Department of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland
Submitted 5 March 2004 ; accepted in final form 23 April 2004
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ABSTRACT |
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cellular excitability; neuronal signaling; pH
Many noxious stimuli are associated with extracellular acidification, such as that caused by injury, inflammation, or ischemia (23, 26). ASICs in central neurons might contribute to the neuronal death associated with brain ischemia and epilepsy, which are accompanied by extracellular acidification (7, 23). In addition, fluctuation in extracellular pH occurs during normal brain function. The content of synaptic vesicles is acidic, and synaptic release during neuronal activity is expected to create extracellular acidification in the synaptic cleft. Consistent with a role for ASICs in physiological function, ASIC1a knockout mice showed a mild defect in spatial learning and fear conditioning (31, 32).
It has been known for a long time that extracellular acidification can induce action potentials (APs) in neurons; however, the acid sensors involved in this process have not been identified (7, 8, 21, 28). The aim of this study was to determine whether ASICs can mediate acid-induced generation and modulation of APs in brain neurons. For this purpose, we compared the pharmacological and biophysical properties of acid-induced AP generation determined under current clamp in cultured hippocampal neurons with those of the ASIC-like currents in these neurons characterized under voltage clamp and with the known properties of cloned ASICs. We show that acid-induced AP generation in hippocampal neurons is essentially due to activation of ASICs. We demonstrate for the first time a direct dependence of the probability of inducing APs on the density of functional ASICs at the cell surface and the pH to which the extracellular solution is changed. We show that ASIC activation can modulate AP generation and, depending on the conditions, can facilitate AP generation or inhibit AP bursts. ASICs are localized in close proximity to voltage-gated Na+ and K+ channels in the neuronal plasma membrane. Thus the functional properties and the localization of ASICs in hippocampal neurons suggest that these channels can sense local extracellular pH changes in hippocampus and transduce them into neuronal activity.
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MATERIALS AND METHODS |
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Recombinant expression of ASICs. To obtain clues about the molecular identity of ASICs that mediate the H+-induced currents in hippocampal neurons, we compared ASIC currents in hippocampal neurons with currents of cloned ASICs in recombinant expression systems. For the expression of heteromeric ASICs, we performed transient transfections in COS cells. We used a COS cell line that was selected for low endogenous expression of K+- and H+-gated channels. These cells were controlled for endogenous H+-gated currents, which were either absent or <150 pA. Equal concentrations of cDNA in the pcDNA3.1 vector (ASIC1a, ASIC2a) were cotransfected at a 10:1 ratio with either the CD8 antigen or green fluorescent protein (for identification of transfected cells) at 3 µg/35-mm dish with the use of PerFectin (Gene Therapy Systems, San Diego, CA) according to the instructions of the manufacturer. Cells were split 1 day after transfection and were studied on days 2 and 3 after transfection. Cells were cultured in DMEM with 10% fetal calf serum, 50 U/ml penicillin, and 50 µg/ml streptomycin at 37°C.
Cell lines that stably expressed ASICs were established for homomultimeric assembly of ASIC1a and of ASIC2a. The cDNAs were subcloned into the pEAK8 expression vector (Edge Biosystems, Gaithersburg, MD). DNA (4 µg/35-mm dish) was used to transfect Chinese hamster ovary (CHO) cells, which have no endogenous transient H+-gated currents, with the use of Lipofectamine 2000 (Invitrogen) according to the instructions of the manufacturer. ASIC-expressing cells were isolated by selection in culture medium (DMEM/NutMix F-12, 3.6% fetal calf serum, 50 U/ml penicillin, and 50 µg/ml streptomycin) containing 10 µg/ml puromycine.
Electrophysiological measurements.
