JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M312145200 on February 11, 2004

J. Biol. Chem., Vol. 279, Issue 18, 18296-18305, April 30, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/18/18296    most recent
M312145200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Askwith, C. C.
Right arrow Articles by Welsh, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Askwith, C. C.
Right arrow Articles by Welsh, M. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Acid-sensing Ion Channel 2 (ASIC2) Modulates ASIC1 H+-activated Currents in Hippocampal Neurons*

Candice C. Askwith{ddagger}§, John A. Wemmie||**, Margaret P. Price{ddagger}, Tania Rokhlina{ddagger}, and Michael J. Welsh{ddagger}§{ddagger}{ddagger}§§

From the Departments of {ddagger}Internal Medicine, {ddagger}{ddagger}Physiology and Biophysics, and ||Psychiatry, §Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242 and **Department of Veterans Affairs Medical Center, Iowa City, Iowa 52242

Received for publication, November 5, 2003 , and in revised form, February 9, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hippocampal neurons express subunits of the acid-sensing ion channel (ASIC1 and ASIC2) and exhibit large cation currents that are transiently activated by acidic extracellular solutions. Earlier work indicated that ASIC1 contributed to the current in these neurons and suggested its importance for normal behavior. However, the specific contribution of ASIC1 and ASIC2 subunits to acid-evoked currents in hippocampal neurons remained uncertain. To decipher the individual role of the ASIC subunits, we studied H+-gated currents in neurons from both ASIC1 and ASIC2 null mice. We found that much of the current was produced by ASIC1a/2a heteromultimeric channels, and individual subunits made distinct contributions. The ASIC1a subunit was key in establishing current amplitude. The ASIC2a subunit had little effect on amplitude but influenced desensitization, recovery from desensitization, pH sensitivity, and the response to modulatory agents. We also found heterogeneity in the contribution of ASIC2 throughout the neuronal population, with individual neurons expressing both ASIC1a homomultimeric and ASIC1a/2a heteromultimeric channels. Studies of neurons heterozygous for disrupted ASIC alleles indicated that the properties of H+-gated currents are dependent on the proportion of the individual subunits. These findings indicate that the absolute and relative amounts of ASIC subunits determine the amplitude and properties of hippocampal H+-gated currents and therefore may contribute to normal physiology and pathophysiology.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although the extracellular pH of the central nervous system is tightly controlled, both localized and global reductions in pH have important physiologic and pathophysiologic consequences (1). For example, repetitive nerve activity transiently reduces the local pH, perhaps by releasing the acidic contents of synaptic vesicles (2–4). Ischemia and seizures also cause extracellular acidosis that may contribute to the pathophysiology of those diseases (1, 5). In situations where the extracellular pH falls, protons may act as ligands, activating H+-gated channels (4, 6). Similarities between the biophysical properties of transient H+-gated cation currents in hippocampal neurons (7, 8) and currents generated by recombinant ASICs1 suggested they might be responsible (6, 8).

Three genes, ASIC1, -2, and -3, and their alternatively spliced transcripts (ASIC1a, -1b, -2a, and -2b) produce subunits that combine to form multimeric channels activated by extracellular acid (9, 10). When the various ASIC subunits are expressed individually, they form homomultimeric channels with distinct electrophysiologic signatures (4, 11–26). When expressed together, the resulting heteromultimeric channels manifest specific characteristics that depend on the subunit composition (12, 14, 19, 24, 26–30). A fourth gene, ASIC4, has also been identified, but its heterologous expression has neither generated nor modified currents from other ASIC subunits (31, 32).

Because different ASIC subunits confer distinct properties to H+-gated channels, the subunit composition of ASIC channels in neurons could dramatically affect H+-gated currents and ASIC-induced neuronal signaling. In the peripheral nervous system, studies of ASIC null animals revealed a contribution of ASIC1, -2, and -3 to acid-evoked currents and showed their contribution to mechanosensation and acid-evoked nociception (24, 33–37). In the brain, neurons express ASIC1a, -2a, and -2b subunits in variable and overlapping distributions (4, 12, 28, 38–40). Studies in mice bearing disruptions of the ASIC1 gene have demonstrated the importance of ASIC1 for normal central nervous system function; ASIC1 –/– mice exhibited defective spatial learning, eye-blink conditioning, fear conditioning, and hippocampal synaptic plasticity (40, 41).

The contribution of individual ASIC subunits to H+-gated currents in central neurons remains uncertain. In an earlier study, we found that the loss of ASIC1 abolished pH 5-evoked currents in hippocampal neurons (41). This result seemed surprising, because ASIC1a is not the only ASIC subunit expressed in the central nervous system; hippocampal neurons also express ASIC2a and -2b (12, 28, 38, 39). Although recombinant ASIC2b generates no current, heterologous expression of ASIC2a does elicit acid-evoked cation currents (12, 42, 43). In addition, Zn2+, which modulates ASIC2a-containing channels but not those produced by ASIC1a, potentiated hippocampal acid-evoked current (8, 30). Therefore, the goal of this work was to learn which ASIC subunits contribute to central H+-gated currents and to determine whether they function as homomultimers or heteromultimers. To accomplish this, we examined the characteristics of whole-cell H+-gated currents in hippocampal neurons from mice with disrupted ASIC1 and ASIC2 genes. We focused on channel properties that differentiate the individual ASIC subunits including pH sensitivity, desensitization, recovery from desensitization, and sensitivity to FMRFamide and zinc. In light of earlier reports, we were particularly interested in testing the hypothesis that ASIC2 contributes to these currents in hippocampal neurons.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Culture—Mouse hippocampal neuron cultures were prepared from postnatal day 1–2 mice as described previously (44) with the addition of insulin-transferrin-sodium selenite (41). Hippocampi from 4–10 pups were pooled and plated in 24-well dishes on 35-mm collagencoated cover slips. Cytosine {beta}-D-arabinofuranoside was added at day 3–4 to inhibit glial growth. Whole-cell patch clamp was performed on large pyramidal cells maintained in culture for 1 to 3 weeks. Neurons from ASIC1 –/– (41), ASIC2 –/– (34), and heterozygous mice were compared with neurons from the corresponding wild-type animals plated and cultured at the same times. We observed no differences between the groups of wild-type neurons, and they were combined for comparison to heterozygous neurons (ASIC1 +/– and ASIC2 +/–).

