The Acid-sensitive Ionic Channel Subunit ASIC and the Mammalian Degenerin MDEG Form a Heteromultimeric H+-gated Na+ Channel with Novel Properties

Proton-gated cation channels are acid sensors that are present in both sensory neurons and in neurons of the central nervous system. One of these acid-sensing ion channels (ASIC) has been recently cloned. This paper shows that ASIC and the mammalian degenerin MDEG, which are colocalized in the same brain regions, can directly associate with each other. Immunoprecipitation of MDEG causes coprecipitation of ASIC. Moreover, coexpression of ASIC and MDEG subunits in Xenopus oocytes generates an amiloride-sensitive H+-gated Na+ channel with novel properties (different kinetics, ionic selectivity, and pH sensitivity). In addition, coexpression of MDEG with mutants of the ASIC subunit can create constitutively active channels that become completely nonselective for Na+ versusK+ and H+-gated channels that have a drastically altered pH sensitivity compared with MDEG. These data clearly show that ASIC and MDEG can form heteromultimeric assemblies with novel properties. Heteromultimeric assembly is probably used for creating a diversity of H+-gated cation channels acting as neuronal acid sensors in different pH ranges. Proton-gated cation channels are acid sensors that are present in both sensory neurons and in neurons of the central nervous system. One of these acid-sensing ion channels (ASIC) has been recently cloned. This paper shows that ASIC and the mammalian degenerin MDEG, which are colocalized in the same brain regions, can directly associate with each other. Immunoprecipitation of MDEG causes coprecipitation of ASIC. Moreover, coexpression of ASIC and MDEG subunits in Xenopus oocytes generates an amiloride-sensitive H+-gated Na+ channel with novel properties (different kinetics, ionic selectivity, and pH sensitivity). In addition, coexpression of MDEG with mutants of the ASIC subunit can create constitutively active channels that become completely nonselective for Na+ versusK+ and H+-gated channels that have a drastically altered pH sensitivity compared with MDEG. These data clearly show that ASIC and MDEG can form heteromultimeric assemblies with novel properties. Heteromultimeric assembly is probably used for creating a diversity of H+-gated cation channels acting as neuronal acid sensors in different pH ranges. H+-gated cation channels are ligand-gated ion channels activated by the simplest possible ligand, the proton. In nociceptive neurons those channels are thought to be responsible for the sensation of pain that accompanies tissue acidosis (1Bevan S. Yeats J. J. Physiol. (Lond .). 1991; 433: 145-161Crossref Scopus (301) Google Scholar, 2Akaike N. Ueno S. Prog. Neurobiol. 1994; 43: 73-83Crossref PubMed Scopus (50) Google Scholar, 3Krishtal O.A. Pidoplichko V.I. Neuroscience. 1981; 6: 2599-2601Crossref PubMed Scopus (153) Google Scholar), particularly during inflammation and ischemic conditions. H+-gated cation channels are also present in neurons of the central nervous system (2Akaike N. Ueno S. Prog. Neurobiol. 1994; 43: 73-83Crossref PubMed Scopus (50) Google Scholar), where their physiological role remains to be established. We have recently cloned a proton-gated cation channel (ASIC,acid sensing ionchannel 1) (4Waldmann R. Champigny G. Bassilana F. Heurteaux C. Lazdunski M. Nature. 1997; 386: 173-177Crossref PubMed Scopus (1127) Google Scholar). The closest structural homologue of ASIC is MDEG (5Waldmann R. Champigny G. Voilley N. Lauritzen I. Lazdunski M. J. Biol. Chem. 1996; 271: 10433-10434Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 6Price M.P. Snyder P.M. Welsh M.J. J. Biol. Chem. 1996; 271: 7879-7882Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). MDEG is a mammalian degenerin. Upon the same mutations that in Caenorhabditis elegans degenerins induce degeneration of specific neurons (7Chalfie M. Wolinsky E. Nature. 1990; 345: 410-416Crossref PubMed Scopus (258) Google Scholar, 8Driscoll M. Chalfie M. Nature. 