INTRODUCTION

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Fast drops in extracellular pH (pH_e) activate proton-gated/acid-sensing ion channels (ASICs) in peripheral sensory neurons (Kovalchuk et al. 1990; Krishtal and Pidoplichko 1980) and various brain neurons (Grantyn et al. 1989; Ueno et al. 1992; Varming 1999). In most brain neurons, the ASIC currents consist of a single, rapidly inactivating component. However, in a subset of dorsal root ganglion neurons, the transient H⁺-activated current is followed by a sustained component (Bevan and Yeats 1991). The differences between H⁺-activated currents in sensory and central neurons are largely due to different subunit compositions of ASICs in central and peripheral neurons.

Six different ASIC subunits have been cloned to date, which are encoded by four genes (ASIC1-ASIC4). The channels cloned all belong to the amiloride-sensitive Na⁺-channel/degenerin family (Corey and Garcia-Anoveros 1996; Waldmann et al. 1999; Waldmann and Lazdunski 1998). ASIC1a (also named ASIC or BNaC2) subunits are highly enriched in primary sensory neurons of dorsal root and trigeminal ganglia but are also expressed in most brain regions (Garcia-Anoveros et al. 1997). Interestingly, a recent study by Gunthorpe et al. has reported that ASIC1a are also expressed in HEK 293 cells (Gunthorpe et al. 2001). These channels respond to low pH_e by mediating an amiloride-sensitive Na⁺-selective transient current, and the pH for half-maximal activation (pH_0.5) is ~6.2 (Waldmann et al. 1997b). In addition to being selective for Na⁺, ASIC1a channels are also permeable to Ca²⁺ ions (Waldmann et al. 1997b). ASIC1b (also named ASIC) is a splice variant of ASIC1a with a restricted expression in sensory neurons (Chen et al. 1998). When expressed in an heterologous system, ASIC1b forms homomultimeric channels that respond to pH drop with a transient inward current and a pH_0.5 of ~5.9 (Chen et al. 1998). Different from ASIC1a, which displays high Ca²⁺-permeability, the ASIC1b is not permeable to Ca²⁺ (Bassler et al. 2001; Chen et al. 1998). Like the ASIC1 gene, the rodent ASIC2 gene is alternatively spliced to code for two variants: ASIC2a and 2b. ASIC2a (also named MDEG, or BNaC1) subunits have a widespread distribution in the nervous system (Garcia-Anoveros et al. 1997; Price et al. 1996; Waldmann et al. 1996). Homomeric ASIC2a channels respond to pH drop with a fast desensitization and a low sensitivity to pH_e (pH_0.5 = 4.1) (Lingueglia et al. 1997; Price et al. 1996; Waldmann et al. 1996). ASIC2b subunits (also named MDEG2) are highly expressed in sensory neurons but are also found in central neurons (Lingueglia et al. 1997). They do not form functional proton-gated channels by themselves but can associate with other ASIC subunits and change their ion selectivity (Lingueglia et al. 1997). ASIC3 (also named DRASIC) was originally found in dorsal root ganglia (Waldmann et al. 1997a) and has recently been found in other neurons (Babinski et al. 1999). Homomeric ASIC3 receptors respond to pH drops by a biphasic current with a fast desensitizing phase followed by a late sustained current (De Weille et al. 1998; Waldmann et al. 1997a). Recently cloned ASIC4 subunits show high level of expression in pituitary gland. They do not seem to form functional proton-gated channels on their own (Akopian et al. 2000; Grunder et al. 2000).

The functional role that ASICs play is not fully understood. In sensory neurons, ASICs are considered to participate in nociception (Askwith et al. 2000; Bevan and Yeats 1991; McCleskey and Gold 1999), and the sustained current is believed to be the base element in the perception of nonadaptive painful stimuli (Bevan and Geppetti 1994). In the lanceolate nerve endings that lie adjacent to and surround the hair follicle, the ASIC2a channels have been shown to be involved in mechanosensation (Price et al. 2000). Since tissue acidosis is a common feature of cerebral ischemia and epilepsy (Siemkowicz and Hansen 1981; Siesjo 1988; Xiong and Stringer 2000), the presence of ASICs in brain neurons suggests that ASICs may play some pathological role. Consistent with this notion, our recent study has shown that expression of ASIC2a is up-regulated in rat brain following global ischemia (Johnson et al. 2001). Although an acidic pH in general may be considered neuroprotective due to proton inhibition of N-methyl-D-aspartate (NMDA) receptors (Giffard et al. 1990; Tang et al. 1990; Traynelis and Cull-Candy 1990; Vyklicky et al. 1990), its adverse effects could be explained by activation of ASICs that might contribute to membrane depolarization and subsequent Ca²⁺ accumulation (Varming 1999).