We used an EPC-9 amplifier and Pulse and PulseFit software (HEKA Electronik, Lambrecht, Germany) for data acquisition and analysis. The sampling interval was 50100 µs for current-clamp experiments, 15 ms for voltage-clamp experiments to measure ASIC currents, and 100 µs for voltage-gated currents, and filtering was set to 5 kHz in all experiments. Experiments were performed in the whole cell and the excised outside-out configuration of the patch-clamp technique (13). For rapid changes of extracellular solutions, we used either an array of nine tubes whose position in front of the cell or the excised patch could rapidly be changed (Rapid Solution Changer RSC-200; Biologic, Grenoble, France) or a micromanifold that brings nine tubes into one outlet tube (Ala Scientific Instruments, Westbury, NY). The solution flow was controlled by computer-driven solenoid valves. For whole cell measurements, the perfusion outlet was positioned close to the cell body. With excised outside-out patches, we used the RSC-200 device exclusively and placed the patch pipette containing the membrane patch in front of the tube array. Extracellular solutions contained (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 MES, 10 HEPES, and 10 glucose, and pH was adjusted to 7.4 or to the values indicated. Pipettes were pulled from borosilicate glass (World Precision Instruments, Sarasota, FL). When filled with the pipette solution, the pipettes used for whole cell measurements had a resistance of 24 M and those used for excised outside-out patches had a resistance of 48 M
. The pipette solution for whole cell measurements contained (in mM) 90 K-gluconate, 60 HEPES, 10 KCl, 10 NaCl, 1 MgCl2, and 10 EGTA, pH 7.3. For outside-out patches, the pipette solution contained (in mM) 130 KF, 2 MgCl2, 10 NMDG, 10 EGTA, 10 HEPES, and 5 HCl, pH 7.35. Hippocampal neurons had a resting potential of 45 ± 14 mV (n = 51). When APs were induced in current-clamp mode by short (3 ms) depolarizing current pulses from a holding potential of 75 mV in current-clamp mode, the AP threshold was 37 ± 7 mV (n = 46), the current injection needed for AP generation was 378 ± 302 pA, and the mean AP duration was 0.85 ± 0.20 ms (measured at 50% of AP amplitude, n = 46; all values shown are means ± SD). An effective holding potential of 75 mV in current-clamp experiments was chosen to reproduce the membrane potential in hippocampal neurons in vivo.
Psalmopoeus cambridgei venom was obtained from Spider Pharm (Yarnell, AZ) and was used in all experiments at a 1:20,000 dilution. The P. cambridgei venom inhibits ASIC1a currents because of the toxin Psalmotoxin-1 contained in the venom (11). Preapplication of the venom at this dilution blocked 94 ± 1% (n = 5) of IpH6 in ASIC1a homomultimers expressed in CHO cells. Within the pH range 64, the current inhibition by the venom was not relieved by the application of more acidic stimuli (Vukicevic M and Kellenberger S, unpublished observations). Under the same experimental conditions, the P. cambridgei venom did not inhibit H+-induced current of all other ASICs tested (ASIC1b, ASIC2a, and ASIC3; Poirot O and Kellenberger S, unpublished observations). The other chemicals were obtained from Sigma-Aldrich or Fluka. The pH activation curves were fit by using the Hill equation
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RESULTS |
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Probability of inducing APs by acidification correlates with amplitude of ASIC inward current.
We tested whether there is a correlation between the density of functional ASICs at the plasma membrane and the probability of inducing APs. As a measure of the density of functional ASICs at the plasma membrane, we determined the IpH6 density, which we defined as the ratio of the IpH6 under voltage clamp to cell capacitance. To establish this correlation, we took advantage of the fact that the IpH6 density showed great cell-to-cell variation (34 ± 31 pA/pF, mean ± SD; n = 52). To calculate the probability of AP induction, we determined in each experiment whether acidification to pH 6 under current clamp induced APs (one or more). Experiments were then grouped according to their IpH6 density, and for each group, the probability of AP induction was calculated as the frequency of experiments with successful pH 6-induced AP generation. In Fig. 2A, we plot the probability of AP induction against the IpH6 density. This graph shows that the probability of AP induction increased with higher IpH6 density and that it was maximal at IpH6 densities >40 pA/pF. For comparison, in Fig. 2A, we also plotted the pH 6-induced depolarization Vm, which illustrates that the capacity of the extracellular acidification to induce APs strictly correlates with its capacity to induce depolarization.
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pH dependence of AP train duration.
In most experiments, ASIC activation induced bursts ("trains") of APs. In the pH range 6.06.8, AP trains were of longer duration at less acidic pH, as shown in Fig. 3 and Table 1. Acidification to pH 6 or 6.4 induced strong depolarizations (Vm of 54 ± 2 and 47 ± 2 mV, respectively) (Table 1) that lasted several seconds. AP trains, however, were short despite continued depolarization. The absence of APs during this plateau phase likely reflects the accumulation of voltage-gated Na+ channels in the inactivated state, in which they remain trapped until the membrane is repolarized. A similar phenomenon was recently observed in hippocampal neurons in which AP trains were induced by activation of heterologously expressed TRPV1 channels (34). Thus, for pH changes from 7.4 to the 76 range, the duration of induced AP trains is inversely correlated with the extent of acidification, while the probability of AP induction correlates with the extent of acidification (Fig. 2B).
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The pH dependence of the venom-resistant ASIC current is shown in Fig. 7B. This current had a pH0.5 of 5.9 ± 0.1 and a Hill coefficient of 1.9 ± 0.3 (n = 7). For comparison, in parallel to the experiments in hippocampal neurons, we determined the pH dependence of peak currents of cloned ASICs that were expressed in COS or CHO cells (Fig. 7B). The comparison indicates that the pH dependence of the venom-resistant H+-induced current is similar but not equal to that of ASIC1a2a heteromers. Therefore, in agreement with the study by Baron et al. (2), the venom-resistant component is most likely mediated by one type or several types of heteromeric ASICs composed of ASIC1a together with ASIC2a and/or ASIC2b. This finding is also consistent with the absence of ASIC currents in hippocampal neurons of ASIC1a knockout mice (32). The functional importance of the ASIC1a homomeric current (>95% of IpH6 in 60% of neurons, 42% of IpH6 in the remaining 40% of neurons) is illustrated by our observation that the presence of the P. cambridgei venom prevented AP generation in the pH range 5.56.8 (n = 8).
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DISCUSSION |
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AP induction and modulation by extracellular acidification. ASICs are not the only neuronal ion channels whose function depends on extracellular pH. Extracellular acidification, for example, has been shown to inhibit N-methyl-D-aspartate channels, voltage-gated Na+ channels, and the two-pore domain TASK channels; to activate the capsaicin receptor TRPV1 and the chloride channel CLC-2; and to be a coactivator for the inhibitory GABAA receptors (8, 14, 15, 17, 25). Thus extracellular acidification might depolarize the membrane by inhibiting the background K+ channels TASK-1 or TASK-3 or by activating TRPV1. ASIC currents are transient, in contrast to the TASK and TRPV1 currents, which do not inactivate. Extracellular acidification induces predominantly a transient inward current in hippocampal neurons (>90% of total pH 6-induced current). These transient, acid-induced currents display pH dependence similar to that of cloned ASICs and are blocked by the ENaC/degenerin inhibitor amiloride, which clearly identifies them as ASIC-mediated currents. Two recent studies that either used a pharmacological approach (2) or took advantage of ASIC1 and ASIC2 knockout mice (1) indicated that ASIC1a homomultimers and heteromers composed of ASIC1a together with ASIC2a and/or ASIC2b mediate ASIC currents in hippocampal neurons. We confirmed these observations and found a higher prevalence of ASIC1a homomultimers than of the heteromers. The presence of functional ASICs in cultured hippocampal neurons is consistent with observations of ASIC1a, ASIC2a, and ASIC2b mRNA in rodent hippocampus and of ASIC-like currents in acutely isolated hippocampal neurons (4, 12, 22, 29).
It previously was shown that extracellular acidification induces APs in hippocampal neurons, and recently it was shown that the induced depolarization correlates with acidification and ASIC current density, suggesting a role for ASICs in acid-induced AP generation (2). The aim of this study was to provide direct evidence for the implication of ASICs in acid-induced AP generation, to show how acidification induces APs by activating ASICs despite a potential inhibition of voltage-gated Na+ channels (14), and to investigate potential modulatory roles of ASICs in AP generation. The following observations in our analysis indicate that acid-induced AP generation in hippocampal neurons is essentially mediated by ASICs. 1) The kinetics of acid-induced depolarization are similar to the kinetics of ASIC currents measured under voltage clamp. 2) Acid-induced membrane depolarization and AP generation are inhibited by amiloride. 3) The probability of successful acid-induced AP generation depends on Na+ entry through ASICs, because it is higher in neurons with an increased density of functional ASICs at the membrane and increases with the extent of acidification in the pH range in which ASIC activity is steeply pH dependent. The observations that the probability of inducing APs by acidification increased with more acidic pH in the range 7.06.0 and that the voltage-threshold for AP generation did not depend on pH indicate that in this pH range, the pH-dependent inhibition of voltage-gated Na+ channels did not affect AP generation. Because homomultimeric ASIC1a is activated at higher pH than the other ASIC types in these neurons [pH0.5 = 6.4 (ASIC1a) compared with 5.9 (venom-resistant, acid-induced currents in hippocampal neurons)], it probably mediates AP generation induced by pH changes to pH 6.5. The important role of ASIC1a homomultimers is further supported by our observation that the presence of the P. cambridgei venom prevented acid-induced AP generation.
We show that ASIC activation induces APs but that the duration of the induced AP bursts decreases with stronger depolarization. Consistent with these observations, ASIC activation facilitates AP generation when combined with subthreshold excitatory stimuli, and if it occurs during bursting activity of a neuron, the ASIC activation can terminate the burst. The mechanism of this inhibitory effect of ASIC activity on neuronal signaling is analogous to that of depolarizing blocking agents at the neuromuscular junction (e.g., suxamethonium) that cause a maintained depolarization. Thus ASICs are modulators that, depending on the conditions present, are excitatory or inhibitory. The type of electrical response of a neuron to acidification thus depends on the extent of the pH change, on the expression level of ASICs present in the neuron, and on the momentary signaling activity of the neuron. ASIC expression is likely to change in response to the (patho)physiological situation. For example, upregulation of ASIC2a expression in brain after global ischemia and downregulation of ASIC1a and ASIC2b after status epilepticus were recently demonstrated (3, 16).
Localization of pH changes and ASICs.
Brain ischemia, hypoxia, and epilepsy are accompanied by acidosis (7, 23). On the basis of the current understanding of ASIC function, during long-lasting acidification such as that induced by brain ischemia or hypoxia, ASICs are expected to be briefly activated and then to inactivate and remain inactive. During global brain ischemia, extracellular pH decreases by 1 pH unit (reviewed in Ref. 23). Under these conditions, ASIC1a homomultimers and ASIC1a-containing heteromers are inactivated. During seizure activity, rapid extracellular acidification by 0.20.5 pH unit has been observed (19, 27, 33). In these studies, pH was measured using pH-sensitive microelectrodes. Because of limitations in spatiotemporal resolution, the actual pH changes may have been underestimated with the use of this approach. Thus ASICs may be activated during seizure activity and are expected to be inhibitory under these conditions. Fluctuations of extracellular pH also occur during normal brain function. Several studies with brain slices have indicated that neuronal activity causes rapid changes in extracellular pH (7, 8, 21). Much interest has been focused on the pH changes in the synaptic cleft during synaptic activity. Direct information regarding the exact pH changes in extracellular microdomains such as the synaptic cleft is currently not available (7). However, the pH in hippocampal synaptic vesicles has been determined to be pH 5.7 (24), and extracellular acidification due to the release of presynaptic vesicular contents has been detected indirectly (10). In addition to synaptic pH changes, there is evidence for glial acid secretion in response to neural activity. This acid secretion is expected to induce pH changes with a time course on the order of seconds (7); thus these pH changes would also be fast enough to activate ASICs. Currently, it is not clear whether the extent of acidification by glial acid secretion would be sufficient to activate ASICs. The pH changes reported during seizure activity were relatively small but might have been underestimated because of the limited spatiotemporal resolution of pH microelectrodes that were used in these studies (8).
For a better appreciation of the role of ASICs with regard to their potential involvement in synaptic functions, it is important to know their subcellular localization with respect to synapses. The subcellular localization in brain neurons has thus far been addressed only for the ASIC1a subunit. Studies conducted at two different laboratories produced somewhat divergent results. Wemmie and colleagues (31, 32) showed evidence for preferential localization of ASIC1a at synapses and for involvement in synaptic functions. De la Rosa et al. (9), however, reported that the ASIC1a protein is equally distributed in plasma membrane of soma, axons, and dendrites of hippocampal and other brain neurons.
While functional roles of synaptic ASICs can be imagined and were proposed in recent studies (31, 32), the potential roles of extrasynaptic ASICs are less clear. Extrasynaptic ASICs might be activated by glial cell-dependent acid secretion or during seizure activity, or they may play as yet undefined roles in signaling during ischemia. To elucidate the role of the venom-resistant ASIC current in hippocampus, it is important to determine in addition the subcellular and synaptic vs. extrasynaptic localization of ASIC2a and ASIC2b.
In conclusion, we have shown in this study that acid-induced AP generation in hippocampal neurons is due to the activation of ASICs. Our study suggests that ASICs in hippocampus are likely to change the electrical properties of neurons in response to even small pH changes. The effect on hippocampal neuron excitability due to ASIC activation depends on the extent of the pH change, on the expression level of ASICs present in the neuron, and on the activity of the neuron at the moment of ASIC activation.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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