Concentration Dependence of Bicarbonate-Induced Calcium Current Modulation

C. Bruehl,1 W. J. Wadman,2 and O. W. Witte1

 1Department of Neurology, Heinrich-Heine-University, 40225 Duesseldorf, Germany; and  2Institute for Neurobiology, University of Amsterdam, 1098 SM Amsterdam, The Netherlands


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Bruehl, C., W. J. Wadman, and O. W. Witte. Concentration Dependence of Bicarbonate-Induced Calcium Current Modulation. J. Neurophysiol. 84: 2277-2283, 2000. High-voltage-activated calcium currents (HVA) of CA1 neurons are prominently attenuated following a switch from HEPES-buffered solution to one buffered with CO2/HCO3-. In the present study we investigated whether bicarbonate ions or the dissolved CO2 induce this alteration in current characteristic. The study was carried out on freshly isolated CA1 neurons using the whole cell patch-clamp technique. Maximal calcium conductance and the mean peak amplitude of the currents showed a concentration-dependent decrease when cells were consecutively bathed in solutions containing increasing amounts of bicarbonate and CO2. This decrease is best described by the Hill equation, yielding a maximal attenuation of 69%, a half-maximal concentration (EC50) of 7.4 mM HCO3-, and a Hill coefficient of 1.8. In parallel, the potentials of half-maximal activation (Vh,a) and inactivation (Vh,i) were linearly shifted in hyperpolarizing direction with a maximal shift, in the 10% CO2/37 mM HCO3- containing solution of 10 ± 1 mV for Vh,a (n = 23) and 17 ± 1.4 mV for Vh,i (n = 18). When currents were evoked in solutions containing equal concentrations of bicarbonate but different amounts of CO2, only nonsignificant changes were observed, while marked alterations of the currents were induced when bicarbonate was changed and CO2 held stable. The experiments suggest that bicarbonate is the modulating agent and not CO2. This bicarbonate-induced modulation may be of critical relevance for the excitation level of the CNS under pathological situation with altered concentration of this ion, such as hyperventilation and metabolic acidosis.


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In a previous study we have shown that CO2/HCO3--buffered solutions have modulatory effects on whole cell calcium currents in neurons from the hippocampal area CA1 of the rat (Bruehl et al. 1998, 1999). When current characteristics were compared between a CO2/HCO3--free bathing medium (HEPES buffering) and a CO2/HCO3--containing solution, we could demonstrate that the so-called high-voltage-activated (HVA) calcium current decreases in amplitude and maximal conductance in the latter bath solution. Furthermore, the potentials for half-maximal activation and inactivation were strongly shifted to more negative values. This kind of modulation may be of importance for the excitability of the single neuron and, more generally to the whole neuronal network, especially under conditions when bicarbonate concentrations are strongly altered, e.g., during hypocapnia and increased metabolism accompanying enhanced neuronal activity.

Changes in the membrane potential and excitability of neurons induced by the de novo introduction of CO2/HCO3- or by varying the concentration of both have been demonstrated in cell cultures and slice preparations previously (Church 1992; Church and McLennan 1989; Cowan and Martin 1995, 1996). Increasing the amounts of CO2/HCO3- led to a switch of the activity mode from a spike-train to a burst-generating mode. Moreover, calcium spikes (sodium currents were blocked by TTX) could be evoked at more negative potential than in solutions without CO2 and bicarbonate. Both phenomena indicate a modulation of calcium currents by bicarbonate ions or the gas CO2.

Both, bicarbonate and CO2 are end products of metabolism. A direct interaction of bicarbonate with calcium currents may therefore act as a negative feedback mechanism between excitability and energy consumption. Strong neuronal activity increases CO2 and bicarbonate, which in turn may reduce or even stop this activity by attenuation of calcium currents.

The present study addressed the following questions. First, it was investigated whether CO2 or the bicarbonate ion or even both are the modulating agents. Second, we tested whether the modulation is dependent on the concentration of the modulator. And finally, it was examined whether this modulator acts preferentially on intra- or extracellular sites of the membrane. The investigations were carried out on freshly isolated pyramidal neurons from the hippocampal CA1 area of the rat using the whole cell voltage-clamp technique.