1991; 349: 588-593Crossref PubMed Scopus (454) Google Scholar), MDEG can also acquire constitutive Na+ channel activity that becomes toxic for the cells in which it is expressed (5Waldmann R. Champigny G. Voilley N. Lauritzen I. Lazdunski M. J. Biol. Chem. 1996; 271: 10433-10434Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). In addition, native MDEG also behaves as a H+-gated Na+ channel. The variety of H+-gated cation channels with different ion selectivities, pH dependencies, and kinetics described in sensory neurons (1Bevan S. Yeats J. J. Physiol. (Lond .). 1991; 433: 145-161Crossref Scopus (301) Google Scholar, 9Krishtal O.A. Pidoplichko V.I. Brain Res. 1981; 214: 150-154Crossref PubMed Scopus (69) Google Scholar, 10Davies N.W. Lux H.D. Morad M. J. Physiol. (Lond .). 1988; 400: 159-187Crossref Scopus (74) Google Scholar), as well as in neurons of the central nervous system (2Akaike N. Ueno S. Prog. Neurobiol. 1994; 43: 73-83Crossref PubMed Scopus (50) Google Scholar), suggests that ASIC and MDEG are probably only the first two members of this novel ion channel family and that other genes for new H+-activated Na+ channels remain to be discovered. However, the existence of different genes is probably only one of the ways used to create a diversity of H+-gated cation channels. Many other types of ion channels are known to form heteromultimers of structurally related subunits, resulting in the assembly of channels with novel properties (11Barnard E.A. Trends Pharmacol. Sci. 1996; 17: 305-309Abstract Full Text PDF PubMed Google Scholar). Furthermore, other structural homologues of the ASIC and MDEG channels, such as the epithelial amiloride-sensitive Na+ channel (12Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1764) Google Scholar, 13Canessa C.M. Horisberger J.D. Rossier B.C. Nature. 1993; 361: 467-470Crossref PubMed Scopus (824) Google Scholar, 14Lingueglia E. Renard S. Waldmann R. Voilley N. Champigny G. Plass H. Lazdunski M. Barbry P. J. Biol. Chem. 1994; 269: 13736-13739Abstract Full Text PDF PubMed Google Scholar, 15Lingueglia E. Voilley N. Waldmann R. Lazdunski M. Barbry P. FEBS Lett. 1993; 318: 95-99Crossref PubMed Scopus (316) Google Scholar, 16Waldmann R. Champigny G. Bassilana F. Voilley N. Lazdunski M. J. Biol. Chem. 1995; 270: 27411-27414Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 17Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1035) Google Scholar, 18Garty H. FASEB J. 1994; 8: 522-528Crossref PubMed Scopus (113) Google Scholar) and the degenerins of the nematode C. elegans, which are believed to be mechanosensitive channels (19Huang M. Chalfie M. Nature. 1994; 367: 467-470Crossref PubMed Scopus (343) Google Scholar, 20Gu G. Caldwell G.A. Chalfie M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6577-6582Crossref PubMed Scopus (91) Google Scholar, 21Herman R.K. Bioessays. 1996; 18: 199-206Crossref PubMed Scopus (10) Google Scholar), require heteromultimeric subunit assembly for their function. All these considerations taken together suggest that new channels might be formed by heteromultimers of ASIC and MDEG subunits. This paper provides both electrophysiological and biochemical evidence that ASIC, together with MDEG, form heteromultimeric channels with properties distinct from those of the parental subunits. Experiments were performed on adult Wistar rats by using standard procedures (4Waldmann R. Champigny G. Bassilana F. Heurteaux C. Lazdunski M. Nature. 1997; 386: 173-177Crossref PubMed Scopus (1127) Google Scholar). Brains were fixed in ice-cold 4% (w/v) paraformaldehyde, 0.1 m sodium phosphate buffer solution (PBS, 1The abbreviations used are: PBS, phosphate-buffered saline; BSA, bovine serum albumin. pH 7.4) for 8 h and then immersed overnight at 4 °C in a 20% sucrose/PBS solution. Frozen sections (10 μm) were cut on a cryostat (Leica) at −25 °C, collected on 3-aminopropylethoxysilane-coated slides and stored at −20 °C until use. Four antisense oligonucleotides, complementary to the rat cDNA sequences of ASIC and MDEG, were used to detect ASIC and MDEG transcripts, respectively. The sequences of the oligonucleotides were: ASIC, 5′-GGTGTAGAAGAGGATACTCGCCGACAGAGACTTC-3′ or 5′-AACAGACACCACTCTTTCCTCAGAACTGGTATCTCAGGA-3′ and MDEG, 5′-ATGAGCACCTGTGACACAATGCCAAACCAC3′ or 5′-GTCTACATGTTTCGTGGACGTACGTACGAGTCTGAAGAATGTCGC-3′. Probes were 3′-end-labeled with α-33PdATP (3000 Ci/mmol, ICN Radiochemicals) by terminal deoxynucleotidyltransferase. Sections were treated consecutively with 0.1 m glycine in PBS