Mouse ASIC1a, -2a, and -2b were cloned into pMT3 for heterologous expression as described (24). Chinese hamster ovary (CHO) cells were transfected with 1–3 µg of DNA using the Transfast lipid reagent (Promega, Madison, WI). CHO cells were used because of the ease of studying by patch clamp, the efficiency of transfection, and their lack of native H+-gated currents. The properties of ASICs expressed in CHO cells, COS-7 cells, Xenopus oocytes, and lipid bilayers can vary (4, 11–16, 22, 24, 26, 28, 30, 42, 45). However, mouse ASICs studied in CHO cells produced properties very similar to those we reported previously in COS cells (26); compare data in Ref. 24 to that in this manuscript. The pC1EGFP vector encoding enhanced green fluorescent protein (Clontech, Palo Alto, CA) was added at 20% the DNA concentration to identify transfected cells using epifluorescence microscopy. For experiments assessing the function of ASIC2b with ASIC2a, pMT3 was added to maintain a constant final DNA concentration. Cells were studied 2–3 days following transfection.

Patch Clamp Electrophysiology—Electrodes were filled with the intracellular solution containing (in mM): 120 KCl, 10 NaCl, 2 MgCl2, 5 EGTA, 10 HEPES, and 2 ATP. The pH was adjusted to 7.3 with tetramethylammonium hydroxide. Extracellular solutions contained (in mM): 128 NaCl, 5.4 KCl, 5.55 glucose, 10 HEPES, 10 2-(4-morpholino)-ethanesulfonic acid, 2 CaCl2, and 1 MgCl2. The pH was adjusted with tetramethylammonium hydroxide, and osmolarity was adjusted with tetramethylammonium chloride. For neuronal cultures, inhibitors of synaptic activity were routinely added to the extracellular solution. These included 1 µM tetrodotoxin, 10 µM 6-cyano-7-nitroquinoxaline-2, 3-dione, 50 µM DL-2-amino-5-phosphonopentanoic acid (dAP5), and 30 µM (–)-bicuculline methiodide. Tetrodotoxin has been shown to alter the kinetics of H+-gated channels in some neurons (46, 47). However, the concentration of tetrodotoxin used in our experiments had no effect on H+-gated currents in cultured mouse hippocampal neurons. The peptide FRRFamide was synthesized by Research Genetics (Huntsville, AL). All other chemicals were obtained from Sigma.

Electrodes had a resistance of 3–5 megohms, and series resistance was compensated by 70% after establishing the whole-cell configuration. Neurons were held at –70 mV, and solutions were changed by gravity-fed perfusion pipes. Data were acquired using an AXOPATCH 200B amplifier with pCLAMPex 8.1 software (Axon Instruments, Foster City, CA). Data were analyzed using Clampfit (Axon Instruments) and IGOR Pro (WaveMetrics, Lake Oswego, OR). Amplitude was determined by subtracting the baseline current at pH 7.4 from the peak current amplitude determined in Clampfit (Axon Instruments). Capacitance was measured for each neuron in Clampex (Axon Instruments). The tau of desensitization ({tau}d) was calculated by fitting the data to a single exponential using IGOR Pro (WaveMetrics). Because of the complex nature of current desensitization with FRRFamide application, the Td 1/2 of desensitization was used as a quantitative measurement of desensitization. The Td 1/2 of desensitization was recorded as the time from the peak current amplitude to half-maximal peak current (as described above).

Because of current run-down, data for pH dose-response, zinc potentiation, and recovery from desensitization were normalized to the average of the preceding and following control pH applications. All data were obtained from at least two different culture preparations representing 8–20 mice. Because current amplitude can vary depending on the time in culture, neurons of different genotypes were prepared within 24 h of one another, cultured identically, and analyzed on the same day after culture. Statistical significance was evaluated using the paired or unpaired Student's t test as appropriate.

Northern Analysis and RT-PCR—Hippocampi of 14-day-old mice were dissected and placed into RNAlater solution (Qiagen, Valencia, CA). The hippocampi from four animals were pooled, and RNA was isolated using the RNeasy Mini purification kit (Qiagen) and quantified. Total RNA (10 µg per lane) was run on a formaldehyde-agarose gel using the NorthernMax system (Ambion, Austin, TX) and transferred to BrightStar-Plus membrane (Ambion, Austin, TX). The probes used for these studies were generated from PCR products and included the following: an 528-bp product specific to ASIC2a (primers m2AF, 5'-atggacctcaaggagagcccc-3'; and m2AR, 5'-ggtgaagtcttgatgcccaca-3'), an 495-bp product specific to ASIC2b (primers m2BF, 5'-tgtggcccgcacaacttctcc-3'; and m2BR, 3'-ctggccttctgcacgtccctt-3'), and an ~900-bp product common to both transcripts (m2ABF, 3'-gatggcaagcctctgctcacc-3'; and m2ABR, 5'-aatctcctccagggtgcccaa-3'). The radioactive probes were generated using the Random Primed DNA Labeling Kit (Roche Applied Science) and purified using NucAwayTM Spin Columns (Ambion). Blots were visualized by phosphorscreen (STORM; Amersham Biosciences).

For RT-PCR analysis, first strand cDNA was synthesized with Superscript II (Invitrogen) with random hexamer primers. Primers used for PCR amplification of sequences specific to ASIC2a or ASIC2b, and common to ASIC2a/2b, were designed to cross intron/exon boundaries. The following primers were used: ASIC2a, 5'-aacctcaatggcttccggttctcc-3' and 5'-cccccccttgaccgtggtgagcag-3' (360-bp band); ASIC2b, 5'-cgagtgcaccgcgagtggagccgc-3' and 5'-cccccccttgaccgtggtgagcag-3' (430-bp band); and ASIC2a/2b, 5'-tccgagaacattcttgttctggat-3' and 5'-gttctcatcatggctcccttcctc-3' (210-bp band). The cycling parameters using the RoboCycler thermocycler (Stratagene, La Jolla, CA) consisted of 40 cycles of 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 2 min.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Extracellular pH 4 Solutions Activate H+-gated Currents in Some ASIC1 –/– Hippocampal Neurons—Central neurons express ASIC1a, -2a, and -2b subunits (4, 12, 25, 28, 38–40, 48, 49). To determine the contribution of individual ASICs to hippocampal H+-gated currents, we first studied neurons from ASIC1 –/– animals. Consistent with our earlier work (41), pH 5 solutions failed to activate current (Fig. 1A). However, previous findings suggested that recombinant ASIC2a homomultimeric channels require a very low pH for activation, and ASIC2a/2b heteromultimeric channels require an even greater reduction in pH (12, 43). Therefore, we applied a more acidic solution (pH 4) and uncovered amiloride-sensitive currents in ASIC1 –/– neurons (Fig. 1A shows some examples). The average peak current was dramatically reduced compared with wild-type neurons (Fig. 1, A and B), and not every cell responded. Approximately 43% of the ASIC1 –/– neurons showed a transient amiloride-sensitive current during pH 4 application. In contrast, acidic solutions (pH 4 or 5) elicited transient currents in >95% of wild-type neurons (41).