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Cell preparation

CA1 pyramidal neurons were isolated enzymatically from male Wistar rats (75-85 g) as described in detail previously (Vreugdenhil and Wadman 1992). From both hippocampi, 500-µm-thick slices were cut, and the CA1 area was dissected. These tissue pieces were incubated 75 min at 32°C in dissociation solution (in mM: 120 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 20 PIPES, and 25 D-glucose; pH 7.0) containing 1 mg/ml trypsin (Bovine Type XI), which was equilibrated with oxygen. Following enzymatic treatment, tissue was rinsed twice and kept in the dissociation solution without trypsin at 19°C. Directly before measurements, tissue pieces were dispersed in HEPES-buffered bath solution by trituration through Pasteur pipettes with decreasing tip diameter, and cells were allowed to settle in the perfusion chamber.

To assure total solution exchange, we used a bath with a volume of ~120 µl, which was perfused with a constant flow rate of 1 ml per minute. Bath solutions contained 110 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 5 mM 4-aminopyridine (4-AP), 25 mM TEA-Cl, 25 mM D-glucose 25, and 0.5 µM tetrodotoxin (TTX); pH was set at 7.4 (unless otherwise stated). For seal formation all cells were patched in the above-mentioned solution, plus 10 mM HEPES. Bath solutions containing CO2/HCO3- as the pH-buffering system were thoroughly gassed with different amounts of CO2 (2.5; 5; 10%) before HCO3- was added. Special care was taken to assure that the solutions were always equilibrated with CO2 throughout the course of the experiment, since otherwise CaCO3 would have precipitated.

All chemicals were obtained from Sigma (Deisenhofen, Germany) and Merck (Darmstadt, Germany).

Current recording

Currents were measured under whole cell voltage-clamp conditions at room temperature using patch pipettes of 2-4 MOmega resistance. Electrode solution contained 80 mM CsF, 2 mM MgCl2, 0.5 mM CaCl2, 15 mM TEA-Cl, 10 mM EGTA, 5 mM phosphocreatine, 50 units/ml phosphocreatine kinase, 2 mM MgATP, 0.1 mM NaGTP, and 0.1 mM leupeptin; pH set at 7.3 (unless otherwise stated). The solution was strongly buffered by 50 mM HEPES, to prevent intracellular pH changes following the introduction of CO2/HCO3--buffered solution. The ATP regenerating system reduced the "run down" of the calcium current; the gradual decrease in current amplitude never exceeded 10% within the recording period of 10-15 min. Currents were measured with an Axopatch 200A amplifier (Axon Instruments) and stored on an Atari ST computer (1-kHz sample frequency). Capacitive transients and series resistances were compensated on-line. Data were evaluated off-line using a custom-made computer program. All current traces were corrected for aspecific linear leak (reversal potential 0 mV) determined at holding potential.

Experimental protocols

Calcium currents were activated using 200-ms voltage steps to voltage levels between -40 and +40 mV. Holding potential was kept at -80 mV. The steady-state inactivation of the calcium current was determined using a standard depolarization to +10 mV after the cell was polarized for 3 s at various levels between -105 and 0 mV. During solution changes a ramp protocol was carried out (from -100 to 50 mV; within 150 ms) to monitor current changes and to ensure the stability of the changes.

Neurons were bathed first in HEPES-buffered saline, and voltage protocols that determine activation and inactivation properties were performed. Next, the HEPES-buffered saline was replaced by a CO2/HCO3--buffered saline while the ramp protocol was applied. When a stable condition in the presence of the CO2/HCO3--buffered saline was achieved, currents were examined again using the same set of voltage-clamp protocols as during the HEPES condition.

