA currents

LVA or T-type currents were detected in a subset of cells (21/65 = 32%) by both ramp and step depolarization (Fig. 11). These currents activated below -50 mV and peaked near -40 mV, inactivated rapidly, were amiloride sensitive (500 µM) (Tombaugh and Somjen 1995), and exhibited little if any rundown, as described elsewhere (Fox et al. 1987; Karst et al. 1993; Rorsman 1988; Tang et al. 1988). When evoked by voltage pulses at or below -50 mV, the rapidly inactivating LVA currents could be enhanced by external alkalosis (pHo = 8.0; 24 ± 4%, n = 4) and depressed by external acidosis (pHo = 6.4; 64 ± 11%, n = 4; Fig. 12). In contrast, LVA current amplitude was relatively unaffected by exposure to either NaAC or NH4Cl (Fig. 12).


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FIG. 11. Some CA1 neurons express low-voltage activated Ca currents. Examples of ramp-evoked currents recorded within 5-10 s after break-in (to) and at 20-s intervals thereafter (Vh = -100 mV; for voltage protocol, see Fig. 2). Prominent low-threshold (LVA) currents (asterisk) were evoked at membrane potentials negative to -50 mV in 32% of the cells examined. Inset: LVA currents evoked by a series of depolarizing voltage steps from the same cell (Vh -100 mV). Scale bars: 100 pA, 50 ms.


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FIG. 12. LVA currents are pHo sensitive but pHi insensitive. LVA currents were evoked repeatedly (10-s intervals) with test pulses between -50 and -60 mV (n = 4/group). Cells were exposed to NH4Cl (A), Na-acetate (B), or to solutions adjusted to pHo 8.0 (C) or 6.4 (D). Sustained, noninactivating currents measured after 195-200 ms were subtracted from the peak. Current amplitudes are expressed as a percent of baseline values and are presented as means ± SE.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

In isolated rat CA1 neurons, we find that 1) internal acidosis depresses whereas internal alkalosis enhances HVA Ca currents; 2) L-type Ca currents are less sensitive to alkaline pHi shifts than N-type currents; and 3) low-threshold Ca currents are insensitive to both acidic and alkaline pHi shifts even though they are sensitive to external pH shifts.

Mechanisms of action of NH4Cl

The enhancement of HVA currents by NH4Cl was sensitive to the level of internal pH buffering, confirming that this effect occurred via shifts in pHi. In the absence of CO2/HCO-3 buffering, the normal intrinsic pH buffering capacity of neurons has been estimated to be 15-25 mM (Chesler 1990), due primarily to large immobile anions and proteins that are resistant to dialysis during whole cell recording. The existence of a rigid source of cytosolic H+ buffering provides a plausible explanation for why a 50-fold decrease in nominal pH buffering resulted in a relatively modest (3-fold) enhancement of NH4Cl's effect on HVA currents.

Microfluorometry confirmed that cells were readily alkalinized with NH4Cl. However, because in vivo SNARF1 calibrations were not performed and because the precise pH buffering capacity in our cells is unknown, we can only estimate the changes in pHi. In an earlier study with fish retinal neurons, NH4Cl (20 mM) evoked an increase of 0.4 pH units occurred with little or no pH buffering in the internal solution (Dixon et al. 1993). In our cells, pHi shifts were presumably smaller due to the presence of 10 mM HEPES in the pipette, a concentration reported by Dixon et al. to blunt the effects of NH4Cl on HVA currents.

In the present study, EGTA was used as the principal intracellular Ca2+ buffer. Because EGTA's affinity for Ca2+ is highly pH sensitive, the effects of NH4Cl and Na-acetate on ICa could have simply reflected changes in intracellular Ca2+ buffering, which could have altered the extent of Ca-induced channel inactivation, the driving force for Ca2+, or both. However, we detected no difference in the NH4Cl-mediated increase in HVA currents between cells dialyzed with EGTA or the pH-insensitive Ca2+ buffer BAPTA (Tsien 1980). Similar results have been reported in both chick spinal neurons (Mironov and Lux 1991) and fish retinal neurons (Takahashi et al. 1993). Moreover, pHi effects on HVA Ca currents in retinal neurons are preserved when Ba2+ is used as the charge carrier (Dixon et al. 1993). These findings argue that passive changes in intracellular Ca2+ associated with pHi shifts play a minor role in mediating the effects on HVA Ca currents.

Under physiological conditions, NH4Cl wash out leads to a rapid and reversible intracellular acidosis. This acidosis, however, is dependent on the influx of NH4+ through K channels (Thomas 1984), which are blocked in the presence of TEA+. Fluorometric measurements using the pH-sensitive dye SNARF1 confirmed that bath-applied TEA+ was sufficient to abolish the rebound acidification after NH4Cl wash out while preserving the alkaline shift. Changes in HVA currents were consistent with this pHi change, showing no abrupt decrease upon NH4Cl wash out. In the absence of extracellular TEA+, Ca currents in both fish retinal and chick dorsal root ganglion (DRG) neurons become transiently depressed upon NH4Cl wash out (Dixon et al. 1993; Mironov and Lux 1991).

Differences in pHi sensitivity among calcium currents

At a negative holding potential (-100 mV), NH4Cl enhanced the sustained (195-200 ms) current proportionally more than the peak current. We considered three possible explanations for this effect. First, a rise in pHi could have slowed current inactivation without affecting peak conductance. However, when HVA currents were fitted with double exponentials, the decay rate for either component was not slowed significantly during NH4Cl exposure. Similar findings have been reported in chick DRG neurons (Mironov and Lux 1991).

A second possibility was that this asymmetric increase in HVA currents reflected the depression of a slowly, or noninactivating K+ or H+ outward current by NH4Cl (Byerly and Moody 1986). This would also explain the apparent positive shift seen in the ascending limb of ramp I-V curves. However, when HVA Ca currents were abolished with Cd2+, we failed to find any change in the residual outward current caused by NH4Cl. Outward currents that persist in the presence of TEA+ (0 mM K+) are known to contaminate Ca currents in a variety of cells and probably reflect Cs+ ions flowing through Ca channels (Fenwick et al. 1982; Lee and Tsien 1982).

A third possibility was that a rise in intracellular pH selectively enhanced one of several HVA Ca channel types. In cases where the holding potential was raised to -50 mV to inactivate the N-type current (Fox et al. 1987), a more slowly inactivating, nifedipine-sensitive current could be evoked. This L-type current represented a large fraction of the sustained current and was enhanced by 40% with NH4Cl (slightly smaller than the 50% increase observed for the sustained current evoked from -100 mV). This initially suggested to us that a slowly inactivating L-type current was selectively enhanced by internal alkalosis, a suspicion reinforced by the fact that 1) hippocampal neurons contain both N- and L-type channels (Fisher et al. 1990) and 2) L-type currents are blunted by internal H+ in many cell types (Dixon et al. 1993; Irisawa and Sato 1986; Umbach 1982).

In an earlier study, we reported that two overlapping but distinct HVA current components in CA1 neurons could be resolved during external acidosis (Tombaugh and Somjen 1996). In the present study, we were able to separate these components to some degree on the basis of differences in sensitivity to voltage and nifedipine, but a detailed comparison was hampered by the fact that the two currents had overlapping I-V profiles and underwent different rates of rundown. The L-type (nifedipine-sensitive) current typically represented a large fraction of the whole cell current shortly after break-in but was usually lost within 15-30 min. A rapid loss of the L-type current in hippocampal neurons has been reported previously (Takahashi et al. 1989) and, in our study, was evident by the fact that after long periods of dialysis (15-30 min) 1) very little inward current could be evoked from a holding potential of -50 mV, and 2) nifedipine had a much smaller effect on currents evoked from -100 mV. Under either of these conditions, the rapidly inactivating N-type current that remained, although often smaller in size, showed little rundown. This omega -conotoxin-sensitive current, which typically represented a small fraction of the initial whole cell current, was more dramatically enhanced by NH4Cl than the L-type current, a difference masked at early time points by the greater fractional current carried by L-type channels. As L-type currents ran down, a larger percentage of the N-type current remained, and its greater sensitivity to NH4Cl was revealed.

In contrast to an earlier report (Mironov and Lux 1991), NH4Cl also caused a reversible depolarizing shift in the voltage dependence of L-type currents. This shift could have arisen from a smaller net negative surface charge on the inner face of the cell membrane, or from reduced H+ titration of specific negative groups on the channel itself (Hille 1968). However, it should be pointed out that a reduction in the Ca2+ driving force could have occurred during NH4Cl exposure due to increased Ca influx and may have blunted Ca tail currents, leading to an overestimate of the size of the voltage shift. Because intracellular Ca was not measured, corrections for changes in driving force could not be performed.

In addition to HVA currents, low-threshold T-type currents were occasionally seen. Their presence in only one-third of the cells examined may reflect variation in the extent of dentritic membrane loss (Karst et al. 1993). Earlier studies have questioned the existence of these channels in CA1 neurons (Kay and Wong 1987). However, our findings, combined with those of Karst et al., should put to rest any remaining doubt that adult CA1 neurons express functional LVA Ca channels. These LVA currents were insensitive to internal pH changes even though they could be reversibly enhanced or depressed by raising or lowering external pH. This finding is essentially identical to that reported for T-type currents in cardiac myocytes (Tytgat et al. 1990). In CA1 neurons, the pHo sensitivity of LVA and HVA currents appears quite similar (Barnes and Bui 1991; Tombaugh and Somjen 1996), although reliable Hill plots must await simultaneous ICa and pHi measurements.

Possible physiological significance

Although H+ modulation of Ca channels has been observed in many cell types, its physiological significance remains speculative. Intracellular H+ ions are widely thought to compete with negative binding sites in the cytosol and thereby modulate free Ca2+ levels. A slight rise in baseline Ca2+ has been reported in cultured cortical neurons during acidification (Ou-Yang et al. 1994), although the opposite effect has been observed in other cell types (cf. Busa and Nuccitelli 1984). Less emphasized has been the idea that cytosolic pH shifts may themselves act as long-range signals. In this idea, endogenous pH buffers and transporters act to restrict pHi transients both spatially and temporally much in the same way that analogous systems limit the extent of Ca2+ signals. However, it has been suggested that pHi transients are less rapidly buffered than Ca2+ transients and may therefore modulate pH-sensitive targets over greater distances (Morris et al. 1994). Cytosolic pH shifts could either alter protein function directly by titrating critical residues, or indirectly by affecting the duration or magnitude of a coincident Ca2+ transient. In addition, the strong H+ sensitivity of calmodulin's affinity for Ca2+ indirectly confers a pH dependence on the many Ca2+-dependent processes that calmodulin normally regulates (Trachuk and Men'shikov 1981).

In general, intracellular pH changes that function as biological signals (e.g., during sea urchin fertilization) (Busa and Nuccitelli 1984) are not known to affect ion channels selectively. Such selectivity could arise by limiting the range or physical compartment in which a pH change occurs. For example, if pHi shifts should occur in synaptic terminals, they would presumably influence neurotransmitter release, either via their action on Ca channels or by some other mechanism (Drapeau and Nachshen 1988; White et al. 1989). A related scheme might involve the spatial segregation of pH-sensitive targets, one that may be relevant to neuronal Ca channels given that they are thought to have distinct cellular distributions (Christie et al. 1995). However, our results provide evidence for a third way of deriving specificity, namely by encoding differences in the H+ sensitivity of Ca channels. In this case, a widespread shift in internal pH might still be capable of selectively modulating certain aspects of cell function. As an example, intradendritic acidosis associated with synaptic activity may depress HVA channels without affecting LVA channels, thereby preventing an excessive rise in cytosolic Ca2+ while preserving LVA channel activity for normal integration and amplification of synaptic input.

In summary, we report that HVA Ca currents in hippocampal neurons are sensitive to pHi shifts induced by exposure to a weak acid or base. The magnitude of these shifts was not determined but was probably on the order of a few tenths of a pH unit. Whether such pHi shifts actually occur in vivo has not yet been reported, although they remain theoretically possible in the submembrane space given the calculated buffering capacity of mammalian neurons and size of pHo transients known to occur in the mammalian CNS. Perhaps more importantly, we find that different Ca channel types expressed in CA1 neurons exhibit a wide range of sensitivities to internal pH, providing a possible source of target specificity for changes in cytosolic pH during normal and pathological states.

    ACKNOWLEDGEMENTS

  We thank Drs. Mitchell Chesler and Peter Aitken for critical readings of this manuscript. Expert technical assistance was provided by D. Poe.

  Support for this work was provided to G. C. Tombaugh by the National Stroke Association, the American Heart Association, and the Carl J. Herzog Foundation and to G. G. Somjen by National Institute of Neurological Disorders and Stroke Grant 5R01-NS-18670.

    FOOTNOTES

  Address for reprint requests: G. C. Tombaugh, Dept. of Cell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710.

  Received 12 July 1996; accepted in final form 21 October 1996.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society