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 1.
Amiloride-sensitive current in ASIC1 –/– neurons. A, representative recordings of pH 4 and 5 applications in the presence and absence of amiloride (250 µM). Data are from a wild-type neuron (above) and three ASIC1 –/– neurons (below) with varying responses to pH 4. B, peak amiloride-sensitive currents elicited by pH 5 and 4 for wild-type (n = 9) and ASIC1 –/– neurons (n = 38). The asterisk indicates p < 0.05 compared with pH 5. C, rate of desensitization ({tau}d) of pH 4-evoked currents in ASIC1 +/+ and –/– neurons (n = 7; the asterisk indicates p < 0.0005). In all figures, error bars represent S.E.

 
The presence of ASIC2a in the hippocampus made it a candidate to generate the H+-gated currents in ASIC1 –/– cells. One feature that distinguishes heterologously expressed ASIC2a and ASIC2a/2b channels from channels containing ASIC1 subunits is their slower desensitization (4, 11, 12, 42, 43). We found that transient currents from ASIC1 –/– neurons desensitized more slowly than those in wild-type neurons (Fig. 1, A and C). Thus, the expression of ASIC2a and -2b in the hippocampus, the failure of ASIC2b expression to generate current, the requirement for very low pH solutions to activate, and the slow desensitization ({tau}d) suggest that ASIC2a (either alone or as a heteromultimeric channel with ASIC2b) generated currents in ASIC1 –/– neurons.

Loss of ASIC2 Enhances pH Sensitivity and Slows Desensitization of Acid-evoked Currents in Hippocampal Neurons—To further test the hypothesis that ASIC2 subunits contribute to H+-gated currents, we compared wild-type and ASIC2 –/– neurons; disruption of the ASIC2 gene eliminates both ASIC2a and ASIC2b subunits (34). A pH 5 application generated currents with comparable peak amplitudes in wild-type and ASIC2 –/– neurons (Fig. 2, A and B). This result suggests that ASIC2 made little contribution to current amplitude. Therefore, we studied additional properties. Earlier work showed that currents from heterologously expressed ASIC2a channels required more acidic solutions for activation than ASIC1a channels, and ASIC2a/1a heteromultimers had intermediate pH sensitivity (24, 28). In wild-type hippocampal neurons, reducing extracellular pH increased current amplitude (Fig. 2C), consistent with earlier work (8). Eliminating ASIC2 increased the pH sensitivity; for example, pH 6.3 solutions stimulated 59% of the pH 5-induced current in ASIC2 –/– neurons versus only 26% in wild-type neurons.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 2.
Proton-gated currents from ASIC2 +/+ and –/– hippocampal neurons. A, representative trace of pH 5-induced current in ASIC2 +/+ and –/– neurons. B, average peak amplitude of pH 5-induced current (ASIC2 +/+, n = 67; ASIC2 –/–, n = 53). C, relationship between pH and current. Holding pH was 7.4. Data were normalized to currents of flanking pH 5 applications (n >= 7). D, desensitization rate of pH 5-induced currents. Data are from ASIC2 +/+ (n = 58) and ASIC2 –/– (n = 49) hippocampal neurons (the asterisk indicates p < 0.00005 compared with ASIC2 +/+). The absolute desensitization rates for wild-type neurons differ in Fig. 1C and Fig. 2D as expected based on previous reports of pH-dependent differences in desensitization rates (26).

 
Loss of ASIC2 also prolonged desensitization of H+-gated currents as measured by the desensitization rate ({tau}d) (Fig. 2, A and D). Because homomultimeric ASIC2a desensitizes more slowly than other ASIC subunits, removing its contribution might have been expected to shorten, rather than lengthen {tau}d. To investigate the basis of this change, we measured the {tau}d of recombinant ASIC1a and ASIC2 subunits expressed in heterologous cells (Fig. 3, A and B). For this and all other studies, we used mouse ASIC subunits to facilitate comparison with the properties of ASICs in the mouse neurons. Consistent with earlier reports, ASIC1a currents desensitized more rapidly than ASIC2a (24, 28), and ASIC1a/2a currents had a {tau}d shorter than either subunit alone (24). Because the ASIC1a/2a combination generated currents that desensitized faster than the sum of the components, these data indicated that heteromultimeric channels generated the current. The fact that ASIC2 –/– neurons desensitized more slowly than wild-type further suggests that the acid-evoked current in wild-type hippocampal neurons arose, at least in part, from heteromultimeric channels composed of ASIC1a and ASIC2a. Although ASIC2b is expressed in hippocampus, earlier reports indicated that it neither generated current nor altered ASIC1a currents (12). Consistent with that observation, adding ASIC2b failed to alter {tau}d compared with ASIC1a alone (Fig. 3B). These results suggest that the faster desensitization of wild-type neurons is because of the ASIC2a subunit.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 3.
Proton-gated currents from CHO cells expressing ASIC1a and -2a. A, examples of current traces from CHO cells that were untransfected and that were expressing ASIC1a, ASIC2a, and ASIC1a plus ASIC2a. B, desensitization rate of pH 5-induced currents from CHO cells expressing ASIC1a + ASIC2a (n = 12), ASIC1a (n = 21), ASIC1a + ASIC2b (n = 8), and ASIC2a (n = 5) (the asterisk indicates p < 0.0005 compared with ASIC1a).

 
ASIC2 Contributes to the Modulatory Effects of FRRFamide and Zinc—To further test the hypothesis that ASIC2 contributes to acid-evoked currents in hippocampal neurons, we tested the effect of agents that selectively modulate channels containing ASIC1a or ASIC2a. In heterologous expression systems, FRRFamide, FMRFamide, and related neuropeptides potentiate ASIC1a-mediated currents, whereas ASIC2a is unaffected (20, 29). In wild-type hippocampal neurons, FRRFamide slowed desensitization and in some cases generated sustained acid-evoked currents, consistent with the presence of ASIC1a (Fig. 4, A and B). Surprisingly, eliminating ASIC2 attenuated this response. These results suggest that the presence of ASIC2 subunits enhanced peptide modulation of ASIC1a-containing channels. To investigate this possibility, we expressed the subunits in CHO cells and found that adding ASIC2a enhanced the response of ASIC1a (Fig. 4B). These data suggest that heteromultimeric channels are responsible for H+-gated currents in hippocampal neurons and that the normal response to FRRF-amide requires both ASIC1a and ASIC2a subunits. These results have a parallel in a previous study (29) showing that heteromultimeric channels composed of ASIC3 and ASIC2a responded more robustly to FMRFamide-related peptides than ASIC3 alone.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 4.
FRRFamide and zinc modulation of H+-gated currents. A, representative traces showing effect of FRRFamide (200 µM) on pH 5-evoked currents in ASIC2 +/+ and –/– neurons. B, -fold change in Td1/2 induced by FRRFamide. Left, ASIC2 +/+ neurons (n = 12) and ASIC2 –/– neurons (n = 10). The asterisk indicates p < 0.01 by unpaired Student's t test. Right, ASIC1a (n = 8) and ASIC1a + ASIC2a (n = 9) expressed in CHO cells. The asterisk indicates p < 0.005. C, representative traces showing effect of zinc (300 µM) on pH 6.3-evoked currents in ASIC2 +/+ and –/– neurons. D, amplitude of pH 6.3-evoked current in the presence of 300 µM zinc as a percentage of current in the absence of zinc in ASIC2 +/+ (n = 23) and ASIC2 –/– (n = 13) neurons. Data were normalized to currents of flanking pH 6.3 applications. The asterisk indicates p < 0.005 compared with absence of zinc by unpaired Student's t test.