Like other ligand-gated ion channels, ASICs are believed to assemble from homomultimeric or heteromultimeric subunits (Waldmann et al. 1997b). The exact subunit combination of ASICs in native neurons, however, is not known. In the past 3 years, the electrophysiological properties and pharmacological profiles of recombinant homomeric and heteromeric ASICs in heterologous expression systems have been extensively investigated (Babinski et al. 2000; Bassilana et al. 1997; Waldmann et al. 1997b). These studies have provided information critical for elucidating what subunits might compose the native channels, since different homomeric and heteromeric ASICs have distinct pH sensitivity, ion selectivity, and channel kinetics. Furthermore, the recent findings that tarantula toxin PcTX1 specifically blocks ASIC1a homomeric channels (Escoubas et al. 2000) and Gd³⁺ preferentially blocks ASIC3-containing channels (Babinski et al. 2000) have provided additional means by which to investigate the subunit composition of native ASICs. Determining the subunit composition and the pharmacological profile of ASICs in native neurons is an important step toward defining the physiological and pathological roles of these channels in the neuronal system.

PC12 is a commonly used clonal neuronal cell line derived from a pheochromocytoma of the rat adrenal medulla (Greene and Tischler 1976). When cultured under normal conditions, PC12 cells resemble adrenal chromaffin cells in morphology and physiology. However, when cultured in the presence of nerve growth factor (NGF), they differentiate to resemble neurons. Like most neuronal cells, PC12 contain Na⁺, K⁺, and Ca²⁺ channels and other membrane receptors (Shafer and Atchison 1991). Recent northern blots by Chen et al. have also shown that mRNA for ASIC1a is expressed in PC12 cells (Chen et al. 1998). Using a combination of patch-clamp, Ca²⁺-imaging, and RT-PCR techniques, we have investigated the existence of a functional ASIC in PC12 cells. Our data indicate that PC12 cells likely contain a single population (ASIC1a) of proton-gated channels. It might be an ideal cell line for the study of functional role and the modulation of this key subunit of ASICs.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

PC12 cell culture

PC12 cells were purchased from ATCC (Cat No. CRL-1721). Undifferentiated PC12 cells were maintained in F-12K medium (GIBCO) with 15% heat-inactivated horse serum (GIBCO) and 2.5% fetal bovine serum (Hyclone). Medium was replaced every 2-3 days. For electrophysiological recording, cells were subcultured at a ratio of 1:4 in 35 × 35 mm Petri dishes. For differentiation, 50 ng/ml of NGF- (Sigma) was added to the culture medium. Cells were allowed to differentiate for 7 days before use in experiments. During this time, medium was changed every 2-3 days and replaced with the same concentration of fresh NGF-.

Electrophysiology

Whole cell recordings were performed as described previously (Xiong et al. 1997, 1999). Patch electrodes were constructed from thin-walled borosilicate glass (1.5 mm diam, WPI, Sarasota, FL) on a two-stage puller (PP83, Narishige, Tokyo). The tips of the electrodes were normally heat polished on a Narishige microforge (Scientific Instruments Laboratory, Tokyo, model MF-83) to a final diameter of 1-2 µm. The patch electrodes had resistance between 3 and 5 M when filled with intracellular solution. Whole cell currents were recorded using Axopatch 1-D amplifiers (Axon Instruments, Foster City, CA) in the voltage-clamp mode. Data were filtered at 2 kHz and digitized on-line using Digidata 1320A DAC units (Axon Instruments). The on-line acquisition was done using pClamp software pClamp 8.0 (Axon Instruments). Unless otherwise stated, the cells were voltage-clamped at 60 mV.

