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Originally published In Press as doi:10.1074/jbc.M605122200 on August 1, 2006

J. Biol. Chem., Vol. 281, Issue 39, 29369-29378, September 29, 2006
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Modulation of Acid-sensing Ion Channel Currents, Acid-induced Increase of Intracellular Ca2+, and Acidosis-mediated Neuronal Injury by Intracellular pH*

Wei-Zhen Wang, Xiang-Ping Chu1, Ming-Hua Li1, Joshua Seeds, Roger P. Simon, and Zhi-Gang Xiong2

From the Robert S. Dow Neurobiology Laboratories, Legacy Research, Portland, Oregon 97232

Received for publication, May 30, 2006 , and in revised form, July 6, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Acid-sensing ion channels (ASICs), activated by lowering extracellular pH (pHo), play an important role in normal synaptic transmission in brain and in the pathology of brain ischemia. Like pHo, intracellular pH (pHi) changes dramatically in both physiological and pathological conditions. Although it is known that a drop in pHo activates the ASICs, it is not clear whether alterations of pHi have an effect on these channels. Here we demonstrate that the overall activities of ASICs, including channel activation, inactivation, and recovery from desensitization, are tightly regulated by pHi. In cultured mouse cortical neurons, bath perfusion of the intracellular alkalizing agent quinine increased the amplitude of the ASIC current by ~50%. In contrast, intracellular acidification by withdrawal of NH4Cl or perfusion of propionate inhibited the current. Increasing pH buffering capacity in the pipette solution with 40 mM HEPES attenuated the effects of quinine and NH4Cl. The effects of intracellular alkalizing/acidifying agents were mimicked by using intracellular solutions with pH directly buffered at high/low values. Increasing pHi induced a shift in H+ dose-response curve toward less acidic pH but a shift in the steady state inactivation curve toward more acidic pH. In addition, alkalizing pHi induced an increase in the recovery rate of ASICs from desensitization. Consistent with its effect on the ASIC current, changing pHi has a significant influence on the acid-induced increase of intracellular Ca2+, membrane depolarization, and acidosis-mediated neuronal injury. Our findings suggest that changes in pHi may play an important role in determining the overall function of ASICs in both physiological and pathological conditions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Acid-sensing ion channels (ASICs)3 are H+-gated cation channels that belong to the Deg/ENaC superfamily (1). In peripheral sensory neurons, ASICs are implicated in nociception (2, 3), mechanosensation (4, 5), and taste transduction (6). In central neurons, ASICs play an important role in physiological processes such as synaptic transmission, learning, and memory (7-9). In pathological conditions including brain ischemia, ASICs are involved in acidosis-mediated glutamate-independent neuronal injury (10-12), disclosing a novel therapeutic target for stroke intervention.

The activities of ASICs are subjected to modulation by endogenous signaling molecules and biochemical changes associated with pathological conditions. For instance, Zn2+, an endogenous trace element released during neuronal activity, inhibits ASIC1a-containing channels with high affinity (13), whereas neurochemical components associated with tissue inflammation (e.g. Phe-Met-Arg-Phe (FMRF) amide) (14) and ischemia (e.g. lactate and arachidonic acid) (15-18) potentiate the ASIC currents. Delineating detailed modulation of ASICs by endogenous signaling molecules and biochemical changes is important for better understanding the exact and precise role these channels play in various physiological and pathological conditions.

During hypoxia/ischemia, increased anaerobic glycolysis leads to lactic acid accumulation (19), causing dramatic decreases in tissue pH. For example, pHo typically falls to ~6.5 under normoglycemic conditions and to below 6.0 under hyperglycemic conditions (19, 20). These drops of pHo are expected to activate homomeric ASIC1a and heteromeric ASIC1a/ASIC2a channels that are highly expressed in the brain (10, 21).

Along with pHo, changes in pHi also take place in both physiological and pathological conditions (19, 20). It is clear that a drop of pHo is required to activate the ASICs. However, it is not known whether changes in pHi have any effect on the activities of these channels. Although pHi also drops during ischemia in general, the degree of pHi changes is not homogeneous across all brain regions, and an alkalization of pHi has been demonstrated in cortical penumbra region following the ischemia (22). Here we demonstrate that the amplitude of ASIC current, its pH-dependent activation and inactivation, as well as current desensitization all are tightly regulated by the level of pHi; intracellular acidification inhibits whereas alkalization potentiates the activities of ASICs. Consistent with the changes in ASIC current, acid-induced increases of intracellular Ca2+ concentration ([Ca2+]i), membrane depolarization, and acidosis-mediated neuronal injury are dramatically affected by the level of pHi.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Primary Neuronal Culture—Primary cortical neuronal cultures were prepared from embryonic Swiss mice at 16 days of gestation according to our previously described method (10, 23). In brief, the brains of embryos were rapidly removed and incubated in Ca2+- and Mg2+-free ice-cold phosphate-buffered saline. Cerebral cortices from 10-12 embryos were dissected and incubated with 0.05% trypsin-EDTA for 10 min at 37 °C, dissociated by trituration with fire-polished glass pipettes, and plated on poly-L-ornithine-coated dishes at 1.0 x 106 cells/dish. The neurons were cultured with Neurobasal medium supplemented with B27 and glutamax and maintained at 37 °C in a humidified 5% CO2 atmosphere incubator. The cultures were fed twice a week and used for electrophysiological recording 12-21 days after plating.