 
Earlier work showed that zinc selectively increased H+-gated currents from ASIC2a and ASIC2a-containing heteromultimeric channels (8, 30). We found that zinc potentiated H+-gated currents in wild-type neurons, whereas it slightly inhibited current in ASIC2 –/– neurons (Fig. 4, C and D). These results support the conclusion that ASIC2 contributes to hippocampal H+-gated currents and is required for zinc potentiation.

ASIC2a Determines the Fast Recovery from Desensitization in Wild-type Neurons—Because ASIC2a influences recovery from desensitization, we also tested this property. ASIC2a and ASIC1a/2a channels recover rapidly following a pH 5 application, whereas ASIC1a homomultimers recover more slowly (24). We found that after 1 s at pH 7.4, wild-type hippocampal neurons recovered 61% of the pH 5-evoked current (Fig. 5, A and B). Deleting ASIC2 extended the recovery time, so that in 1 s, only 4% of the current had recovered, and even by 10 s, recovery was only 58%. Because ASIC2b is reported to not affect ASIC1a current (12), we assumed that ASIC2a was the subunit responsible for speeding recovery. To test this assumption, we expressed recombinant ASIC1a with ASIC2a or -2b and measured recovery rate. Fig. 5C shows that ASIC2a but not -2b accelerated the recovery of ASIC1a currents. These striking differences indicate that ASIC2a determined the fast recovery from desensitization in hippocampal neurons.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 5.
Effect of subunit composition on recovery from desensitization. Recovery from desensitization was measured by applying pH 5 for 10 s and then pH 7.4 for the indicated length of time (1 to 30 s), followed by a second pH 5 application. A, representative tracings showing recovery from desensitization during 1 s at pH 7.4 in ASIC2 +/+ and –/– neurons. B, percentage recovery from desensitization in ASIC2 +/+ and –/– hippocampal neurons. Data were normalized to currents of flanking pH 5 applications (n = 9–13). C, percentage recovery from desensitization in CHO cells expressing ASIC1a, ASIC1a + ASIC2b, or ASIC1a + ASIC2a (n = 6–12).

 
Channels with Heterogeneous Subunit Composition Contribute to H+-gated Hippocampal Currents—The results suggested that hippocampal H+-gated current reflect predominantly ASIC1a/2a heteromultimers. However, they did not exclude the presence of ASIC1a homomultimeric channels. Several observations are consistent with this possibility. First, the peptide toxin PCTX1, which was reported to selectively inhibit ASIC1a homomultimeric channels, reduced H+-gated current in central nervous system neurons (8, 19). Second, recovery from desensitization by wild-type neurons (Fig. 5B) was slower than recombinant ASIC1a/2a heteromultimers (Fig. 5C). Third, wild-type neurons (Fig. 2C) were slightly more pH-sensitive than ASIC1a/2a heteromultimers (24, 28).

Therefore, we tested for heterogeneity of channel types within individual neurons. We reasoned that, because ASIC1a homomultimers recover much more slowly from desensitization than heteromultimers containing ASIC2a (see Fig. 5C and Ref. 24), we could largely eliminate the contribution of ASIC1a homomultimers by limiting the time of recovery from desensitization. This would uncover currents from other homo- and heteromultimeric channels, which we might recognize by a change in {tau}d. To test this, we applied a pH 5 solution, measured the {tau}d ({tau}d0) and then continued the perfusion for 10 s to desensitize acid-evoked currents. We then stopped the pH 5 solution, allowed 2 s at pH 7.4 for recovery, and reapplied pH 5 solution to measure the {tau}d a second time ({tau}d2). Fig. 6A shows the ratio of {tau}d after 2 s of recovery relative to that prior to desensitization ({tau}d2/{tau}d0). We expected that if all the channels expressed on a cell were of identical subunit composition, then the {tau}d would be unchanged after desensitization and the ratio would be 1. To test this notion, we expressed recombinant ASIC1a, -2a, and -1a/2a channels and found a ratio of ~1 in all cases. Thus, with a homogeneous population of channels, {tau}d was the same irrespective of whether the channels showed fast or slow recovery. In contrast, wild-type neurons had a {tau}d2/{tau}d0 ratio less than 1, suggesting that these neurons contain a population of rapidly recovering channels with a short {tau}d (consistent with ASIC1a/2a channels) and a population of slowly recovering channels with a longer {tau}d (consistent with ASIC1a homomultimers). Supporting this conclusion, ASIC2 –/– neurons had a {tau}d2/{tau}d0 ratio of 1. Thus, individual wild-type neurons appeared to express ASIC1a/2a heteromultimeric channels plus channels with properties consistent with ASIC1a homomultimers.



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 6.
Heterogeneity of H+-gated currents in individual neurons and in the population of hippocampal neurons. A, data are ratio of the {tau}d of desensitization 2 s after recovery at pH 7.4 ({tau}d2) versus before desensitization ({tau}d0). Measurements were made from pH 5-activated currents in CHO cells expressing ASIC1a (n = 12), ASIC2a (n = 5), and ASIC1 + ASIC2a (n = 13) and from ASIC2 +/+ (n = 25) and ASIC2 –/– (n = 17) hippocampal neurons. The asterisk indicates value different from 1.0, p < 0.02. B, recovery from desensitization was measured as shown in Fig. 5A by applying pH 5 for 10 s, pH 7.4 for 2 s, and then a second pH 5 application. We measured the percentage recovery with the second acid stimulus for 32 ASIC2 +/+ and 17 ASIC2 –/– neurons. The graph shows percentage current recovery (y axis) for a series of individual neurons (x axis). Each data point indicates an individual neuron; squares indicate ASIC2 +/+, and circles indicate ASIC2 –/– neurons.