for 10 min, PBS for 3 min, 5 μg/ml proteinase K diluted in 0.1m Tris, 50 mm EDTA (pH 8.0) for 15 min at 37 °C, 4% paraformaldehyde/PBS (pH 7.2) for 5 min. Slides were then rinsed 10 min in PBS, acetylated for 10 min in 0.25% acetic anhydride in 0.1 m triethanolamine, and dehydrated. Hybridization was carried out overnight at 37 °C in hybridization buffer (50% deionized formamide, 10% dextran sulfate, 500 μg/ml denatured salmon sperm DNA, 1% Denhardt, 5% Sarcosyl, 250 mg/ml yeast tRNA, 20 mm dithiothreitol, 20 mm NaPO4 in 2 × SSC, and the radiolabeled probe (0.2 ng/ml, 8 × 108 dpm/μg)). After hybridization, slides were washed in 1 × SSC at room temperature for 30 min before dehydration, drying, and exposure to Hyperfilm-βmax (Amersham Corp.) for 6 days. Slides were then dipped in Ilford K5 nuclear emulsion (diluted 1:1 with water) and exposed 4 weeks. Sections were then stained with cresyl violet and coverslipped. The specificity of labeling was verified by cold displacement of the radioactive probe with a 500-fold excess of unlabeled oligonucleotide and by the use of two specific oligonucleotide probes for each subunit complementary to nonconserved regions in either the coding or the noncoding sequences of the cDNAs. Primers were designed to add the FlagM2 epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (Eastman Kodak Co.) to the NH2 terminus of MDEG and the T7tag epitope (Met-Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly) (Novagen) to the NH2 terminus of ASIC. The tagged ASIC and MDEG coding sequences were amplified by polymerase chain reaction and subcloned in the pVL 1393 vector (PharMingen). SF9 cells were transformed with either pVL-ASIC-T7tag or pVL-MDEG-Flag vector, and recombinant baculovirus was isolated according to the “baculoGold” protocol (PharMingen). 7 × 106 SF9 cells were infected with 108 recombinant baculovirus particles (ASIC-T7tag, MDEG-Flag, or both). Four days later, cells were harvested, washed in PBS, sonicated at 4 °C in solubilization buffer (50 mm Tris-Cl (pH 7.5), 150 mm NaCl, 1% Triton X-100, 0, 1% desoxycholate, 1 mmphenylmethylsulfonyl fluoride, 0.1 mm iodoacetamide) and subsequently centrifuged for 30 min at 80,000 × g. An aliquot of the supernatant containing 50 μg of protein was incubated overnight with 10 μl of M2 anti-Flag mouse monoclonal antibody immobilized on Sepharose (Eastman Kodak Co.). After a 30 s 8000 × g spin, the supernatant was saved and the pellet was washed six times in 500 μl of solubilization buffer and twice with PBS. Both supernatant and pellet were resolved by SDS-polyacrylamide gel electrophoresis (9% acrylamide) and transferred onto nitrocellulose membranes (Hybond C extra Amersham). The blots were saturated 30 min with TBST (10 mm Tris-Cl pH 7.5, 50 mm NaCl, 0, 1% Tween 20) containing 4% BSA, then incubated overnight at 4 °C with either M2 anti-Flag antibody (anti MDEG-Flag) or anti-T7tag monoclonal antibody (anti-ASIC-T7tag) in TBST containing 2% BSA. After 5 washes with TBST, the blots were incubated with goat anti-mouse horseradish peroxidase-conjugated secondary antibodies (Jackson Immunotech) 1:10,000 in TBST containing 2% BSA for 1 h at room temperature, washed with TBST and revealed using a substrate for enhanced chemiluminescence detection (ECL, Pierce). Oocytes were injected with 0.1 or 0.25 ng of cRNA and microelectrode voltage-clamp and patch-clamp recordings were performed two days after injection. For outside-out patch the pipettes contained: 140 mm KCl, 2 mmMgCl2, 5 mm EGTA, 10 mm Hepes (pH 7.4) (with KOH). The bath medium contained: 140 mm NaCl (or 140 mm LiCl or 140 mm N-methyl-d-glucamine chloride), 2 mmMgCl2, 1.8 mm CaCl2, 10 mm Hepes (pH 7.4) (adjusted with HCl, NaOH, LiOH, or tetramethyl ammonium hydroxide). Rapid pH changes from the initial pH of 7.4 were produced by rapid perfusion with bath solution adjusted to the pH values indicated in Figs. 2, 3, 4. Data were sampled at 2 kHz and filtered at 500 Hz for analysis (Biopatch software).Figure 3Selectivity and single channel conductance of the ASIC/MDEG channel. A, H+-activated currents recorded at various potentials from an outside-out patch bathed in Na+ containing solution. B, meanQ-V relationships of the H+-gated current recorded in Na+ rich solution (reversal potential = +50 mV), Li+-rich solutions (reversal potential = +50 mV) and Na+-free solution containing 1.8 mm Ca2+ (reversal potential = −75 mV). C, outside-out multichannel currents recorded after a pH pulse from pH 7.4 to pH 5. Current levels are indicated byhorizontal dashed lines. D, I-Vrelationship with Na+ as conducting ion. E, properties of ASIC, MDEG, and ASIC + MDEG channels. Permeability ratios and single channel conductances for Na+ were determined from outside-out patches. pS, picosiemens.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Effect of ASIC mutations on the activity of the ASIC and ASIC/MDEG channel. A, histogram of the mean basal currents at pH 7.4 measured at −70 mV. B andC, mean I-V relationships of the whole oocyte currents measured by voltage ramps from −150 mV to +100 mV at pH 7.4. D, current responses induced by a drop of pH from 7.4 to 5 recorded from whole oocytes. E, pH dependence of the H+-activated ASIC(G431V)/MDEG and ASIC(G431F)/MDEG currents. Points represent mean values from 15 oocytes. The added curve indicates the pH dependence of MDEG for comparison.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The in situ experiments performed on whole brain sections shows a widely overlapping expression of ASIC and MDEG mRNAs (Fig. 1 A). The highest expression levels were detected in the olfactory bulb, neo and allocortical regions, dentate granule cells, as well as in pyramidal cells of CA1–CA3 subfields of the hippocampal formation, habenula, basolateral amygdaloid nuclei, and in the Purkinje and granule cells of the cerebellum. A diffuse expression was observed over most other regions of the basal ganglia, including thalamic nuclei, substantia nigra, striatum and globus pallidus, hypothalamus, midbrain, pons, and medulla. ASIC and MDEG transcripts were expressed at low levels in choroid plexus. Coexpression of both transcripts in the same type of neurons was observed in all regions of gray matter, which are strongly labeled. Fig. 1 A, panels c–d, show an example of the high degree of colocalization of ASIC and MDEG in the Purkinje and granule cells of the cerebellar cortex. These results are consistent with observations made by others (22Garcia-Añoveros J. Derfler B. Neville-Golden J. Hyman B.T. Corey D.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1459-1464Crossref PubMed Scopus (291) Google Scholar). This colocalization led us to analyze whether MDEG and ASIC could interact to form a proton-activated channel and whether this new channel had different properties from those of the parental subunits expressed alone. The direct demonstration of an association of ASIC and MDEG was obtained from co-immunoprecipitation experiments. ASIC and MDEG were tagged with short epitopes (“T7tag™” and “Flag™”) introduced at their NH2 terminus. The tagged proteins ASIC-tag and MDEG-Flag were expressed either alone or coexpressed in SF9 cells using a baculovirus system. Electrophysiological analysis of the infected cells was carried out to show that the introduction of the epitope does not alter the channel properties (data not shown). The detergent solubilized membranes of SF9 cells expressing either ASIC-tag or MDEG-Flag or both subunits were immunoprecipitated with an anti-Flag(MDEG) antibody, and both the immunoprecipitated fractions and the supernatants were analyzed by Western blot. Analysis of the blots with the anti-Flag(MDEG) antibody (Fig. 1 B, top panel) revealed that at least 50% of MDEG-Flag was immunoprecipitated (lane 2) and that the anti-Flag(MDEG) antibody did not cross-react with ASIC-tag (lane 1). We then incubated the same blots with an anti-tag(ASIC) antibody (Fig. 1 B, bottom panel) to analyze whether immunoprecipitation of MDEG-Flag caused coprecipitation of ASIC-tag. The anti-tag(ASIC) antibody did not cross-react with MDEG-Flag, since a signal was only obtained with samples that contained ASIC-tag. When both ASIC-tag and MDEG-Flag were co-expressed in the same cells, immunoprecipitation with the anti-Flag(MDEG) antibody caused co-precipitation of ASIC-tag (lane 3), indicating that ASIC-tag associates with MDEG-Flag to form a heteromultimeric channel. The heteromultimeric association occurs in vivo and not after solubilization of the cells. No co-immunoprecipitation of ASIC-tag by the anti-Flag(MDEG) antibody was used a of membranes from cells expressing either ASIC-tag or MDEG-Flag (lane bottom Both ASIC (4Waldmann R. Champigny G. Bassilana F. Heurteaux C. Lazdunski M. Nature. 1997; 386: 173-177Crossref PubMed Scopus (1127) Google Scholar) and MDEG (5Waldmann R. Champigny G. Voilley N. Lauritzen I. Lazdunski M. J. Biol. Chem. 1996; 271: 10433-10434Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar) expressed in Xenopus form amiloride-sensitive H+-gated cation channels that are activated the pH (4Waldmann R. Champigny G. Bassilana F. Heurteaux C. Lazdunski M. Nature. 1997; 386: 173-177Crossref PubMed Scopus (1127) Google Scholar). J. and R. J. Biol. Chem. in The ASIC and the MDEG channels have different kinetics, ion selectivities, and pH 2, B, E, and and Coexpression of both ASIC and MDEG an amiloride-sensitive H+-gated current (Fig. 2 with single kinetics that are those of the ASIC and the MDEG current and of the kinetics that be for two channels. The pH dependence of the H+-activated current obtained after coexpression of ASIC and MDEG (Fig. 2 is with either subunit alone and the pH at which occurs = is that obtained with ASIC = or MDEG alone. the pH dependence curve of the ASIC/MDEG channel is a curve is for expression of the two subunits (Fig. 2 B, dashed The channel formed after coexpression of ASIC and MDEG is for Na+ either the ASIC or the MDEG channel A, B, and that most of the channel activity recorded in oocytes expressing both ASIC and MDEG is to a heteromultimeric channel. the ASIC/MDEG channel as it ASIC and MDEG activity (Fig. 2, C and for this is 20 The single channel conductance of the ASIC/MDEG channel is 10 (Fig. 3, C of an before the of the degenerin of the nematode C. elegans by such as or causes degeneration of mechanosensitive neurons M. Nature. 1994; 367: PubMed Scopus Google Scholar). This was to be to a of a channel of subunits, and (19Huang M. Chalfie M. Nature. 1994; 367: 467-470Crossref PubMed Scopus (343) Google Scholar). of the acid in MDEG also causes constitutive activity and (5Waldmann R. Champigny G. Voilley N. Lauritzen I. Lazdunski M. J. Biol. Chem. 1996; 271: 10433-10434Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). We that mutations or introduced ASIC not constitutive channel activity (4Waldmann R. Champigny G. Bassilana F. Heurteaux C. Lazdunski M. Nature. 1997; 386: 173-177Crossref PubMed Scopus (1127) Google Scholar). a of the basal currents of ASIC mutants revealed a low constitutive channel activity for or a activity for the A and These constitutive channels were not by 1 mm and have a potential of mV for and mV for the to were also altered by in did not induce channel of the and the of the constitutive current of the (Fig. 4 and were to alter the properties of MDEG (Fig. C, D, Coexpression of the ASIC with MDEG oocytes induced an nonselective current (Fig. A and with an which is the of MDEG or currents (Fig. 4 indicating that a novel heteromultimeric channel has been formed by association of both subunits. from pH 7.4 to pH 7 activated the ASIC(G431V)/MDEG = or = channels (Fig. the subunits and form proton-activated channels by and since the of MDEG an to pH values 5 (Fig. the activated current recorded at pH values pH in oocytes or together with MDEG can only be by the of a heteromultimeric channel. other ligand-gated cation channels, such as the and (11Barnard E.A. Trends Pharmacol. Sci. 1996; 17: 305-309Abstract Full Text PDF PubMed Google Scholar), ASIC as well as MDEG subunits can form both and heteromultimeric channels. The of heteromultimeric channels with novel properties the of H+-gated channels and new of For ASIC a for protein A in and two for protein C in and with MDEG a new for protein C in H+-gated cation currents with kinetics and pH to those of the heteromultimeric ASIC/MDEG channel were recorded in brain neurons S. N. J. Physiol. (Lond .). Scopus Google Scholar). Rapid pH activity and were to be in O.A. Brain Res. PubMed Scopus Google Scholar, M. Prog. Neurobiol. 1990; PubMed Scopus Google Scholar). is to that H+-gated cation channels present in such as ASIC and MDEG, also the heteromultimeric ASIC/MDEG channels, have a role and changes in neuronal We are to for and to and for their for and for with the