PC12 cells were washed three times and bathed in extracellular solution containing (in mM) 140 NaCl, 5.4 KCl, 25 HEPES, 33 glucose, 1.3 CaCl₂, and 1.0 MgCl₂, pH 7.4 using NaOH or HCl; 320-335 mOsm (Na⁺-rich solution). As a maximal response was achieved with pH of 5.0 in most PC12 cells, MES was not used for the buffering of lower pH solution. Patch electrodes contained (in mM) 140 CsF, 2.0 MgCl₂, 1.0 CaCl₂, 10 HEPES, 11 EGTA, and 4 MgATP, pH 7.3, using NaOH (the final Na⁺ concentration is about 10 mM), 300 mosmolar. Na⁺-free Ca²⁺-rich solution contained (in mM): 10 CaCl₂ and 25 HEPES, with pH adjusted with tris base and osmolarity adjusted to ~320 mosmolar with sucrose. For the study of relative Ca²⁺ permeability, pipette solutions contained Cs⁺ as the only cation.

Psalmotoxin-1 (PcTX1) from tarantula Psalmopoeus cambridgei was purchased from Invertebrate Biologics (Los Gatos, CA). Other chemicals were purchased from Sigma.

All electrophysiological experiments were done at room temperature (22-24°C). A multi-barrel perfusion system (SF-77B, Warner Instrument) was employed to achieve a rapid exchange of solutions.

RT-PCR

PC12 cells were transferred to a 15-ml Falcon tube and collected by centrifugation. Cells were washed twice and resuspended in 100 µl PBS. Total RNA was collected with the RNA SafeKit (Bio 101, Vista, CA). Poly(A⁺) mRNA was then isolated with the mRNA Kit Oligo [dT]30 (Bio 101, Vista, CA). The concentration of mRNA was determined by spectrophotometry.

cDNA was prepared from 0.2 µg PC12 mRNA (Clontech, Palo Alto, CA) using the Thermoscript RT-PCR Kit (Life Technologies, Rockville, MD) with oligo dT primers. Reaction was performed in 50-µl volume containing 1 times buffer, 1.5 mM MgCl₂, 0.2 mM dNTP, 0.4 µM sense and antisense primers (Table 1), 2 µl cDNA, and 2 U Platinum Taq DNA Polymerase (Life Technologies). PCR was carried out for 35 cycles in general, using the following programs: ASIC 1a (30 s at 94°C; 2 min at 70°C); ASIC 2a (30 s at 94°C; 2 min at 67°C; 1 min at 68°C); ASIC 3 (30 s at 94°C; 1 min at 60°C; 1 min at 72°C). The cDNA from rat brain was used as positive control for ASIC1a amplification. For ASIC 2a and ASIC 3, transcripts cloned into PC^DNA3 plasmids were used as the positive control. Reactions were analyzed on a 2% agarose gel.

Ca²+ imaging

Ca²⁺-imaging experiments were performed as previously described with slight modification (Jarvis et al. 1997; Xiong et al. 2000). PC12 cells grown on 25 × 25 mm glass coverslips were washed 3 times with extracellular solution and incubated with 5 µM fura-2-acetoxymethyl ester for 40-50 min at room temperature. Coverslips with fura-2-loaded cells were then transferred to a perfusion chamber on an inverted microscope (Nikon). Cells were illuminated using a xenon lamp (75 W) and observed with a ×40 UV fluor oil-immersion objective lens (Nikon). Video images were obtained using a cooled charge-coupled device (CCD) camera (Sensys KAF 1401, Photometrics). Digitized images were acquired, stored, and analyzed in a PC-type computer controlled by Axon Imaging Workbench software (AIW2.1, Axon Instruments). The shutter and filter wheel were also controlled by AIW to allow timed illumination of cells at either 340 or 380 nm excitation wavelengths. Fura-2 fluorescence was detected at an emission wavelength of 510 nm. Ratio images were analyzed by averaging pixel ratio values in circumscribed regions of cells in the field of view. The values were exported from AIW to SigmaPlot 2000 and then plotted.

All data are expressed as means ± SE. The paired and unpaired t-tests were employed where appropriate to examine the statistical significance of the difference between groups of data.