 
We also tested for heterogeneity in the contribution of ASIC2a throughout the population of neurons. The rate of recovery from desensitization is one of the characteristics most strikingly influenced by ASIC2a; ASIC1a/2a heteromultimeric channels recover quickly, whereas ASIC1a homomultimeric channels recover more slowly. Therefore, we desensitized current with a 10-s pH 5 perfusion and then measured the percentage of current recovery 2 s after returning to a pH 7.4 solution. Wild-type neurons showed substantial variability (Fig. 6B). For example, within 2 s, some neurons had recovered ~20% of the initial current, whereas others had recovered all of the initial current. In contrast, when ASIC2 was eliminated, none of the neurons showed more than 25% recovery at 2 s. These results suggest that ASIC2a subunits contributed to the properties of H+-gated currents in the majority of neurons.

ASIC2b Is Expressed in Excess of ASIC2a in Neonatal Hippocampal Neurons—The results described above suggested a paradox. On one hand, only 43% of ASIC1 –/– neurons possessed transient currents evoked by pH 4. Yet, on the other hand, ASIC2a influenced the properties of acid-evoked currents in the vast majority of wild-type hippocampal neurons. If ASIC2a were present in most neurons, why were ASIC2a currents not more common in ASIC1 –/– neurons? A potential explanation for the discrepancy would be that ASIC1 disruption reduces ASIC2 expression. Although an earlier study showed that ASIC2 expression was the same in wild-type and ASIC1 –/– brains, hippocampal expression was not specifically measured (41). Using RT-PCR, we found ASIC2a and -2b transcripts in hippocampi of both wild-type and ASIC1 –/– animals (Fig. 7A). Northern analysis indicated that ASIC2 transcripts were present equally in wild-type and ASIC1 –/– hippocampi (Fig. 7B). Interestingly, the Northern blot also revealed that ASIC2b transcripts were much more abundant than ASIC2a transcripts. A greater abundance of ASIC2b mRNA compared with ASIC2a mRNA has also been reported in several other brain regions (34, 38, 39, 50). This observation raised the possibility that ASIC2b might affect ASIC2a current in ASIC1 –/– neurons.



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 7.
Effect of ASIC2b on ASIC2a-induced currents. A, RT-PCR from ASIC1 +/+ and ASIC1 –/– hippocampus using primers that detected both ASIC2a and -2b or either alone. B, Northern analysis of RNA from ASIC1 +/+ and ASIC1 –/– hippocampus using probes that recognize the indicated transcript. C, relationship between pH and H+-gated current in CHO cells expressing ASIC2a alone (n = 10–17) or ASIC2a and -2b at ratios of 1:1 (n = 9–14), 1:2 (n = 6–13), and 1:3 (n = 6–8). In all cases the amount of ASIC2a cDNA was constant, and pMT3 DNA was varied to maintain a constant amount of total DNA. Data were normalized to response to pH 3 stimulus. D, pH 3-induced current amplitude in cells expressing ASIC2a with different amounts of ASIC2b (n = 17–19).

 
ASIC2b subunits do not generate H+-gated currents when expressed alone, but they were reported to form heteromultimeric channels with ASIC2a (12, 42, 43). We reasoned that if ASIC2b were present in excess of ASIC2a, then in ASIC1 –/– neurons most channels would be ASIC2a/2b heteromultimers. To test the consequences of such a situation, we expressed ASIC2a with varying amounts of ASIC2b in CHO cells. Co-expressing ASIC2b with ASIC2a tended to reduce the pH sensitivity, although the effects were small (Fig. 7C); this result is consistent with earlier studies (12, 43). However, ASIC2b reduced current amplitude produced by pH 3 application (Fig. 7D). Moreover, as the relative amount of ASIC2b increased, current amplitude fell. These results may explain the low percentage of ASIC1 –/– neurons with H+-gated currents and their relatively small current amplitude. In those neurons, ASIC2b likely dampens current from channels mediated by ASIC2a, and perhaps currents were only observed in neurons expressing a higher proportion of ASIC2a.

ASIC1 and ASIC2 Heterozygote Neurons Show Altered H+-gated Currents—The contribution of ASIC1 and -2 subunits to H+-gated currents suggested that hippocampal neurons heterozygous for the ASIC1 and ASIC2 alleles might have altered acid-evoked currents. We found that compared with wild-type controls, the amplitude of pH 5-evoked currents fell by approximately half in ASIC1 +/– neurons but remained unchanged in ASIC2 +/– neurons (Fig. 8A). ASIC1 +/– neurons also showed an accelerated {tau}d and ASIC2 +/– neurons a slowed {tau}d (Fig. 8B). H+-gated currents from ASIC2 +/– neurons showed little shift in pH sensitivity, but recovery from desensitization was delayed (Fig. 8, C and D). Despite the dramatic reduction in current amplitude in ASIC1 +/– neurons (Fig. 8A), the pH dose-response and recovery from desensitization were not altered significantly (Fig. 8, C and D). Together, these results further support the conclusion that both ASIC1a and ASIC2 subunits influence acid-evoked currents in hippocampal neurons. ASIC1 seems to be important in determining current amplitude, whereas the predominant role of ASIC2 appears to be in influencing the properties of the current.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 8.
H+-gated currents from ASIC1 +/– and ASIC2 +/– neurons. A, average peak amplitude with pH 5 application in wild-type (WT; n = 10), ASIC1 +/– (n = 24), and ASIC2 +/– (n = 28) hippocampal neurons. The asterisk indicates p < 0.002 versus wild-type. B, desensitization rate ({tau}d) for wild-type (n = 24), ASIC1 +/– (n = 27), and ASIC2 +/– (n = 27) hippocampal neurons. The asterisk indicates p < 0.005 versus wild-type. C, relationship between pH and H+-gated current from wild-type (n = 13–17), ASIC1 +/– (n = 10–13), and ASIC2 +/– (n = 14–16) hippocampal neurons. D, recovery from desensitization of wild-type (n = 16–18), ASIC1 +/– (n = 12–15), and ASIC2 +/– (n = 14–17) neurons.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It has been known for many years that acidic extracellular solutions activate transient cation currents in hippocampal neurons (7). The biophysical properties of those currents (8) and the expression of ASIC1a and ASIC2 subunits in the hippocampus (4, 12, 26, 28, 38, 39) suggested that ASIC subunits were responsible. ASIC subunits might generate H+-gated currents in two general ways. ASIC1a and ASIC2a could each form homomultimeric channels, with net current representing the sum of the individual currents. Alternatively, the subunits could combine to form heteromultimeric channels. Our data indicate that at least two ASIC subunits, ASIC1a and -2a, contribute to H+-activated currents in mouse hippocampal neurons. They also suggest that both heteromultimeric and homomultimeric channels generate current, and individual ASIC subunits make distinct contributions.