Current analysis

Peak amplitudes of the currents (I) evoked with the activation protocol were plotted as a function of membrane potential (V). The resulting I-V relations were fitted with a combination of Boltzmann activation function and the Goldman-Hodgkin-Katz (GHK) current-voltage relation (Hille 1992; Kortekaas and Wadman 1997)
<IT>I</IT>(<IT>V</IT>)<IT>=</IT><IT>V</IT> <FR><NU><IT>g</IT><SUB><IT>max</IT></SUB></NU><DE><IT>1+exp</IT><FENCE><FR><NU><IT>V</IT><SUB><IT>h</IT></SUB><IT>−</IT><IT>V</IT></NU><DE><IT>V</IT><SUB><IT>c</IT></SUB></DE></FR></FENCE></DE></FR> <FR><NU>[<IT>Ca<SUP>2+</SUP></IT>]<SUB><IT>in</IT></SUB><IT>/</IT>[<IT>Ca<SUP>2+</SUP></IT>]<SUB><IT>out</IT></SUB><IT>−exp</IT>(−<IT>&agr;</IT><IT>V</IT>)</NU><DE><IT>1−exp</IT>(−<IT>&agr;</IT><IT>V</IT>)</DE></FR> (1)
with alpha  = 2F/RT and gmax = 2alpha FP0[Ca2+]out, where gmax is the maximal membrane conductance (which is directly related to the maximal permeability and the extracellular calcium concentration), Vh is the potential of half-maximal activation, and Vc is proportional to the slope of the curve at Vh. F represents the Faraday constant, R the gas constant, P0 the maximal permeability, and T the absolute temperature.

The voltage dependence of steady-state inactivation of the calcium current was estimated from the relation of the amplitude of the current versus the prepotential. This relation was well described by a Boltzmann function, which also normalized the current
<IT>N</IT>(<IT>V</IT>)<IT>=</IT><FR><NU><IT>I</IT>(<IT>V</IT>)</NU><DE><IT>I</IT><SUB><IT>max</IT></SUB></DE></FR><IT> where </IT><IT>I</IT>(<IT>V</IT>)<IT>=</IT><FR><NU><IT>I</IT><SUB><IT>max</IT></SUB></NU><DE><IT>1+exp</IT><FENCE><FR><NU><IT>V</IT><SUB><IT>h</IT></SUB><IT>−</IT><IT>V</IT></NU><DE><IT>V</IT><SUB><IT>c</IT></SUB></DE></FR></FENCE></DE></FR> (2)
where N(V) is the level of steady state inactivation determined from the current amplitude I(V) normalized to Imax, V is the prepulse potential, Vh is the potential of half-maximal inactivation, and Vc is a factor proportional to the slope of the curve at Vh. The concentration-response curves were fitted with the Hill equation
<IT>N</IT>(<IT>c</IT>)<IT>=</IT><FR><NU><IT>N</IT><SUB><IT>max</IT></SUB></NU><DE><IT>1+</IT><FENCE><FR><NU><IT>c</IT></NU><DE><IT>EC<SUB>50</SUB></IT></DE></FR></FENCE><SUP><IT>h</IT></SUP></DE></FR> (3)
where Nmax is the maximal effect, c is the concentration of bicarbonate, EC50 the concentration of half-maximal effect, and h the Hill coefficient.

Statistics

Values are given as means ± SE. Statistical comparisons were made with Student's t-test. P < 0.05 was taken to indicate significant differences.


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Concentration-dependent alteration of HVA currents

In HEPES-buffered bath solution, HVA currents could be evoked in 23 cells by voltage steps more positive than -35 mV (Fig. 1). They showed the typical fast activation (time-to-peak at 0 mV: 9.2 ± 0.5 ms) and a slow and incomplete inactivation (time constant at 0 mV tau : 47.3 ± 3.3 ms). They reached their maximal peak amplitude at around 5 mV with a value of -1.85 ± 0.1 nA (Fig. 1). When current amplitude was plotted as a function of membrane potential, it gives the typical I-V relationship, which could well be fitted by the GHK-current-voltage equation (Eq. 1). In this way the activation properties of the current could be described with only three variables (Vh, Vc, and gmax). The mean value of the potential of half-maximal activation (Vh) was -2.4 ± 0.9 mV and the mean value of the slope parameter (Vc) was 6.5 ± 0.1 mV. The maximal calcium conductance (gmax) also obtained from the GHK fit was calculated to be 257 ± 17 nS under these conditions.



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Fig. 1. Whole cell calcium currents of a CA1 neuron evoked in 4 bath solutions buffered to pHo: 7.3 by increasing concentrations of CO2 and bicarbonate (see top row). Cell was held at -80 mV followed by a test pulse to 10 mV (see inset). The peak amplitude gradually decays with the increase of CO2/HCO3-.