Dose-response curves were fitted to the Hill equation to calculate pH_0.5 and IC₅₀ values using the SigmaPlot 2000 software.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Proton activates inward current in PC12 cells

In both undifferentiated and NGF-differentiated PC12 cells, a rapid drop of extracellular pH ([pH_e]) from 7.4 evoked a transient, rapidly inactivating inward current (Fig. 1A), as previously reported in most central neurons (Ueno et al. 1992; Varming 1999) and a subpopulation of sensory neurons (Escoubas et al. 2000; Konnerth et al. 1987; Krishtal and Pidoplichko 1980). The amplitude of ASIC current in PC12 cells decreased (or "run-down") slightly following the formation of whole cell configuration. However, in most cells, the currents stabilized after 5-10 min and remained stable for more than 30 min. For this reason, most experiments were performed 10 min after the formation of whole cell configuration. The current completely decayed within 3-4 s with a single exponential time course. The time constant of the decay depends on pH drop with faster decay occurring in response to lower pH (Fig. 1D), similar to ASIC currents reported by other investigators (Chen et al. 1998; Kim et al. 1990). The amplitude of peak current increases in a sigmoidal fashion as pH_e decreases. In most PC12 cells, the threshold pH_e to elicit the inward current was about 7.0 and the maximum response appeared at 5.0. A detailed dose-response analysis gave a pH_0.5 of 6.2 ± 0.1 (mean ± SE, n = 5) for undifferentiated PC12 cells (Fig. 1B) and 6.0 ± 0.1 (n = 5) in differentiated cells (Fig. 1C). The kinetics and pH sensitivity of proton-gated currents in PC12 cells are similar to the currents mediated by homomeric ASIC1a channels (Waldmann et al. 1997b).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Electrophysiological properties of acid-sensing ion channels (ASICs) in PC12 cells. Currents were recorded in whole cell configuration at 60 mV. A: representative traces showing the pH-dependent activation of ASIC currents in a differentiated PC12 cell. The amplitude of inward current increases with larger drop of pH with maximal response recorded at pH 5.0. B and C: summary data showing the pH dependence of ASICs in undifferentiated (B) and nerve growth factor (NGF)-differentiated (C) PC12 cells. The pH0.5 for differentiated and undifferentiated PC12 cells were 6.0 ± 0.1 (n = 5) and 6.2 ± 0.1 (n = 5), respectively. D: pH dependence of the decay time constant of ASIC currents in differentiated PC12 cells (n = 6). The decay of the inward current activated by pH drops to 7.0-4.5 can be fitted by a single exponential time constant. The time constant is smaller with a larger drop of pH. Inset: typical decay time course of proton-gated currents fitted with single exponential.

Na+ selectivity of ASIC in PC12 cells

The selectivity of ASIC and relative permeability of Ca²⁺ ions were studied by recording the current-voltage relationship (I-V curve) in the presence of either Na⁺-rich or Ca²⁺-rich Na⁺-free solutions. The I-V curve was constructed by plotting the peak amplitude of the currents activated by a drop of pH_e from 7.4 to 5.0 at different membrane potentials. Cells were voltage clamped initially at 60 mV and then changed at a step of +20 mV every 2 min and the current activated by the same pH drop recorded.

As shown in Fig. 2A, in Na⁺-rich solution, proton-gated current in PC12 cells has a linear I-V relationship with a reversal potential at about +60 mV in both differentiated and undifferentiated PC12 cells. These reversal potentials coincide with the Na⁺ equilibrium potential (E_Na = +68 mV with extra- and intracellular solutions containing 140 and ~10 mM Na⁺), indicating that ASICs in PC12 cells are selective for Na⁺ ions.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Na+ selectivity and Ca2+ permeability of ASICs in PC12 cells. A: representative traces and summary data showing the current-voltage relationship of proton-activated currents in PC12 cells. PC12 cells were bathed in normal Na+-rich solutions. The extrapolated reversal potentials for both differentiated and undifferentiated PC12 cells were at ~60 mV (n = 6-7), which is close to Na+ equilibrium potential of +68 mV with the solutions used. For the plot of current-voltage relationship, the amplitude of currents at different potentials were normalized to that recorded at 60 mV. B: current-voltage relationship of ASIC currents recorded with 10 mM Ca2+ in extracellular solution (Na+-free solution) and Cs+ as the main cation in pipette solution. A junction potential of 15 mV has been corrected. The extrapolated reversal potential is ~10 mV (n = 3), indicating significant Ca2+ permeability of ASICs in PC12 cells. C: intracellular Ca2+ transient in differentiated PC12 cells (n = 7) in response to a 20-s drop of pHe to 5.0 from 7.4 with normal Na+-rich solution containing 1.3 mM CaCl2. Ca2+-imaging was performed as described in our previous study (Xiong et al. 2000). Intracellular free Ca2+ is expressed as the ratio of fura-2 fluorescence emitted at 510 nM following sequential excitation of the dye at 340 and 380 nM. * P < 0.05.

Ca²⁺ permeability was studied with ion-substitution experiments and supported by fluorescent Ca²⁺-imaging technique. With Na⁺-free extracellular solution containing 10 mM Ca²⁺, inward current was recorded with holding potentials negative to 10 mV (Fig. 2B), indicating that the ASIC in PC12 cells is permeable to Ca²⁺. This was supported by fura-2 fluorescent Ca²⁺-imaging experiment (Fig. 2C). In seven cells tested, perfusion of PC12 cells with pH 5.0 solution for 20 s caused a small but significant increase in intracellular Ca²⁺ signal as indicated by an increase in the ratio of fura-2 fluorescence emitted at 510 nm following sequential excitation of the dye at 340 and 380 nm.

Amiloride block of ASIC currents in PC12 cells

The effect of amiloride, a known inhibitor of proton-gated channels, was tested on proton-gated currents in PC12 cells. Inward currents were activated by a pH_e drop from 7.4 to 5.0 at a holding potential of 60 mV. After recording of two to three consecutive traces that had similar amplitude, cumulative concentrations of amiloride were added to both pH 7.4 and pH 5.0 solutions. Enough time was allowed for the effect of each concentration of amiloride to be stabilized. After the effect of final concentration of amiloride had stabilized, wash out of the drug was performed. Similar to proton-gated current in sensory and central neurons, the proton-gated currents in PC12 cells were dose dependently and reversibly inhibited by amiloride (Fig. 3A). Unlike ASICs in other cell types, however, the current in PC12 cells displayed higher sensitivity to amiloride. The IC₅₀ for amiloride inhibition of inward currents in differentiated and undifferentiated PC12 cells were 0.68 ± 0.04 µM (n = 6) and 0.21 ± 0.01 µM (n = 3), respectively (Fig. 3, B and C). These IC₅₀ values for amiloride block of ASIC in PC12 cells are ~10-20 times lower than amiloride block of ASICs in central (Varming 1999) and sensory neurons (Benson et al. 1999).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Amiloride block of ASIC currents in PC12 cells. A: representative traces showing the dose-dependent block of ASIC currents activated by pH drop to 5.0 from 7.4. Holding potential = 60 mV. B and C: summary data showing the amiloride block of ASIC currents in both differentiated (B) and undifferentiated (C) PC12 cells. The IC50 of amiloride block is 0.68 ± 0.05 µM (n = 6) for differentiated and 0.23 ± 0.02 µM (n = 5) for undifferentiated PC12 cells.

ASIC currents in PC12 cells are blocked by the specific homomeric ASIC1a blocker PcTX1

Escoubas and colleagues have shown that PcTX1 from tarantula toxin potently and specifically blocks the proton-gated currents mediated by homomeric ASIC1a without affecting the current mediated by other homomeric or heteromeric ASICs (Escoubas et al. 2000). We have therefore used the same toxin to see whether it can block the proton-gated current in PC12 cells. As shown in Fig. 4, PcTX1 blocked the proton-gated currents in both differentiated and undifferentiated PC12 cells in a dose-dependent manner with IC₅₀ of 1.5 ± 0.3 nM (n = 5) and 2.6 ± 0.4 nM (n = 6), respectively. The block of ASIC current by PcTX1 is reversible on wash out of the drug for ~10 min (Fig. 4A). These data further indicates that the proton-gated currents in PC12 cells are mediated by homomeric ASIC1a.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Proton-activated currents in PC12 cells are blocked by the specific ASIC1a blocker PcTX1. A: example recording showing the dose-dependent block of ASIC currents by PcTX1. Currents were activated by pH drop to 5.0 from 7.4 at membrane potential of 60 mV. B and C: summary data of PcTX1 block of ASIC currents in differentiated (B) and undifferentiated (C) PC12 cells. The IC50 of PcTX1 block is 1.5 ± 0.3 nM (n = 5) for differentiated and 2.6 ± 0.4 nM (n = 6) for undifferentiated PC12 cells.

PCR detection of ASIC1a transcript in PC12 cells

Nonquantitative RT-PCR was performed to verify the presence of ASIC1a transcript in both undifferentiated and NGF-differentiated PC12 cells. Consistent with our eletrophysiological studies and the northern blots by Chen et al. (1998), ASIC1a specific transcript was detected in both differentiated and undifferentiated PC12 cells (Fig. 5). The transcripts for ASIC2a and ASIC3 were not detected in PC12 cells.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Detection of ASIC1a transcript in PC12 cells. Nonquantitative RT-PCR analysis of mRNA isolated from undifferentiated and differentiated PC12 cells revealed the presence of mRNA for ASIC1a subunits. mRNA for ASIC2a and ASIC3 were not detected. D, differentiated PC12 cells; U, undifferentiated PC12 cells; C, positive control.

Up-regulation of ASIC activity in differentiated PC12 cells

Inclusion of NGF (50 ng/ml) in the culture medium induced a characteristic differentiation of PC12 cells. After 7 days in NGF, the size of cell bodies increased, along with the formation of long processes and extensive networks. We measured cell capacitance as an estimation of cell surface area of both differentiated and undifferentiated PC12 cells. The capacitance was obtained by integrating the capacitive transient induced by a 10-mV hyperpolarizing potential and dividing by the voltage step.

The membrane capacitance in undifferentiated cells was 19.2 ± 2.8 pF (n = 40). While in differentiated cells, the capacitance became 32.4 ± 2.0 pF (n = 47), significantly larger than that in undifferentiated cells (P < 0.01). Consistent with an increase in the size of PC12 cells after differentiation, the amplitude of proton-gated current increased significantly (Fig. 6A-C). The peak amplitude of currents activated by a drop of pH from 7.4 to 5.0 was 200.2 ± 24.6 pA (n = 92) in undifferentiated and 581 ± 75.0 pA (n = 72) in differentiated PC12 cells, indicating an increase in total number of proton-gated channels. Furthermore, the current density, which indicates number of channels per unit membrane area, also increased following the differentiation (Fig. 6D). The current density was 13.9 ± 2.8 pA/pF (n = 40) in undifferentiated cells and 26.1 ± 4.3 pA/pF (n = 47) in differentiated PC12 cells (P < 0.05). The up-regulation of ASIC activity in differentiated cells suggests that this channel likely plays some role in neuronal development and differentiation.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Up-regulation of ASICs in differentiated PC12 cells. A and B: representative traces showing the ASIC currents in differentiated (A) and undifferentiated (B) PC12 cells. Currents were activated by fast drops of pH from 7.4 to 5.0 with membrane potential at 60 mV. C: summary data showing the current amplitude recorded in differentiated (n = 72) and undifferentiated (n = 92) PC12 cells. The amplitude of proton-activated currents in differentiated cells is significantly larger than that in undifferentiated cells. D: current density of ASICs in PC12 cells. Current density was calculated by dividing the current amplitude (pA) by the membrane capacitance (pF). The capacitance was obtained by integrating the capacitive transient induced by a 10-mV hyperpolarizing potential and dividing by the voltage-step. U, undifferentiated PC12 cells; D, differentiated PC12 cells. ** P < 0.01.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In the present study we demonstrated that PC12 cells, derived from a clonal neuronal cell line, respond to pH drop with a transient inward current. The current is blocked by submicromolar concentrations of amiloride, and the reversal potential is close to the Na⁺ equilibrium potential, indicating that functional ASICs are present in PC12 cells. Our data further suggest that the proton-gated channels in PC12 cells are likely formed only from ASIC1a subunits based on the following observations. 1) The pH sensitivity of ASIC currents in PC12 cells (pH_0.5 = 6.0 for differentiated and 6.2 for undifferentiated PC12 cells) corresponds to that of homomeric ASIC1a (pH_0.5 = approximately 6.2) (Waldmann et al. 1997b). 2) The kinetics of ASIC current in PC12 cells is identical to that of homomeric ASIC1a, which displays a transient current with a single exponential decay (Waldmann et al. 1997b). Although the currents carried by homomeric ASIC2a and heteromeric ASIC1a/ASIC2a also display transient kinetics, the pH sensitivities of these channels (pH_0.5 of 4.1 for ASIC2a and 4.8 for ASIC1a/ASIC2a) are much lower than that of homomeric ASIC1a (Bassilana et al. 1997). 3) ASICs in PC12 cells display substantial Ca²⁺ permeability, as for homomeric ASIC1a (Waldmann et al. 1997b). Other ASICs, including homomeric ASIC1b, ASIC2a, and heteromeric ASIC1a/ASIC2a, have little Ca²⁺ permeability (Bassilana et al. 1997; Bassler et al. 2001). 4) Most importantly, the currents in PC12 cells are highly sensitive to blockade by PcTX1, a tarantula toxin specific for homomeric ASIC1a (Escoubas et al. 2000). PcTX1 has been shown to block only the homomeric ASIC1a expressed in Xenopus oocytes with an IC₅₀ of ~1 nM. It had no effect on homomeric ASIC2a, ASIC3, or heteromeric ASIC1a/ASIC2a with concentrations as high as 10 nM (Escoubas et al. 2000). All these data strongly suggest that PC12 cells likely contain a single population of functional proton-gated channels, the homomeric ASIC1a. Further studies, however, may be required to confirm that these cells do not contain heteromeric ASIC1a/ASIC2b, as the pharmacology of this heteromeric receptor has not been characterized.

ASICs in peripheral sensory neurons have been suggested to be implicated in the perception of pain during tissue acidosis (Bevan and Yeats 1991; Krishtal and Pidoplichko 1981; McCleskey and Gold 1999). In the ischemic myocardium, for example, ASICs are thought to mediate anginal pain (Sutherland et al. 2001). The presence of ASICs in the brain, which lacks nociceptors, suggests that these channels may have functions in the setting of acidosis beyond nociception. As tissue acidosis is a common feature of cerebral ischemia and epilepsy (Siemkowicz and Hansen 1981; Siesjo 1988; Xiong and Stringer 2000), activation of ASICs in brain neurons may act as mediators of pathological stimuli. However, due to complex subunit composition, possible interaction of protons with other ion channels/receptors (e.g., the glutamate receptors), and the lack of specific blockers for various ASICs, it is difficult to assess the physiological and pathological roles of specific ASICs in native neurons. The fact that PC12 cells are a neuronal cell line, contain only homomeric ASIC1a that can be blocked specifically by PcTX1, suggests that it may be an ideal system for the study of physiological and potential pathological roles of this key subunit of ASICs.

Although expressing specific ASIC subunits in a heterologous system (e.g., HEK 293 and CHO cells) might be a useful way to study the functional role of ASICs in isolation from other ion channels or membrane receptors, a potential problem in using these systems is that they are not neuronal in origin. Cellular responses and signal transduction pathways in heterologous cells are not representative of those in native neurons. We have recently shown, for example, that the mechanism underlying src-kinase potentiation of NMDA receptors expressed in HEK293 cells is different from that in hippocampal and spinal cord neurons (Xiong et al. 1999).

Similar to most central and peripheral neurons, PC12 cells contain various ion channels and membrane receptors. They have been commonly used in the studies of ion channel function and regulation (Hoshi et al. 1988; Vartian and Boehm 2001). Unlike central neurons, however, PC12 cells express hardly any functional NMDA or -amino-3-hydroxy-5-methyl4-isoxazole propionate (AMPA) receptors (Sucher et al. 1993; Sudo et al. 1997), even though mRNAs for some NMDA receptor subunits were detected (Sucher et al. 1993; Leclerc et al. 1995). The absence of functional glutamate receptor channels also suggests that PC12 cell might be a good model for the study of physiological and pathological roles of ASICs. Since a drop of pH inhibits NMDA receptor-mediated response (Giffard et al. 1990; Traynelis and Cull-Candy 1990), while it potentiates AMPA receptor-mediated toxicity (McDonald et al. 1998), it would otherwise be difficult to dissect the effect of ASIC activation during acidosis from the effect of pH modulation of glutamate receptors. The lack of functional glutamate receptors in PC12 cells may therefore provide the ideal setting for the study of ASIC function in isolation from the complex interaction of pH with NMDA and AMPA receptor channels.

Our studies also show that, like voltage-gated Na⁺ channels and T-type Ca²⁺ channels whose expression increases following NGF-induced differentiation (Garber et al. 1989; Pollock et al. 1990), the density of ASICs is up-regulated following NGF-induced differentiation. The up-regulation of ASIC activity in differentiated cells suggests that this channel likely plays some role in neuronal development and differentiation.