Hippocampal Neurons Contain Both Hetero- and Homo-multimeric ASIC Channels—Heteromultimeric ASIC1a/2a channels produced H+-gated current in cultured hippocampal neurons. Support for this conclusion came from the finding that {tau}d in wild-type neurons exceeded that in both ASIC1 –/– and ASIC2 –/– neurons and in heterologous cells expressing either ASIC1a or -2a alone. Thus, the {tau}d could not be explained by the sum of currents from homomultimeric channels. FRRFamide addition also revealed responses that could not be attributed to an aggregate of homomultimeric channels. Although FRRF-amide does not alter ASIC2a currents, it produced a greater response in wild-type neurons and in heterologous cells expressing ASIC1a/2a heteromultimeric channels than in ASIC2 –/– neurons or cells expressing ASIC1a homomultimers alone. These results indicate that ASIC1a/2a channels have unique properties compared with homomultimeric channels, and those channels generate H+-gated currents in hippocampal neurons. An advantage for making this conclusion is that our studies used neurons with specific gene disruptions. Thus, they do not depend on comparing absolute values of channel properties in neurons with those of ASICs expressed in heterologous cells, where values could vary depending on the cell type and associated proteins. Nevertheless, there was complete qualitative agreement between data with neurons and recombinant channels, and the quantitative responses were very similar.

Brain also expresses two other DEG/ENaC subunits, ASIC4 (31, 32) and BLINaC (brain-liver-intestine amiloride-sensitive Na+ channel) (51, 52). However, ASIC4 neither generated H+-gated currents on its own nor affected currents produced by other ASICs (31). BLINaC bearing a gain-of-function mutation generated a sustained Na+ current, but the wild-type protein did not generate current or alter other DEG/ENaC subunits (51). Although recombinant ASIC3 can generate H+-gated currents, it shows little, if any, expression in the mouse brain (15, 16, 36, 53). Thus, ASIC3, ASIC4, and BLINaC would not be expected to influence the properties of hippocampal acid-evoked currents. Although our results cannot exclude the possibility that other DEG/ENaC subunits contribute to H+-gated hippocampal currents in unexpected ways, invoking a contribution from subunits other than ASIC1a, -2a, and -2b is not required to explain currents in neurons from wild-type, ASIC1 –/–, or ASIC2 –/– mice. The contribution of the various DEG/ENaC subunits to sustained H+-gated neuronal currents is also uncertain. Studying mice with additional targeted gene disruptions might reveal such contributions.

By forming heteromultimeric ASIC channels, hippocampal neurons follow a pattern found in other cells expressing DEG/ENaC subunits. Epithelial cells form heteromultimeric channels from {alpha}, {beta}, and {gamma} ENaC subunits (54–56). Caenorhabditis elegans neurons form channels from the combination of Mec4 and Mec10 (57), and dorsal root ganglion neurons combine ASIC1, -2, and -3 subunits to generate H+-gated currents (24, 33). However, differences between subunit desensitization and recovery from desensitization suggested the presence of ASIC1a homomultimeric channels in hippocampal neurons. This conclusion is supported by the observation that the peptide toxin PCTX1 reduced H+-gated current in central nervous system neurons (8). Expression of both heteromultimeric and homomultimeric channels has not been reported in other cell types expressing more than one DEG/ENaC subunit. This finding suggests the opportunity for hippocampal neurons to construct channels with a complex variety of subunit compositions and characteristics.

ASIC Subunits Confer Distinct Properties on Hippocampal Acid-evoked Currents—ASIC1a and -2a subunits made distinct contributions to H+-activated hippocampal currents. ASIC1a seemed to be important in establishing current amplitude. Eliminating ASIC1 from neurons abolished pH 5-induced current, and its absence reduced average pH 4-induced current by ~95%. Most convincingly, removing one ASIC1 allele approximately halved H+-gated current.

In contrast, ASIC2 –/– and +/– neurons generated the same current amplitude as wild-type neurons, indicating that ASIC2 plays a minor role in determining this quantitative aspect of H+-gated current. Instead, ASIC2 influenced the biophysical properties, defining, in part, acid sensitivity, desensitization, the rate of recovery from desensitization, and the response to neuropeptide modulators and zinc. A predominantly modulatory role for ASIC2a subunits may be analogous to the contributions of some {gamma}-aminobutyric acid-A receptor subunits to that channel complex (58).

The contribution of ASIC2b is less certain. Earlier studies reported that co-expressing ASIC2b with ASIC1a had no effect on H+-gated currents (12). When expressed with ASIC2a, ASIC2b reduced pH sensitivity to a small extent (12, 43), consistent with our results. In addition, our data indicate that ASIC2b reduced ASIC2a current amplitude in response to a maximal stimulus. This finding, together with a greater relative abundance of ASIC2b versus -2a transcripts in the hippocampus, provides a potential explanation for the minimal H+-gated current in ASIC1 –/– neurons. An alternative factor that we cannot exclude, but that could influence current properties, is the possibility that other channels or proteins might differentially regulate ASICs in null and wild-type animals (59).

Contribution of ASIC to Function of Hippocampal Neurons— Specific ASIC subunits make distinct contributions to hippocampal acid-evoked currents, and there is heterogeneity in currents within and between individual neurons. This variability could influence the contribution of ASICs to normal physiology and to pathophysiology. For example, in the peripheral nervous system relatively small changes in H+-gated currents alter sensory transduction (34–36). Moreover, in the mouse brain, disrupting ASIC1 impaired synaptic plasticity and performance in several behavioral tests (40, 41). Pathologic conditions may also skew ASIC levels. For example, ischemia is reported to increase ASIC2a levels (60); such an increase could reduce the pH sensitivity of acid-evoked currents, making them less responsive to an acid insult. Following seizures, the relative contribution of various subunits to the complex may also change (50), perhaps altering the responsiveness of hippocampal neurons to seizure-associated acidosis. In addition, if mutations in ASIC genes are discovered in humans, our data suggest that haplo-insufficiency could have a substantial impact on H+-gated currents and perhaps behavior. Finally, these results suggest that molecules targeting specific ASIC subunits might have value as therapeutics.