The voltage dependence of steady-state inactivation of these currents were determined by holding the cells at different prepotentials followed by a voltage step to 10 mV. The amplitudes of the evoked calcium currents were then plotted as a function of prepotentials and fitted for each individual cell with the Boltzmann equation (Eq. 2). The mean values of inactivation parameters in the Boltzmann equation (Vh, Vc) were then calculated. The mean potential of half-maximal inactivation Vh was -32.0 ± 1.4 mV, and the slope parameter (Vc) had a value of -11.0 ± 0.5 mV.

The cells were subsequently subjected to three bath solution changes, in which the HEPES-buffered solution was replaced by solutions containing 2.5% CO2/5.6 mM HCO3-, 5.0% CO2/18 mM HCO3-, and 10% CO2/37 mM HCO3-. All solutions had a pH value of 7.3. A 3-min period was allowed to guarantee total solution exchange, before the same protocols were applied as with HEPES-containing solution. The introduction of CO2 and bicarbonate reduced the current amplitude (Figs. 1 and 2) and the calcium conductance (Fig. 3). The decrease of current amplitude occurred within seconds after solution exchange and reached its final value within the following minute. Since the size of conductance and current amplitude were inversely related to the amount of CO2 and HCO3-, we plotted the relative reduction in gmax as a function of the concentration of dissolved bicarbonate. The data were then fitted with the Hill equation (Eq. 3), which gave as parameters a maximal decrease of 69%, half-maximal concentration of bicarbonate 7.4 mM and a Hill coefficient of 1.8. The potential of half-maximal activation was shifted to more negative potentials, with the largest shift in the 10% CO2/37 mM HCO3- containing solution (-10.0 ± 1.0 mV), while the slope parameter (Vc) at the point Vh was not altered. The concentration-dependent shift of Vh appeared to be linear (Fig. 4) within the concentration range tested. Steady-state inactivation was also modulated in a concentration-dependent manner (Fig. 4). The potential of half-maximal inactivation was shifted to more hyperpolarized potentials, with a maximal shift of -17.0 ± 1.4 mV in the 10% CO2/37 mM HCO3--buffered solution. The different shifts in Vh for activation and inactivation widens the potential range in which a "window current" can exist in the bicarbonate-containing solution, i.e., a current that in that particular voltage range will activate and not completely inactivate.



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Fig. 2. Mean calcium current-voltage relationship of 23 neurons (bottom half), which were subsequently bathed in solutions with increasing concentrations of CO2 and bicarbonate (see also Fig. 1; pHo: 7.3). The high-voltage-activated calcium current was decreased at potentials more positive than -10 mV. Note also the negative shift of the peak amplitude in solutions with higher concentrations of bicarbonate and CO2. The top half of the graph shows the corresponding shift in voltage dependence of the Boltzmann curves of activation.



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Fig. 3. Reduction of the maximal calcium conductance (gmax) with higher concentrations of CO2/HCO3- (pHo: 7.3) was best defined with the Hill equation (R2: 1.00). Note also the decrease in SE with higher buffer concentrations, typical for a saturating binding or screening process.



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Fig. 4. The relative shift of the potentials of half-maximal activation; Vh,a (open circle ) and inactivation; Vh,i () in solutions containing different amount of CO2 (2.5%; 5%, and 10%) and bicarbonate (5.6, 18, and 37 mM) vs. HEPES-buffered solution (0 mM HCO3-) are plotted against the bicarbonate concentrations. Both parameters shifted to more negative membrane potentials in an almost linear manner within the concentration range tested. Since Vh,i was stronger shifted than Vh,a this resulted into a wider range in which a "window current" can exist. The pH value of all solutions were 7.3.

Bicarbonate acts from outside

In the preceding experiments, several conditions were altered to induce the described effects. First the concentration of CO2 and of HCO3- were concomitantly increased, and second the amount of CO2/HCO3- that occurs within the cells by formation and dissociation according to the Henderson-Hasselbalch equation, was different for each bath solution. Therefore additional experiments were performed to unravel the questions whether CO2 or HCO3- is the primarily modulating agent and whether this agent acts on the outer or inner side of the cell membrane.

The latter question was investigated by superfusing cells with a bath solution that contained 5.0% CO2/26 mM HCO3- with a pH value set to 7.4, and intracellular pH of the cells was preset by the electrode solution to three different values (7.0, 7.3, and 7.6). Since the intracellular pH is heavily buffered by 50 mM HEPES, the CO2/H2CO3 that crosses the membrane has to form lower concentrations of bicarbonate in a more acidic milieu and higher concentrations with a more alkaline pH. For each intracellular pH value, 15 cells were measured. In these experiments no relation between the intracellular bicarbonate concentration and the previously observed effects could be observed. There was neither a difference in the reduction of the maximal calcium conductance (41 ± 8%, 38 ± 3%, and 41 ± 6%; Fig. 5), the reduction of the peak amplitude (24 ± 5%, 24 ± 3%, and 31 ± 3%), nor a difference in the shift of the half-maximal potential of activation (6.3 ± 1.6 mV, 4.7 ± 0.9 mV, and 5.1 ± 1.9 mV). Only the voltage shift of the inactivation showed a slight tendency to change with the intracellular bicarbonate concentration (Vh was 6.0 ± 2.2 mV, 7.5 ± 1.1 mV, and 10 ± 2.8 mV), but this difference did not reach significance.



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Fig. 5. There was only a slight sensitivity of the reduction in gmax when the concentration of CO2 was increased alone (left column). Since pHo varies with different CO2 and constant HCO3-, measurements were done in solution pairs (HEPES vs. CO2/HCO3-) with corresponding pH. Increases of the intracellular HCO3- adjusted by the intracellular pHi had no additional effect on the reduction in gmax (right column). The reduction of gmax was highly sensitive to increases in extracellular bicarbonate concentration (middle column). The extracellular solutions contained the following. Left column: 2.5, 5, or 10% CO2 and 26 mM HCO3-; pHi: 7.3. Middle column: 5% CO2 and 12, 26, and 45 mM HCO3-; pHi: 7.3. Right column: 5% CO2 and 26 mM HCO3-. pHi: 7.0, 7.3, and 7.6.

Bicarbonate, but not CO2 modulate HVA currents

To determine whether CO2 or the bicarbonate ions modulate the HVA current, an additional series of experiments was carried out. Neurons (6 cells tested in each solution) were bathed in solutions gassed by three different CO2 concentrations (2.5, 5.0, and 10%), while the concentration of bicarbonate was kept at 26 mM (Fig. 5, left group). This composition leads to different extracellular pH values (7.76, 7.41, and 7.17), which would introduce a second alteration when the superfusion is switched from the HEPES-buffered solution to the CO2/HCO3--buffered solution. The pH values of the HEPES-buffered solution were therefore adjusted to match the pH of the CO2/HCO3--buffered solution. Under these conditions the reduction of maximal calcium conductance showed a weak relation with increased CO2 values (38 ± 5%, 41 ± 3%, and 48 ± 4%; Fig. 5, left panel), but the trend did not reach significance. The same held for the reduction in mean peak amplitude of the calcium currents (21 ± 5%, 28 ± 3%, and 36 ± 6%). Also the potential of half-maximal inactivation showed comparable negative shifts for the three solutions tested (shift in Vh was 8.8 ± 0.7 mV, 9.1 ± 1.0 mV, and 9.8 ± 1.5 mV). The potential of half-maximal activation was shifted but showed no direct relation with the amount of dissolved CO2 (Vh was 5.7 ± 1.0 mV, 3.7 ± 1.5 mV, and 6.7 ± 1.6 mV).

The experiments shown above suggest that bicarbonate ions alone modulate the calcium current properties. To substantiate this assumption, a second set of experiments was carried out. Solutions containing different amounts of HCO3- ions (12, 26, 45 mM) and a constant concentration of CO2 (5%) were tested in respect to their modulating effect (Fig. 5; middle group). The pH of the HEPES-buffered solution was adjusted to the pH of the bicarbonate-containing solution, like in the experiment above, to avoid effects related to a pH-change. Superfusion of neurons (11 cells per solution) with these solutions resulted in a reduction of the conductance (21 ± 4%, 39 ± 3%, 59 ± 5%) and of mean peak amplitude (5 ± 4%, 24 ± 3%, 38 ± 5%) of the measured currents (Fig. 5). Moreover a clear positive correlation with increasing bicarbonate was observed for the negative shift of the half-maximal potential of activation (4.8 ± 0.7 mV, 5.7 ± 0.6 mV, 11.2 ± 0.8 mV) and for the half-maximal potential of inactivation (8.6 ± 0.7 mV, 9.1 ± 0.7 mV, 14.2 ± 2.5 mV). As with all other experiments, no changes in the slope parameters of the Boltzmann curves (Vc) were observed for activation or inactivation. Taken together, the lack of effect with increasing concentrations of CO2 and the prominent alterations induced by increased bicarbonate concentrations demonstrate that bicarbonate ions modulate the calcium current properties.


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The present study shows the modulatory action of bicarbonate ions on neuronal whole cell calcium currents. Current amplitude and maximal calcium conductance decreased in the presence of bicarbonate, and the potentials of half-maximal activation and inactivation were shifted to more negative values. The data show that bicarbonate alone, and not CO2, is capable of inducing these alterations. Furthermore, a concentration-dependent interaction between bicarbonate and calcium currents has been demonstrated. The outer membrane seems to be the site of action of bicarbonate, since changing the concentration of this ion intracellularly did not show any effect.

Bicarbonate modulates calcium currents

A main question of the present study was to decide whether CO2 or the bicarbonate ion is the molecule that induces the observed alterations of whole cell calcium current. Carbon dioxide has indeed the ability to bind with protein, thereby forming so-called carbamino complexes, of which the complex of CO2 and hemoglobin is one of the best known. Binding of a small molecule such as CO2 to a big protein structure often leads to conformational alterations, which could change the chemical and physical properties of such molecules. It therefore was conceivable that binding of CO2 to or close to calcium channel proteins may decrease calcium permeability of the channel pore and reduce the total calcium current. However, increasing the concentration of CO2 up to 10% by leaving the concentration of bicarbonate constant did not yield differences in the maximal whole cell calcium conductance (ref. Fig. 5). Instead, increasing the amount of dissolved bicarbonate, at constant CO2 concentration, had a marked and concentration-dependent effect on calcium conductance, current, and voltage dependence. Together these data demonstrate that bicarbonate is the modulatory agent and not CO2.

The reduction of maximal calcium conductance and of current amplitude followed a nonlinear pattern with a maximum of 69%, when cells where consecutively bathed with solutions containing increasing amounts of bicarbonate. To hold pHo at constant level (7.3), these solutions were also gassed with increasing concentrations of CO2. The resulting concentration response curve was best fitted with the Hill equation, which delivered three independent parameters (maximal effect, EC50, and the Hill coefficient). The EC50 (7.4 mM) is the amount of bicarbonate at which half of the maximal reductions could be achieved, but due to the steep slope, is also the point at which small changes of bicarbonate would have the biggest effect on the calcium currents.

Physiological and pathological relevance

In mammalian brain, extracellular bicarbonate concentrations are in the range of 24-26 mM (Betz et al. 1989). At this value the concentration-dependent reduction is almost at its saturation level, and only strong changes of bicarbonate concentration would induce additional reductions or increases of the currents. Therefore under normal physiological circumstances a noteworthy dynamic influence of bicarbonate on neuronal calcium currents is unlikely.

During pathological situations, strong decreases of bicarbonate concentration may lead to prominent increases of calcium current amplitude and to an altered excitability pattern.

Hyperventilation induces a loss of dissolved CO2 in blood as well as in the extracellular space surrounding neurons. This results in an increased pH and ends up in a so-called respiratoric alkalosis, since in the early stage bicarbonate is still at normal levels. To re-establish normal pH values bicarbonate has to be reduced, which could be done by extruding HCO3- for example by higher extrusion rates for this ion in the kidney. An increase of pHo of only 0.1 units will therefore be followed by a down-regulation of bicarbonate to almost half the original level, which in turn will increase calcium currents, according to our concentration-response curve, by up to 20-30%. In this light, the so-called hyperventilation test, a diagnostic tool to evoke epileptic seizures, might cause increased calcium currents, which play a prominent role in epileptogenesis (Speckmann and Walden 1986; Speckmann et al. 1990; Witte 1987).