Electrophysiology—Whole cell patch clamp recordings were performed as described previously (10, 23). Patch pipettes were pulled by a two-step puller (PP83; Narishige, Tokyo, Japan) from thin wall borosilicated glass (1.5 mm diameter; WPI, Sarasota, FL). The pipettes had a resistance of 3-4 M{Omega} when filled with intracellular solution: 140 mM CsF, 10 mM HEPES, 11 mM EGTA or 1,2-bis(2-aminophenoxy)ethane-N,N,N,'N'-tetraacetic acid (BAPTA), 2 mM tetraethylammonium chloride, 1 mM CaCl2, 2mM MgCl2, pH 7.3 (adjusted with CsOH), 290-300 mOsm adjusted with sucrose. For solutions containing 40 mM HEPES, the amount of sucrose was reduced to maintain the osmolarity. Extracellular solution (ECF) contained: 140 mM NaCl, 5.4 mM KCl, 2.0 mM CaCl2, 1.0 mM MgCl2, 10 mM HEPES, 10 mM glucose, pH 7.4 (320-330 mOsm). 500 nM tetrodotoxin was used in all ECF to block the voltage-gated Na+ current and the firing of action potential. Unless otherwise stated, chemicals were purchased from Sigma.

Whole cell recordings were done with Axopatch 200B amplifier, Digidata 1320 DAC unit, and pClamp 8 software (Axon Instruments, Foster City, CA). The data were analyzed using pClamp 8 and Sigmaplot software. Unless otherwise stated, ASIC currents were activated every 2 min to achieve a complete recovery from desensitization. A multibarrel perfusion system (SF-77; Warner Instruments, Hamden, CT) was used to achieve a rapid exchange of extracellular solutions.

Ca2+ Imaging—Primary cultured neurons grown on 25-mm round glass coverslips were used for Ca2+ imaging experiments as described previously (10). After being washed three times with normal ECF, the neurons were loaded with 5 µM Fura-2-acetoxymethyl ester for 40 min at room temperature, followed by three washes and incubation in normal ECF for additional 30 min. The coverslips were then transferred to a perfusion chamber on the stage of an inverted microscope (Nikon TE300). Neurons were illuminated using a 75 W xenon lamp and observed with a 40x oil immersion UV fluor objective. Video images were taken by a cooled CCD camera (Sensys KAF 1401; Photometrics, Tucson, AZ). Axon Imaging Workbench software (AIW 2.1; Axon Instruments) was used to acquire and analyze digitized images and to control the shutter and filter wheel (Lambda 10-2; Sutter Instruments, Novato, CA) for timed illumination of cells at an excitation wavelength of either 340 or 380 nm. Fura-2 fluorescence was detected at an emission wavelength of 510 nm. 340/380-nm ratio images were acquired by averaging pixel ratio values in circumscribed regions of neurons in the field of view. SigmaPlot software was used for further data analysis and plotting.

Cell Injury Assay—Acidosis-induced injury of neurons was studied as described previously (10). Neurons grown in 24-well culture plates were washed three times with ECF and randomly divided into different treatment groups. The cells were pretreated at pH 7.4 with or without pHi modifying agents for 15 min and then subjected to an acid incubation with the same agents. Following2hof acid incubation in the absence or presence of pHi modifying agents, the neurons were washed with ECF and incubated in Neurobasal medium in a conventional cell culture incubator at 37 °C. LDH released in the culture medium was measured using the LDH assay kit (Roche Applied Science). 6 h after the beginning of the acid treatment, 50 µl of medium was transferred from each culture well to 96-well plates and mixed with an equal volume of reaction solution provided by the kit. After mixing and a 30-min incubation, optical density was measured at 492 nm utilizing a microplate reader (Spectra Max Plus; Molecular Devices). Background absorbance at 620 nm was subtracted. The 6-h LDH release for each well was normalized against the maximum releasable LDH, obtained by 30 min of incubation with 0.5% Triton X-100 at the end of each experiment.

For fluorescein diacetate (FDA) and propidium iodide (PI) staining of live and dead cells respectively, the neurons were incubated in ECF containing FDA (5 µM) and PI (2 µM) for 30 min followed by three washes with dye-free ECF. Alive (FDA-positive) and dead (PI-positive) cells were viewed and counted on a microscope (Nikon) equipped with epifluorescence at 580/630 nm excitation/emission for PI and 500/550 nm for FDA. The images were collected using a digital camera (Nikon Cool-pix 5000).

Data Analysis—All of the data are expressed as the means ± S.E. Student's t test and analysis of variance were used for statistical analysis where appropriate. The criterion of significance was set at p < 0.05. pH dose-response curves and steady state inactivation curves were fitted using the equation: I = a/(1 + (C50/pH)n), where a is the amplitude of ASIC current, C50 is the pH at which a half-maximal response is induced, and n is the Hill coefficient.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Intracellular Alkalization Potentiates the ASIC Current—As shown previously, a rapid reduction of pHo from 7.4 to below 7.0 activates inward, amiloride-sensitive, ASIC currents in all cultured mouse cortical neurons voltage-clamped at -60 mV (10, 13).


Figure 1
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  		FIGURE 1.
Effects of intracellular alkalization and acidification on ASIC currents. A, dose-dependent potentiation of the ASIC current by intracellular alkalizing agent quinine. A1, representative current traces showing the potentiation of the ASIC current in mouse cortical neurons by bath perfusion of 0.5 or 1.0 mM quinine. A2, bar graph summarizing the potentiation of the ASIC current by quinine. Perfusion of 0.5 mM quinine for 2 min induced a 25.8 ± 8.8% increase in the peak amplitude (n = 8; *, p < 0.05) and a 99.3 ± 25.9% increase in the sustained component (n = 6; *, p < 0.05) of the ASIC currents, and 1.0 mM quinine induced an increase in the peak amplitude of the ASIC currents by 47.0 ± 8.9% (n = 4; *, p < 0.05). The difference between the potentiation by 0.5 and 1.0 mM quinine is statistically significant (p < 0.05). B, inhibition of the ASIC current by intracellular acidifying agents. B1, 2 min following withdrawal of 15 mMNH4Cl, the amplitude of the ASIC current is inhibited. B2, bath application of 10 mM propionate inhibits the ASIC current. B3, bar graph summarizing the inhibition of the ASIC current by NH4Cl withdrawal and perfusion of propionate. 2 min following withdrawal of 15 mM NH4Cl, the peak amplitude of the ASIC current was decreased to 74.7 ± 3.5% of the control value (n = 7; **, p < 0.01), whereas 2 min of perfusion of 10 mM propionate decreased the peak amplitude of the ASIC current to 80.6 ± 5.2% of control (n = 6; *, p < 0.05). C, ASIC currents recorded with intracellular solutions directly buffered at different pH values. C1, representative traces showing the ASIC currents recorded with intracellular solutions directly buffered at pH of 8.0, 7.3, or 6.0. C2, summary data showing the change of ASIC current density with pHi in a linear fashion within the range of pH tested (8.0 to 5.5). 40 mM HEPES was used in the intracellular solution for the buffering of pHi to different values, and the amount of sucrose was reduced to maintain the osmolarity.

 
To determine whether a change in pHi affects the ASIC current, we first applied quinine, an agent known to cause intracellular alkalosis (23, 24), to the extracellular solution, following the recording of stable ASIC currents activated by a pHo drop from 7.4 to 6.5. In our previous studies, we have shown that bath perfusion of 0.5 mM quinine for 2 min increased pHi by ~0.5 unit (23). As shown in Fig. 1A, perfusion of quinine (0.5 mM) for 2 min induced a reversible increase in the peak amplitude of ASIC currents by 25.8 ± 8.8% (n = 8, p < 0.05). The potentiation of the ASIC current by quinine is dose-dependent, because increasing the concentration of quinine to 1.0 mM increased the peak amplitude of the ASIC currents by 47.0 ± 8.9% (Fig. 1A; n = 4, p < 0.05). Similar to the peak current, the sustained component of the ASIC currents, measured at the end of 6-s acid application, was dramatically increased by quinine (0.5 mM) by 99.3 ± 25.9% (n = 6, p < 0.05; Fig. 1A2). In addition, the desensitization of the ASIC current was significantly reduced by intracellular alkalization, as reflected by an increase in decay time constant ({tau}). 2 min after 0.5 mM quinine, {tau} was increased from 1.67 ± 0.19 to 2.17 ± 0.28 s (n = 15, p < 0.05).

Changing pHi may affect the buffering capacity of EGTA (25), thus altering the concentration of [Ca2+]i, which might in turn affect the ASIC current. To rule out this possibility, we have performed the same experiment using an intracellular solution containing 1,2-bis(2-aminophenoxy)ethane-N,N,N,'N'-tetraacetic acid (BAPTA) (11 mM), a pH-insensitive Ca2+ chelator (25). Inclusion of high concentration of BAPTA did not affect the potentiation of the ASIC current by quinine. For example, 1 mM quinine still potentiated the peak amplitude of the ASIC currents by 38.0 ± 9.3% (n = 4, p < 0.05), which is not significantly different from its effect with EGTA in the pipette solution. These data suggest that a change of intracellular Ca2+ is not required for the effect of pHi on ASICs (not shown).

Intracellular Acidification Inhibits the ASIC Current—We then examined whether intracellular acidification has an effect opposite to alkalization on the ASIC current. Two methods were used to induce an intracellular acidification: 1) addition and withdrawal of NH4Cl and 2) bath perfusion of the weak acid propionate. Brief exposure (~3 min) to NH4Cl followed by washout is a common method for inducing intracellular acidification (26, 27). Bath perfusion of NH4Cl causes a transient rise of pHi by the formation of NH +4 from NH3 + H+. Upon withdrawal of NH4Cl, a relatively long lasting decrease of pHi takes place because of dissociation of NH +4 into H+ + NH3. In hippocampal neurons, a 3-min perfusion of 20 mM NH4Cl followed by washout induced up to a 0.5-unit decrease in pHi (28). Our own studies showed that perfusion/withdrawal of 15 mM NH4Cl induced ~0.4 unit decrease in pHi (23). To induce intracellular acidification in cultured mouse cortical neurons, 15 mM NH4Cl was perfused for 3 min followed by washout with normal ECF. As shown in Fig. 1B, 2 min following NH4Cl withdrawal, the peak amplitude of the ASIC current was decreased by 25.3 ± 3.5% from its control value (n = 7, p < 0.01).

The weak acid propionate is known to transport protons across membranes and decrease pHi in a dose-dependent manner (29, 30). To further test the effect of changing pHi on the ASIC current, ECF containing 10 mM propionate was perfused to the neurons following the recording of stable ASIC currents. Consistent with NH4Cl removal, 2 min of perfusion of propionate decreased the peak amplitude of the ASIC current by 19.4 ± 5.2% (n = 6, p < 0.05; Fig. 1B).

To provide additional evidence that the levels of pHi regulate the activities of ASICs, we then compared the amplitude of the ASIC current recorded with intracellular solutions directly buffered at different pH values. To minimize the variation of the current amplitude in different neurons, current density was used. It is expected that the density of the ASIC current recorded with a high pHi (e.g. 8.0) should be larger than the one recorded with a low pHi (e.g. 6.0). As shown in Fig. 1C, the density of the ASIC current recorded at pHi of 8.0 (30.3 ± 3.2 pA/pF, n = 32) is significantly larger than the one recorded at pHi of 7.3 (20.8 ± 2.0 pA/pF, n = 30, p < 0.05), and the density of the ASIC current recorded at pHi of 6.0 (14.1 ± 1.5 pA/pF, n = 29) is significantly smaller than the current recorded at pHi of 7.3 (p < 0.01). Further studies demonstrated that the density of ASIC currents change with pHi in a linear fashion within the range of pH tested from 8.0 to 5.5, with a slope of 8.1 pA/pF per unit pH change (Fig. 1C2). The rate of desensitization of the ASIC current was also affected by the level of pHi. At 7.3, the decay time constant {tau} was 2.22 ± 0.48 s (n = 22). However, it was 2.73 ± 0.19 s at pH 8.0 (n = 32, p < 0.05). Together, these findings strongly indicate that changes in intracellular proton concentration or pHi modulate the activity of ASICs; intracellular alkalization increases, whereas acidification decreases the activity of ASICs.

Increasing HEPES Concentration in the Intracellular Solution Eliminates or Reduces the Changes of ASIC Currents Induced by Alkalinizing and Acidifying Agents—To further determine whether the effects of quinine, NH4Cl, and propionate on the ASIC current are indeed due to the changes in pHi, we performed additional experiments with intracellular solutions containing higher concentration of HEPES (40 mM). It is expected that increasing the concentration of HEPES in the pipette solution will increase its pH buffering capability, thus reducing the degree of pHi change induced by alkalizing or acidifying agents and their effects on the ASIC current. With 40 mM HEPES in the pipette solution, application of 0.5 mM quinine no longer potentiated the ASIC current. 2 min following the perfusion of quinine, the peak amplitude of the ASIC current was 1.02 ± 0.02 times of the control value (n = 6, p > 0.05). Similarly, withdrawal of 15 mM NH4Cl did not induce a significant inhibition of the ASIC current (1.03 ± 0.05 times of the control value, n = 5, p > 0.05, not shown).

Intracellular Alkalization Potentiates the ASIC Current by Increasing the Affinity of ASICs to H+—To explore the potential mechanism(s) underlying the modulation of the ASIC current by pHi, pH dose-response curves were constructed before and after bath perfusion of quinine. As shown in Fig. 2 (A and B), application of 0.5 mM quinine resulted in a significant leftward shift of the pH dose-response curve (pH0.5 before quinine: 6.10 ± 0.21; after quinine: 6.49 ± 0.08; n = 5, p < 0.05), indicating an increase in the affinity of channels to H+. No significant change of the Hill coefficient was observed (before quinine, 1.16 ± 0.04; after quinine, 1.36 ± 0.03). To determine whether the shift of pH dose-response by quinine was indeed due to a change of pHi, we performed the same experiment in the presence of 40 mM HEPES in pipette solution. No significant shift in pH dose-response curve could be induced by quinine when 40 mM HEPES was included in the intercellular solution (pH0.5 values were 6.12 ± 0.05 before and 6.06 ± 0.11 after 0.5 mM quinine, n = 3, p > 0.05; Fig. 2C).

To further prove that changing pHi plays an important role in the shift of pH dose-response, we have also constructed pH dose-response curves with pHi directly buffered at high and low values (8.0 and 5.5). As shown in Fig. 2D, the pH0.5 for dose-response curve at pHi of 5.5 (5.52 ± 0.04, n = 5) was significantly lower than that recorded with a pHi of 8.0 (6.21 ± 0.05, n = 5, p < 0.01). Together, these findings strongly suggest that the potentiation of ASIC current by intracellular alkalization is attributed to an increase in the apparent affinity of ASICs to H+.


Figure 2
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  		FIGURE 2.
Effects of changing pHi on pH dose-response curve. A, representative traces showing pH-dependent activation of the ASIC currents in mouse cortical neurons before and after bath application of 0.5 mM quinine. B, summary data from five neurons showing the effect of 0.5 mM quinine on pH dose-response curve. pH0.5 values before and after quinine were 6.10 ± 0.21 and 6.49 ± 0.08 respectively (n = 5, p < 0.05). C, increasing pH buffering capacity with 40 mM HEPES in the pipette solution abolished the effect of quinine. D, pH dose-response curves with pHi directly buffered at high (8.0) and low (5.5) values. The dose-response curve with pHi at 8.0 (pH0.5 = 6.21 ± 0.05, n = 5) was shifted to the left of that obtained with pHi at 6.0 (pH0.5 = 5.52 ± 0.04, n = 5, p < 0.01).

 
Modulation of ASIC Activity by pHi Is Voltage-independent—To determine whether intracellular protons act at a site within the transmembrane electric field, e.g. within the channel pore, we examined whether the effect of pHi on the ASIC current is voltage-dependent. Currents activated by decreasing pHo from 7.4 to 6.5 were recorded at various holding potentials ranged from -60 to +40 mV, and the effects of quinine (0.5 mM) or propionate (10 mM) on the amplitude of the ASIC current were compared at different membrane potentials.

Quinine and propionate only changed the slope of the I-V curve, with little effect on the reversal potential of the ASIC currents (not shown). The potentiation/inhibition of the current amplitude by quinine/propionate was not affected by the membrane potential. For example, at -60 mV, quinine increased the current to 1.34 ± 0.11 times of the control value and propionate decreased the current to 0.71 ± 0.05 of the control value, although at -20 mV, quinine increased the current amplitude to 1.30 ± 0.01 of the control value and propionated decreased the current to 0.64 ± 0.09 (n = 4, p > 0.05). Together, these data indicate that the modulation of ASICs by pHi is voltage-independent.

Effects of pHi on Steady State Inactivation of the ASICs—To further explore the mechanism underlying the potentiation/inhibition of the ASIC current by changing pHi, the effect of quinine on steady state inactivation of ASICs was also examined. Neurons were incubated in extracellular solutions at various conditioning pH values between 7.8 and 6.6 for ~4 min before the ASIC currents were activated by a drop in pH to 6.0. The amplitude of the current activated with different conditioning pH values was normalized to the one activated with a conditioning pH of 7.8 (where there is no apparent inactivation) and plotted against the value of conditioning pH. Fig. 3 (A and B) shows the steady state inactivation curves before and after bath perfusion of 1 mM quinine. Application of quinine shifted the inactivation curve to more acidic pH (pH0.5 before quinine, 7.22 ± 0.03; after quinine, 7.05 ± 0.03; n = 5, p < 0.01), with little change in Hill coefficient (2.24 ± 0.26 before and 2.53 ± 0.10 after quinine, n = 5, p > 0.05). With 40 mM HEPES in the pipette solution, this shift was attenuated (pH0.5 before quinine, 7.22 ± 0.08; after quinine, 7.14 ± 0.05; n = 3, p > 0.05; Fig. 3C). The apparent shift of the inactivation curve to more acidic pH by quinine suggests that intracellular alkalization reduces the steady state inactivation of ASICs at a given pHo, thus making more channels available for activation.

Intracellular Alkalization Increases the Recovery Rate of ASICs from Desensitization—We then determined whether a change in pHi affects the recovery rate of ASICs from desensitization. ASIC currents were activated consecutively by pairs of low pH pulses (from 7.4 to 6.5) with various time intervals (e.g. 1, 3, 5, 15, 25, 50, and 100 s) between the end of the first and the beginning of the second acid exposure. The peak amplitude of the second current is then normalized to the first one and plotted against the time intervals between the two acid exposures. As shown in Fig. 4, intracellular alkalization by quinine dramatically increased the recovery rate of the ASIC current from desensitization. The time constant for recovery of the ASIC current was reduced from 1.6 ± 0.4 s in control to 0.7 ± 0.3 s in the presence of 1 mM quinine (n = 5, p < 0.05). With intracellular solution containing 40 mM HEPES, however, application of quinine had little effect on the recovery of the ASIC current (time constants for recovery were 1.2 ± 0.1 s in control and 1.0 ± 0.1 s in the presence of quinine, n = 4, p > 0.05; not shown).


Figure 3
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  		FIGURE 3.
Effects of changing pHi on steady state inactivation of ASICs. A, example of traces showing the steady state inactivation of ASICs before and after bath perfusion of 1 mM quinine. The neurons were perfused with ECF at various conditioning pH (e.g. 7.8, 7.4, 7.2, 7.0, and 6.6) for 4 min before activating the ASICs by a pH drop to 6.0. B, summary graph showing the steady state inactivation curves before and after quinine. The pH0.5 of the inactivation curve shifted from 7.22 ± 0.03 in control to 7.05 ± 0.03 after quinine (n = 5, p < 0.01). C, increasing HEPES to 40 mM in the pipette solution attenuated the effect of quinine. The pH0.5 values of the steady state inactivation before and after quinine were 7.22 ± 0.08 and 7.14 ± 0.05, respectively (n = 3, p > 0.05).

 
To further determine whether changing pHi plays an important role in the recovery of the channels from desensitization, we have also compared the recovery rate from desensitization recorded with intracellular solutions directly buffered at high and low pH values (8.0 and 5.5). As shown in Fig. 4C, the time constant for the recovery from desensitization recorded at a pHi of 8.0 (0.87 ± 0.09 s, n = 6) was significantly smaller than the one recorded at a pHi of 5.5 (1.72 ± 0.34 s, n = 6, p < 0.05; Fig. 4C).

Effect of pHi on ASIC-mediated Increase of [Ca2+]i—Accumulation of intracellular Ca2+ is essential for neuronal injury in a variety of neurological disorders including brain ischemia (31). Our previous study showed that activation of ASICs by acidosis is involved in glutamate receptor-independent increases of intracellular Ca2+ and ischemic brain injury (10). To know whether a change in pHi also affects ASIC-mediated increase of [Ca2+]i, we have performed Ca2+ imaging experiments to record acid-induced increase of [Ca2+]i in the absence and presence of intracellular alkalizing or acidifying agents. The blockers of voltage-gated Ca2+ channels (5 µM nimodipine) and glutamate receptors (10 µM (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK801) and 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)) were added in the extracellular solutions to avoid potential Ca2+ entry through those channels. As shown in Fig. 5, increase of [Ca2+]i induced by lowering pH to 6.0 was significantly potentiated by bath perfusion of 0.5 mM quinine (340/380 nm ratio was increased from 2.23 ± 0.30 to 3.16 ± 0.45, n = 8, p < 0.05). In contrast, withdrawal of 15 mM NH4Cl inhibited the acid-induced increase of [Ca2+]i (340/380 nm ratio was reduced from 4.29 ± 0.63 to 3.33 ± 0.54, n = 5, p < 0.05).

Effect of pHi on Acid-induced Membrane Depolarization—Activation of ASICs is also known to induce membrane depolarization (13), which may facilitate glutamate receptor-mediated excitatory neurotransmission (7). We next examined whether a change in pHi affects acid-induced membrane depolarization. Current clamp recording was used to record the membrane potential in cultured mouse cortical neurons. In five neurons tested, the resting membrane potential was -52.42 ± 2.97 mV at pH of 7.4. Decreasing pHo to 6.5 induced a depolarization of membrane potential to -5.84 ± 2.82 mV in the absence of quinine (p < 0.01). In the presence of 1mM quinine, however, decreasing pHo to 6.5 induced a much greater depolarization to +26.28 ± 2.29 mV (p < 0.01, n = 5; Fig. 6).

Effect of pHi on Acid-induced Neuronal Injury—Activation of Ca2+-permeable ASICs by acidosis is known to induce glutamate-independent ischemic brain injury (10). Our next experiment was to determine whether a change in pHi has any effect on acidosis-induced injury of cultured mouse cortical neurons. Neurons grown in 24-well plates were used for cell toxicity studies 12 days after plating. The wells were randomly divided into different treatment groups incubated with solutions at a pH of 7.4 or 6.0 in the absence and presence of quinine or propionate. Neuronal injury was analyzed by LDH measurement 6 h following the beginning of the acid incubation. As shown in Fig. 7 (A and B), 2 h of incubation of neurons with ECF at pH 6.0 increased LDH release to 39.4 ± 1.6% as compared with 8.9 ± 0.5% for neurons treated at 7.4 (n = 12 wells, p < 0.01), indicating acid injury. Incubation of neurons with 10 mM propionate did not affect the base-line LDH release at 7.4 (7.2 ± 0.3%) but significantly reduced the acid-induced LDH release to 18.4 ± 0.5% (n = 8, p < 0.01 compared with LDH release at pH 6.0 alone). In contrast to the reduction of LDH release by propionate, treating neurons with acid solution in the presence of 0.5 mM quinine dramatically increased the LDH release from 12.6 ± 0.6 to 50.2 ± 1.8% (n = 12, p < 0.01 compared with neurons treated at pH 6.0 alone). Because an addition of 0.5 mM quinine to the pH 7.4 solution slightly increased base-line LDH release to 12.6 ± 0.6 (n = 12, p < 0.01; Fig. 7A), a simple comparison between LDH release induced by acid alone and acid with quinine may not be appropriate. For this reason, we also compared the net LDH release induced by acid incubation in the absence and presence of quinine or propionate. In this regard, LDH release at 6.0 was subtracted by its corresponding base-line LDH release at 7.4 in the absence or presence of quinine/propionate (Fig. 7B). The net LDH release with 6.0 alone was 29.7 ± 1.7% (n = 12). However, it was 11.2 ± 0.5% in the presence of 10 mM propionate (n = 8, p < 0.01) and 36.8 ± 1.5% in the presence of quinine (n = 12, p < 0.05). The potentiation or inhibition of acid-induced injury by quinine or propionate was also confirmed by analyzing acid-induced injury of neurons grown in 35-mm-diameter culture dishes. In these experiments, the percentage of neuronal injury was determined by fluorescent staining of live neurons with FDA and dead neurons with PI. Fluorescent staining was performed 6 h following the 2-h acid (pH 6.0) incubation in the absence or presence of either quinine or propionate. Consistent with the LDH data, propionate inhibited while quinine potentiated the acid-induced neuronal injury (Fig. 7C; n = 3-4 in each treatment).


Figure 4
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  		FIGURE 4.
Effects of changing pHi on the rate of recovery of ASICs from desensitization. A, example of traces showing the time-dependent recovery of the ASIC current from desensitization in the absence and presence of 1 mM quinine. The currents were activated by pairs of pH drops from 7.4 to 6.5 with different time intervals (1, 3, 5, 15, and 25 s) between the end of the first acid application (15 s in duration) and the beginning of the second acid application (3 s in duration). B, summary graphs showing an increase in the recovery rate of ASICs from desensitization by alkalized pHi. B1, the peak current amplitude activated by the second acid application is normalized to the amplitude of the first current in each pair and plotted against the time interval before and after quinine application. The solid lines are single exponential fits to the data points. B2, summary graph showing an increase in the rate of recovery (as indicated by the reduction in time constant) of the ASIC current from desensitization. The time constants before and after 1 mM quinine were 1.55 ± 0.38 and 0.70 ± 0.28 s, respectively (n = 5; *, p < 0.05). C, summary data and exponential fitting demonstrating the difference in the recovery rate of the ASIC current from desensitization recorded with pHi directly buffered at 8.0 and 5.5. The time constant for exponential fitting at pHi 5.5 (1.72 ± 0.34, n = 6) was significantly larger than that at pHi 8.0 (0.87 ± 0.09, n = 6, p < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
ASICs are highly expressed in neurons of the central nervous system where they are believed to play an important role in normal synaptic transmission (7) and in the pathology of brain ischemia (10). Although ASICs are activated exclusively by a drop of pHo, our present studies demonstrate that the overall activities of these channels including channel activation, inactivation, and desensitization are tightly regulated by the level of pHi.

The first evidence suggesting a modulation of ASICs by pHi was the finding that bath perfusion of quinine, an agent known to cause intracellular alkalization (23, 24), dramatically increased the amplitude of the ASIC current. In contrast, the manipulations that cause intracellular acidification, e.g. the addition and withdrawal of NH4Cl (13, 23, 26, 27) and the bath perfusion of propionate (29, 30), produced an opposite effect on the ASIC current. Consistent with a change of pHi as the mechanism underlying the effects of quinine and propionate, increasing pH buffering capability in the intracellular solution (i.e. with 40 mM HEPES) attenuated the effects of these agents on the ASIC currents. Further evidence supporting the role of pHi in modulating the activities of ASICs was the finding that the enhancement or inhibition of the ASIC current by alkalizing/acidifying agents can be reproduced by using intracellular solutions directly buffered at high (e.g. 8.0) or low (e.g. 6.0) pH values. Consistent with its effect on the ASIC current, changing pHi also affected the acid-induced increase of [Ca2+]i, membrane depolarization, and acidosis-mediated neuronal injury.


Figure 5
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  		FIGURE 5.
Effect of changing pHi on acid-induced increase of [Ca2+]i. A and B, example of change in 340/380 nm ratio and summary graph showing the potentiation of acid (pH 6.0) induced increase in [Ca2+]i by an increase in pHi. Bath perfusion of 0.5 mM quinine significantly increased the 340/380 ratio from 2.23 ± 0.30 to 3.16 ± 0.45 (n = 8; p < 0.05). C and D, example of change in 340/380 nm ratio and summary graph showing the inhibition of acid (pH 6.0) induced increase of [Ca2+]i by a decrease in pHi. Brief bath perfusion followed by withdrawal of 15 mM NH4Cl significantly decreased the 340/380 ratio from 4.29 ± 0.63 to 3.33 ± 0.54 (n = 5, p < 0.05).

 


Figure 6
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  		FIGURE 6.
Changing pHi affects acid-induced membrane depolarization in cultured mouse cortical neurons. A, representative traces showing the membrane depolarization induced by a drop of pHo from 7.4 to 6.5 in the absence and presence of 1 mM quinine. B, summary graph showing the amplitude of membrane depolarization by acid before and after 1 mM quinine. In the absence of quinine, lowering pHo to 6.5 depolarized the membrane potential from approximately -55 mV to -5.84 ± 2.82 mV. However, in the presence of 1 mM quinine, the same pH decrease depolarized the membrane potential to +26.28 ± 2.29 mV (n = 5; **, p < 0.01).

 
Changes in the affinity of ASICs for H+ and alterations in steady state inactivation have been reported for the modulation of ASICs by some endogenous signaling molecules/elements (13, 32). To determine the potential mechanism(s) responsible for the modulation of ASICs by pHi, we have studied the effects of changing pHi on H+ dose-response relationship and the steady state inactivation of the channels. Our finding that increasing pHi by bath perfusion of quinine or by using intracellular solutions with pHi buffered at 8.0 induced a significant shift of H+ dose-response curve toward less acidic pH suggests that alkalizing pHi can increase the affinity of ASICs for H+. In addition to affecting the apparent affinity of these channels to H+, changing pHi also affects the steady state inactivation of ASICs. Intracellular alkalization, for example, reduced the inactivation through a shift of the steady state inactivation curve toward more acidic pH. The shifts in pH-dependent activation and steady state inactivation by alkalized pHi would make more channels available for activation and can thus explain the dramatic potentiation of the ASIC current.

In addition, our studies demonstrated that changing pHi also influences the recovery of ASICs from desensitization. Alkalization by quinine, for example, increased the rate of recovery from desensitization. In the absence of quinine, the time constant for recovery from desensitization was ~1.6 s. In the presence of quinine, however, it was reduced to ~0.7 s. The fast recovery of the ASIC current from desensitization indicates that these channels can be activated repeatedly on the condition that pHo is briefly restored following the drop of pH. This condition may indeed be satisfied during the process of cortical spreading depression, a common phenomena associated with pathological conditions such as seizures, migraine aura, head injury, and brain ischemia and an important determinant of the degree and extent of ischemic damage (33, 34).

In ischemia, for example, cortical tissue surrounding acute ischemic infarcts undergoes repetitive spontaneous depolarizations and repolarizations, likely elicited by an elevated interstitial [K+]o, particularly in the ischemic core (33, 34). It has been demonstrated that cortical spreading depression is often associated with bi- or triphasic brain pH shifts (35, 36), i.e. a transient tissue acidosis during depolarization followed by an alkaline overshoot during repolarization and in some cases followed by prolonged acidosis (36).


Figure 7
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  		FIGURE 7.
Effect of pHi on acid-induced injury of mouse cortical neurons. A, summary graph showing relative LDH release induced by acid incubation in the absence and presence of 0.5 mM quinine or 10 mM propionate. The neurons were washed three times with normal ECF before a 2-h incubation with ECF at a pH of 7.4 or 6.0 in the absence or presence of quinine or propionate. The LDH sample was taken at 6 h following the acid incubation and normalized to the maximal releasable LDH induced by permeating neurons with 0.5% Triton X-100. The relative LDH release was 8.9 ± 0.5% for 7.4 alone and 39.4 ± 1.6% for 6.0 alone (n = 12, p < 0.01); 7.2 ± 0.3% for pH 7.4 with propionate and 18.4 ± 0.5% for 6.0 with propionate (n = 8, p < 0.01). Incubation of neurons with acid solution in the presence of 0.5 mM quinine increased the LDH release from 12.6 ± 0.6% to 50.2 ± 1.8% (n = 12, p < 0.01). B, the net LDH release induced by acid alone (pH 6.0) or acid in the presence of 10 mM propionate or 0.5 mM quinine. Base-line LDH release at 7.4 alone and 7.4 with propionate or quinine has been subtracted from the corresponding acid-induced LDH release with and without propionate or quinine. C, FDA (green) and PI (red) staining of alive and dead neurons taken at 6 h following2hof acid treatment in the absence or presence of quinine or propionate.

 
ASICs, especially the ASIC1a channels, desensitize rapidly during persistent acid incubation, raising the question of whether these channels can play an important role in brain ischemia where the pHo drop is generally believed to be persistent. The large fluctuation of pHo associated with cortical spreading depression as described above suggests that in cortical regions of the brain, ASICs could be activated repeatedly during ischemia. Even in the regions where pHo drop is persistent, ASICs might still be partially functioning because ischemia itself dramatically reduces the desensitization of these channels (10).

A stable pHi is critical for cellular functions. Normal brain maintains pHi through H+ transporting mechanisms including Na+/H+ and Cl-/HCO-3 systems (37). In cortex or striatum, normal pHo is at ~7.3 and pHi is at ~7.0 (20, 22, 29). In brain ischemia, increased lactate generation and ATP hydrolysis result in increased H+ concentration in brain tissue (19, 20). Although it is generally believed that both pHo and pHi decrease during ischemia, studies by Nedergaard et al. (20) have demonstrated that focal ischemia in rats preferentially causes decreases in pHo rather than in pHi. In their studies, pHo decreased to below 6.0 in hyperglycemic conditions, whereas pHi remained relatively stable at ~6.8). Similar uncoupling of pHi from pHo was reported in trout erythrocytes at the onset of hypoxia (38). Thus, it is conceivable that in regions of the brain where H+ transporting mechanisms are not severely compromised, the cells may continue to extrude H+ to maintain a relatively stable pHi. In these regions of the brain, pHo may drop significantly with relatively normal pHi (20, 22). In other regions where the tight regulation of extracellular and intracellular pH via Na+/H+ exchange and Cl-/HCO-3 and the availability of intracellular anionic buffers are disturbed, greater diversity of the extracellular and intracellular pH may occur. In some studies, an alkaline tissue pH is reported. For example, studies by Hossmann and co-workers (22) group have demonstrated that in the cortical penumbra region of the rats surrounding the ischemic core, alkalization of tissue pH (predominantly the pHi) to as high as ~7.3 may occur.

Because of these variable, nonuniform changes of pHo and pHi in ischemic brain, our studies may suggest that the activities of ASICs in ischemic brain and ASIC-mediated neuronal injury are nonuniform in different brain regions. In regions where the drop of pHo is accompanied by a normal or even alkaline pHi, the activities of ASICs and ASIC-mediated cell injury would be greater than the regions where both pHo and pHi decrease. It is also interesting to speculate that a moderate intracellular acidification should be self-protective in reducing the acidosis-mediated ischemic brain injury.

Our studies suggest that multiple mechanisms may be involved in the potentiation of the ASIC current by intracellular alkalization: an increase in apparent affinity of the channels to H+, a reduction in steady state inactivation, and an increased rate of recovery from desensitization. However, the exact mechanism for the changes in channel activation and steady state inactivation by changing pHi remains to be determined. Babini et al. (32) have demonstrated that increasing the concentration of extracellular divalent or polyvalent cations induces a shift in the steady state inactivation of ASICs to more acidic pH. This leads to a potentiation of the channel response caused by a stabilization of the resting state. Our studies show that reducing the intracellular H+ concentration (e.g. by quinine) induced a similar shift in the steady state inactivation. It is not clear whether these two effects share a similar mechanism. Increasing the concentration of extracellular divalent cation is known to have a charge shielding effect (39), which may affect the local potential that the channels sense across the cell membrane. Because H+ is also known to have some charge shielding effect, reducing the concentration of intracellular H+ might have the same effect as increasing the concentration of extracellular divalent cations.

The studies by Babini et al. (32) also suggested that the sensitivity of ASIC1a activation and inactivation to H+ are intimately linked, because point mutation of either K105 or N106, two amino acids located in the extracellular domain, shifted the H+ sensitivity of activation and inactivation in the same direction. Interestingly, our studies showed that changing pHi, for example by quinine, can induce shifts of activation and inactivation of the ASIC channels in exactly the opposite directions; activation is shifted toward less acidic pH, whereas steady state inactivation is shifted toward more acidic pH. Both effects can increase the number of ASICs available to be activated at a given pHo, thus increasing the amplitude of the ASIC current. Different from the experiments by Babini et al. that studied the ASIC1a current expressed in Xenopus oocytes, ASIC currents in mouse cortical neurons are largely mediated by a combination of homomeric ASIC1a and heteromeric ASIC1a/ASIC2a channels (13). Further studies are required to determine whether the modulation of activation and inactivation of ASICs by pHi is subunit-dependent.


   FOOTNOTES
 
* This work was supported by research grants from the National Institutes of Health, the American Heart Association, and the Legacy Research Advisory Committee. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Robert S. Dow Neurobiology Laboratories, Legacy Clinical Research Center, 1225 NE 2nd Ave., Portland, OR 97232. Tel.: 503-413-2086; Fax: 503-413-5465; E-mail: zxiong{at}Downeurobiology.org.

3 The abbreviations used are: ASIC, acid-sensing ion channel; ECF, extracellular solution; LDH, lactate dehydrogenase; FDA, fluorescein diacetate; PI, propidium iodide. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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