   FOOTNOTES
 
* This work was supported in part by a Veterans Administration Research Career Development Award (to J. A. W.) and the Howard Hughes Medical Institute Biomedical Research Support Program (to J. A. W.). The In Vitro Models and Cell Culture Core were supported by the NHLBI, National Institutes of Health (Grant HL61234), the Cystic Fibrosis Foundation (Grants ENGLH9850 and R458-CR02), and the NIDDK, National Institutes of Health (Grant DK54759). 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

Associate of the Howard Hughes Medical Institute. Present address: Dept. of Neuroscience, The Ohio State University, Columbus, OH 42310. Back

§§ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Inst., Roy J. and Lucille A. Carver College of Medicine, 500 EMRB, Iowa City, IA 52242. Tel.: 319-335-7619; Fax: 319-335-7623; E-mail: michael-welsh{at}uiowa.edu.

1 The abbreviations used are: ASIC, acid-sensing ion channel; CHO, Chinese hamster ovary; RT, reverse transcriptase. Back


   ACKNOWLEDGMENTS
 
We thank Ejvis Lamani, Lisa Momchilov, and Theresa Mayhew for excellent assistance. We thank Phil Karp, Pary Weber, and Tami Nesselhauf of the University of Iowa. We thank the University of Iowa Animal Care Unit.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Chesler, M., and Kaila, K. (1992) Trends Neurosci. 15, 396–402[CrossRef][Medline] [Order article via Infotrieve]
  2. Miesenbock, G., De Angelis, D. A., and Rothman, J. E. (1998) Nature 394, 192–195[CrossRef][Medline] [Order article via Infotrieve]
  3. Krishtal, O. A., Osipchuk, Y. V., Shelest, T. N., and Smirnoff, S. V. (1987) Brain Res. 436, 352–356[CrossRef][Medline] [Order article via Infotrieve]
  4. Waldmann, R., Champigny, G., Bassilana, F., Heurteaux, C., and Lazdunski, M. (1997) Nature 386, 173–177[CrossRef][Medline] [Order article via Infotrieve]
  5. Siesjo, B. K. (1988) Neurochem. Pathol. 9, 31–88[Medline] [Order article via Infotrieve]
  6. Varming, T. (1999) Neuropharm. 38, 1875–1881[CrossRef][Medline] [Order article via Infotrieve]
  7. Vyklicky, L. J., Vlachova, V., and Krusek, J. (1990) J. Physiol. (Lond.) 430, 497–517[Abstract/Free Full Text]
  8. Baron, A., Waldmann, R., and Lazdunski, M. (2002) J. Physiol. 539, 485–494[Abstract/Free Full Text]
  9. Welsh, M. J., Price, M. P., and Xie, J. (2002) J. Biol. Chem. 277, 2369–2372[Free Full Text]
  10. Bianchi, L., and Driscoll, M. (2002) Neuron 34, 337–340[CrossRef][Medline] [Order article via Infotrieve]
  11. Champigny, G., Voilley, N., Waldmann, R., and Lazdunski, M. (1998) J. Biol. Chem. 273, 15418–15422[Abstract/Free Full Text]
  12. Lingueglia, E., de Weille, J. R., Bassilana, F., Heurteaux, C., Sakai, H., Waldmann, R., and Lazdunski, M. (1997) J. Biol. Chem. 272, 29778–29783[Abstract/Free Full Text]
  13. de Weille, J. R., Bassilana, F., Lazdunski, M., and Waldmann, R. (1998) FEBS Lett. 433, 257–260[CrossRef][Medline] [Order article via Infotrieve]
  14. Babinski, K., Catarsi, S., Biagini, G., and Seguela, P. (2000) J. Biol. Chem. 275, 28519–28525[Abstract/Free Full Text]
  15. Babinski, K., Le, K. T., and Séguéla, P. (1999) J. Neurochem. 72, 51–57[CrossRef][Medline] [Order article via Infotrieve]
  16. Waldmann, R., Bassilana, F., de Weille, J. R., Champigny, G., Heurteaux, C., and Lazdunski, M. (1997) J. Biol. Chem. 272, 20975–20978[Abstract/Free Full Text]
  17. Chen, C. C., England, S., Akopian, A. N., and Wood, J. N. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 10240–10245[Abstract/Free Full Text]
  18. Bassler, E. L., Ngo-Anh, T. J., Geisler, H. S., Ruppersberg, J. P., and Grunder, S. (2001) J. Biol. Chem. 276, 33782–33787[Abstract/Free Full Text]
  19. Escoubas, P., De Weille, J. R., Lecoq, A., Diochot, S., Waldmann, R., Champigny, G., Moinier, D., Menez, A., and Lazdunski, M. (2000) J. Biol. Chem. 275, 25116–25121[Abstract/Free Full Text]
  20. Askwith, C. C., Cheng, C., Ikuma, M., Benson, C. J., Price, M. P., and Welsh, M. J. (2000) Neuron 26, 133–141[CrossRef][Medline] [Order article via Infotrieve]
  21. Deval, E., Baron, A., Lingueglia, E., Mazarguil, H., Zajac, J. M., and Lazdunski, M. (2003) Neuropharmacology 44, 662–671[CrossRef][Medline] [Order article via Infotrieve]
  22. de Weille, J., and Bassilana, F. (2001) Brain Res. 900, 277–281[CrossRef][Medline] [Order article via Infotrieve]
  23. Immke, D. C., and McCleskey, E. W. (2001) Nat. Neurosci. 4, 869–870[CrossRef][Medline] [Order article via Infotrieve]
  24. Benson, C. J., Xie, J., Wemmie, J. A., Price, M. P., Henss, J. M., Welsh, M. J., and Snyder, P. M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 2338–2343[Abstract/Free Full Text]
  25. Berdiev, B. K., Xie, J., McLean, J. A., Markert, J. M., Gillespie, G. Y., Mapstone, T. B., Naren, A. P., Jovov, B., Bubien, J. K., Ji, H. L., Fuller, C. M., Kirk, K. L., and Benos, D. J. (2003) J. Biol. Chem. 278, 15023–15034[Abstract/Free Full Text]
  26. Hesselager, M., Timmermann, D. B., and Ahring, P. K. (2004) J. Biol. Chem. 279, 11006–11015[Abstract/Free Full Text]
  27. Zhang, P., and Canessa, C. M. (2001) J. Gen. Physiol. 117, 563–572[Abstract/Free Full Text]