Moreover, metabolic acidosis, following ischemic events, may also result in an increase of calcium current and conductance, since the elevation of proton concentration stimulates the medulla oblongata followed by an increased ventilation rate and finally by a loss of CO2. This loss of CO2 again starts the same regulatory mechanism following respiratoric alkalosis as mentioned above.

Relation between HCO3-, pH, and membrane potential

The observed effects will be strengthened in normal brain, since pHi is not as heavily buffered as in our experiments, in which pH of intracellular solutions (i.e., electrode solution) were strongly buffered with the artificial proton buffer HEPES (50 mM). This high concentration of HEPES has been demonstrated to avoid acidification of the intracellular space following superfusion with weak acids containing solutions, comparable to our CO2/HCO3--buffered solution (Tombaugh and Somjen 1997). Furthermore, it has been shown that acidification of the soma plus superfusion with bicarbonate enhances the reduction of current amplitude by almost 30% (Bruehl et al. 1998, 1999).

In invertebrate preparations (leach and snail) a superfusion with CO2/HCO3--buffered solution leads not only to an acidification of the intracellular pH, but also to a negative deflection (~2-5 mV) of the membrane potential (Schlue and Thomas 1985; Thomas 1976). This may be explained by the bicarbonate-dependent attenuation of calcium currents, which are small but present at resting potential, especially when cells frequently generate action potentials. This assumption is further substantiated since the negative shifts of resting potential in leach neurons could be diminished by adding the potent calcium channel blocker Cd2+ during superfusion with CO2/HCO3--buffered solution (unpublished observation).

Artificially induced hypercapnia (i.e., increased pCO2 plus decreased pHo) can induce hyperpolarizations but also depolarizations on rat CA3 neurons (Lehmenkuhler et al. 1989). The variability between membrane responses was related to the distance of the neurons from the bath fluid: cells in the innermost layers of the slice hyperpolarized while cells close to the bath solution depolarized. The concentration of bicarbonate in the tissue close to the bath fluid resembles more or less the concentration in the surrounding saline (usually 26 mM), while in the inner portions of the slice, additional bicarbonate will be formed from the increased pCO2. With the concentration dependence of the HCO3- effects on the calcium currents it is unlikely that this alteration of membrane potential is due to changes of calcium currents. It is, however, conceivable that HCO3- ions affect also potassium or sodium conductances, and this may cause such alterations. Alternatively, these effects may be attributed to pH-related current modulation (Tombaugh and Somjen 1996, 1997).

In the present study hippocampal CA1 neurons were taken for the measurements simply as a test probe, since they are easily prepared and well characterized. Nevertheless this should not limit the present findings to the hippocampus, and in particular to the CA1 area, but may be of relevance for all neurons in areas throughout the brain, as in cortical and subcortical structures. This is likely because the affected HVA calcium current is present and equals the one of CA1 neurons, in all those cells (Bruehl and Wadman 1999; Hille 1992).

At present we cannot determine whether the observed processes are due to a direct binding of the bicarbonate ion to the outer membrane, which interferes with calcium channels, or whether it is based on surface charge screening effects. The latter may cause a shielding of the calcium-attractive site in the pore mouth, which would decrease the permeability of calcium ions. Such a screening has been postulated for protons (Hille 1992), which compete with calcium ions for the docking site on the outer cell surface and can change the electric field around the channel pore that attracts calcium ions.


    ACKNOWLEDGMENTS

The authors thank D. Steinhoff for perfect technical assistance.

The investigations were supported by Sonderforschungsbereich 194 B2.


    FOOTNOTES

Address for reprint requests: C. Bruehl, Heinrich-Heine-University, Dept. of Neurology, Geb.: 22.22/TVA, 40225 Duesseldorf, Germany (E-mail: bruehl{at}uni-duesseldorf.de).

Received 16 March 2000; accepted in final form 28 July 2000.


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