  28. Bassilana, F., Champigny, G., Waldmann, R., de Weille, J. R., Heurteaux, C., and Lazdunski, M. (1997) J. Biol. Chem. 272, 28819–28822[Abstract/Free Full Text]
  29. Catarsi, S., Babinski, K., and Seguela, P. (2001) Neuropharm. 41, 592–600[CrossRef][Medline] [Order article via Infotrieve]
  30. Baron, A., Schaefer, L., Lingueglia, E., Champigny, G., and Lazdunski, M. (2001) J. Biol. Chem. 276, 35361–35367[Abstract/Free Full Text]
  31. Grunder, S., Geissler, H. S., Bassler, E. L., and Ruppersberg, J. P. (2000) Neuroreport 11, 1607–1611[Medline] [Order article via Infotrieve]
  32. Akopian, A. N., Chen, C. C., Ding, Y., Cesare, P., and Wood, J. N. (2000) Neuroreport 11, 2217–2222[Medline] [Order article via Infotrieve]
  33. Xie, J., Price, M. P., Berger, A. L., and Welsh, M. J. (2002) J. Neurophysiol. 87, 2835–2843[Abstract/Free Full Text]
  34. Price, M. P., Lewin, G. B., McIlwrath, S. L., Cheng, C., Xie, J., Heppenstall, P. A., Stucky, C. L., Mannsfeldt, A. G., Brennan, T. J., Drummond, H. A., Qiao, J., Benson, C. J., Tarr, D. E., Hrstka, R. F., Yang, B., Williamson, R. A., and Welsh, M. J. (2000) Nature 407, 1007–1011[CrossRef][Medline] [Order article via Infotrieve]
  35. Price, M. P., McIllwrath, S. L., Xie, J., Cheng, C., Qiao, J., Tarr, D. E., Sluka, K. A., Brennan, T. J., Lewin, G. R., and Welsh, M. J. (2001) Neuron 32, 1071–1083[CrossRef][Medline] [Order article via Infotrieve]
  36. Chen, C. C., Zimmer, A., Sun, W. H., Hall, J., and Brownstein, M. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 8992–8997[Abstract/Free Full Text]
  37. Sluka, K. A., Price, M. P., Breese, N. M., Stucky, C. L., Wemmie, J. A., and Welsh, M. J. (2003) Pain 106, 229–239[CrossRef][Medline] [Order article via Infotrieve]
  38. Price, M. P., Snyder, P. M., and Welsh, M. J. (1996) J. Biol. Chem. 271, 7879–7882[Abstract/Free Full Text]
  39. García-Añoveros, J., Derfler, B., Neville-Golden, J., Hyman, B. T., and Corey, D. P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1459–1464[Abstract/Free Full Text]
  40. Wemmie, J. A., Askwith, C. C., Lamani, E., Cassell, M. D., Freeman, J. H. J., and Welsh, M. J. (2003) J. Neurosci. 23, 5496–5502[Abstract/Free Full Text]
  41. Wemmie, J. A., Chen, J., Askwith, C. C., Hruska-Hageman, A. M., Price, M. P., Nolan, B. C., Yoder, P. G., Lamani, E., Hoshi, T., Freeman, J. H. J., and Welsh, M. J. (2002) Neuron 34, 463–477[CrossRef][Medline] [Order article via Infotrieve]
  42. Ugawa, S., Ueda, T., Takahashi, E., Hirabayashi, Y., Yoneda, T., Komai, S., and Shimada, S. (2001) Neuroreport 12, 2865–2869[CrossRef][Medline] [Order article via Infotrieve]
  43. Ueda, T., Ugawa, S., Yamamura, H., Imaizumi, Y., and Shimada, S. (2003) J. Neurosci. 23, 7376–7380[Abstract/Free Full Text]
  44. Mennerick, S., Que, J., Benz, A., and Zorumski, C. F. (1995) J. Neurophysiol. 73, 320–332[Abstract/Free Full Text]
  45. Berdiev, B. K., Mapstone, T. B., Markert, J. M., Gillespie, G. Y., Lockhart, J., Fuller, C. M., and Benos, D. J. (2001) J. Biol. Chem. 276, 38755–38761[Abstract/Free Full Text]
  46. Akaike, N., Krishtal, O. A., and Maruyama, T. (1990) J. Neurophysiol. 63, 805–813[Abstract/Free Full Text]
  47. Akaike, N., and Ueno, S. (1994) Prog. Neurobiol. 43, 73–83[CrossRef][Medline] [Order article via Infotrieve]
  48. Alvarez de la Rosa, D., Krueger, S. R., Kolar, A., Shao, D., Fitzsimonds, R. M., and Canessa, C. M. (2003) J. Physiol. 546, 77–87[Abstract/Free Full Text]
  49. Jovov, B., Tousson, A., McMahon, L. L., and Benos, D. J. (2003) Histochem. Cell Biol. 119, 437[CrossRef][Medline] [Order article via Infotrieve]
  50. Biagini, G., Babinski, K., Avoli, M., Marcinkiewicz, M., and Seguela, P. (2001) Neurobiol. Dis. 8, 45–58[CrossRef][Medline] [Order article via Infotrieve]
  51. Sakai, H., Lingueglia, E., Champigny, G., Mattei, M. G., and Lazdunski, M. (1999) J. Physiol. (Lond.) 519, 323–333[Abstract/Free Full Text]
  52. Schaefer, L., Sakai, H., Mattei, M., Lazdunski, M., and Lingueglia, E. (2000) FEBS Lett. 471, 205–210[CrossRef][Medline] [Order article via Infotrieve]
  53. Ishibashi, K., and Marumo, F. (1998) Biochem. Biophys. Res. Commun. 245, 589–593[CrossRef][Medline] [Order article via Infotrieve]
  54. McDonald, F. J., Price, M. P., Snyder, P. M., and Welsh, M. J. (1995) Am. J. Physiol. 268, C1157–C1163
  55. Canessa, C. M., Schild, L., Buell, G., Thorens, B., Gautschi, I., Horisberger, J. D., and Rossier, B. C. (1994) Nature 367, 463–467[CrossRef][Medline] [Order article via Infotrieve]
  56. Kosari, F., Sheng, S., Li, J., Mak, D. O. D., Foskett, J. K., and Kleyman, T. R. (1998) J. Biol. Chem. 273, 13469–13474[Abstract/Free Full Text]
  57. Huang, M., and Chalfie, M. (1994) Nature 367, 467–470[CrossRef][Medline] [Order article via Infotrieve]
  58. Mehta, A. K., and Ticku, M. K. (1999) Brain Res. Rev. 29, 196–217[CrossRef][Medline] [Order article via Infotrieve]
  59. Ji, H. L., Jovov, B., Fu, J., Bishop, L. R., Mebane, H. C., Fuller, C. M., Stanton, B. A., and Benos, D. J. (2002) J. Biol. Chem. 277, 8395–8405[Abstract/Free Full Text]
  60. Johnson, M. B., Jin, K., Minami, M., Chen, D., and Simon, R. P. (2001) J. Cereb. Blood Flow Metab. 21, 734–740[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg