A Modulatory Subunit of Acid Sensing Ion Channels in Brain and Dorsal Root Ganglion Cells

MDEG1 is a cation channel expressed in brain that belongs to the degenerin/epithelial Na+channel superfamily. It is activated by the same mutations which cause neurodegeneration in Caenorhabditis elegans if present in the degenerins DEG-1, MEC-4, and MEC-10. MDEG1 shares 67% sequence identity with the recently cloned proton-gated cation channel ASIC (acid sensing ion channel), a new member of the family which is present in brain and in sensory neurons. We have now identified MDEG1 as a proton-gated channel with properties different from those of ASIC. MDEG1 requires more acidic pH values for activation and has slower inactivation kinetics. In addition, we have cloned from mouse and rat brain a splice variant form of the MDEG1 channel which differs in the first 236 amino acids, including the first transmembrane region. This new membrane protein, which has been called MDEG2, is expressed in both brain and sensory neurons. MDEG2 is activated neither by mutations that bring neurodegeneration once introduced in C. elegansdegenerins nor by low pH. However, it can associate both with MDEG1 and another recently cloned H+-activated channel DRASIC to form heteropolymers which display different kinetics, pH dependences, and ion selectivities. Of particular interest is the subunit combination specific for sensory neurons, MDEG2/DRASIC. In response to a drop in pH, it gives rise to a biphasic current with a sustained current which discriminates poorly between Na+and K+, like the native H+-gated current recorded in dorsal root ganglion cells. This sustained current is thought to be required for the tonic sensation of pain caused by acids. MDEG1 is a cation channel expressed in brain that belongs to the degenerin/epithelial Na+channel superfamily. It is activated by the same mutations which cause neurodegeneration in Caenorhabditis elegans if present in the degenerins DEG-1, MEC-4, and MEC-10. MDEG1 shares 67% sequence identity with the recently cloned proton-gated cation channel ASIC (acid sensing ion channel), a new member of the family which is present in brain and in sensory neurons. We have now identified MDEG1 as a proton-gated channel with properties different from those of ASIC. MDEG1 requires more acidic pH values for activation and has slower inactivation kinetics. In addition, we have cloned from mouse and rat brain a splice variant form of the MDEG1 channel which differs in the first 236 amino acids, including the first transmembrane region. This new membrane protein, which has been called MDEG2, is expressed in both brain and sensory neurons. MDEG2 is activated neither by mutations that bring neurodegeneration once introduced in C. elegansdegenerins nor by low pH. However, it can associate both with MDEG1 and another recently cloned H+-activated channel DRASIC to form heteropolymers which display different kinetics, pH dependences, and ion selectivities. Of particular interest is the subunit combination specific for sensory neurons, MDEG2/DRASIC. In response to a drop in pH, it gives rise to a biphasic current with a sustained current which discriminates poorly between Na+and K+, like the native H+-gated current recorded in dorsal root ganglion cells. This sustained current is thought to be required for the tonic sensation of pain caused by acids. The protein MDEG (or BNaC1) was cloned from rat and human brain (1Waldmann R. Champigny G. Voilley N. Lauritzen I. Lazdunski M. J. Biol. Chem. 1996; 271: 10433-10436Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 2Price M.P. Snyder P.M. Welsh M.J. J. Biol. Chem. 1996; 271: 7879-7882Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 3Garcı́a-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 (296) Google Scholar). It is a member of the degenerin/epithelial Na+channel superfamily which includes sodium-permeable ion channels such as the epithelial Na+ channel (ENaC) 1The abbreviations used are: ENaC, epithelial Na+ channel; ASIC, acid sensing ion channel; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; MES, 4-morpholinoethanesulfonic acid. (4Lingueglia E. Voilley N. Waldmann R. Lazdunski M. Barbry P. FEBS Lett. 1993; 318: 95-99Crossref PubMed Scopus (321) Google Scholar, 5Lingueglia 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, 6Canessa C.M. Horisberger J.D. Rossier B.C. Nature. 1993; 361: 467-470Crossref PubMed Scopus (835) Google Scholar, 7Canessa C. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1789) Google Scholar), involved in sodium homeostasis as well as in taste perception (8Barbry P. Hofman P. Am. J. Physiol. 1997; 273: G571-G587PubMed Google Scholar), and the FMRFamide-activated Helix aspersa channel FaNaC (9Lingueglia E. Champigny G. Lazdunski M. Barbry P. Nature. 1995; 378: 730-733Crossref PubMed Scopus (353) Google Scholar), involved in neurotransmission. It also includes the degenerins ofCaenorhabditis elegans, involved in mechano-transduction (10Driscoll M. Chalfie M. Nature. 1991; 349: 588-593Crossref PubMed Scopus (461) Google Scholar, 11Huang M. Chalfie M. Nature. 1994; 367: 467-470Crossref PubMed Scopus (347) Google Scholar). Rat MDEG did not display detectable currents after expression in Xenopus oocytes or HEK cells (1Waldmann R. Champigny G. Voilley N. Lauritzen I. Lazdunski M. J. Biol. Chem. 1996; 271: 10433-10436Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). In contrast, a large sodium-selective current was observed in cells that expressed mutated MDEG (1Waldmann R. Champigny G. Voilley N. Lauritzen I. Lazdunski M. J. Biol. Chem. 1996; 271: 10433-10436Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). The mutations correspond to substitution of a glycine,i.e. a small amino acid for a large amino acid, such as a valine or a phenylalanine, just before the second hydrophobic region. These mutations are equivalent to those which in the C. elegans degenerins cause cell swelling and neuronal death (10Driscoll M. Chalfie M. Nature. 1991; 349: 588-593Crossref PubMed Scopus (461) Google Scholar, 11Huang M. Chalfie M. Nature. 1994; 367: 467-470Crossref PubMed Scopus (347) Google Scholar, 12Hall D.H. Gu G. Garcı́a-Añoveros J. Gong L. Chalfie M. Driscoll M. J. Neurosci. 1997; 17: 1033-1045Crossref PubMed Google Scholar). MDEG shares 67% sequence identity with the recently cloned ASIC channel (for acid sensing ion channel), another member of the ENaC/FaNaC/degenerin family (13Waldmann R. Champigny G. Bassilana F. Heurteaux C. Lazdunski M. Nature. 1997; 386: 173-177Crossref PubMed Scopus (1155) Google Scholar). ASIC expression yields a transient amiloride-sensitive inward current in response to a rapid drop in extracellular pH, that is mostly carried by Na+ ions (13Waldmann R. Champigny G. Bassilana F. Heurteaux C. Lazdunski M. Nature. 1997; 386: 173-177Crossref PubMed Scopus (1155) Google Scholar). It is found in both the central and peripheral nervous system (3Garcı́a-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 (296) Google Scholar, 13Waldmann R. Champigny G. Bassilana F. Heurteaux C. Lazdunski M. Nature. 1997; 386: 173-177Crossref PubMed Scopus (1155) Google Scholar). More recently, a second H+-gated Na+ channel, called DRASIC, was found to be expressed specifically in dorsal root ganglion cells (14Waldmann R. Bassilana F. de Weille J. Champigny G. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 20975-20978Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar). Expression of DRASIC in either Xenopusoocytes or COS cells gives rise to a H+-inducible current with a dual time course comprising a rapidly inactivating current, followed by a slowly activating and sustained current (14Waldmann R. Bassilana F. de Weille J. Champigny G. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 20975-20978Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar). Both components are essentially Na+ currents. Fast drops in extracellular pH have been shown to activate transient sodium currents in peripheral sensory neurons (15Krishtal O.A. Pidoplichko V.I. Neuroscience. 1980; 5: 2325-2327Crossref PubMed Scopus (384) Google Scholar) and various central neurons (16Grantyn R. Perouansky M. Rodriguez-Tebar A. Lux H.D. Dev. Brain Res. 1989; 49: 150-155Crossref PubMed Scopus (29) Google Scholar, 17Ueno S. Nakaye T. Akaike N. J. Physiol. 1992; 447: 309-327Crossref PubMed Scopus (50) Google Scholar). Whereas the proton-gated current in brain neurons consists of a single rapidly inactivating component, a dual current is found in polymodal nociceptive dorsal root ganglion neurons (18Bevan S. Yeats J. J. Physiol. 1991; 433: 145-161Crossref PubMed Scopus (304) Google Scholar). In these neurons, the transient H+-induced Na+current is followed by a sustained component. This sustained current is most likely the base element in the perception of non-adaptive painful stimuli (19Bevan S. Geppetti P. Trends Neurosci. 1994; 17: 509-512Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 20Steen K.H. Steen A.E. Reeh P.W. J. Neurosci. 1995; 15: 3982-3989Crossref PubMed Google Scholar). Although the biphasic kinetics closely resemble those of the DRASIC channel, differences remain concerning the selectivity of the sustained component of the current, the native one being a non-selective rather than a Na+-selective current. In this study we will show that MDEG (now designated as MDEG1) expressed in COS cells corresponds to a proton-gated cation channel with properties different from ASIC or DRASIC. We will also describe the molecular cloning, localization, and functional expression of a splice variant of the MDEG1 subunit, MDEG2, which has major structural differences with MDEG1. MDEG2 is not active by itself, but it can associate with either MDEG1 or DRASIC, modifying their properties. In particular it will be demonstrated that this splice variant confers non-selectivity to the late H+-induced current. A fragment of the expressed sequence tag (GenBank accession number W50528) was amplified by PCR and used to screen a mouse brain cDNA library (Stratagene). A clone of 3062 base pairs was sequenced on both strands. It displays an open reading frame of 1689 nucleotides preceded by stop codons. The rat cDNA was obtained by PCR with the Expand High fidelity PCR System (Boehringer Mannheim) using a primer flanking the start codon (GCCTCGGGCTGAATGAATG) and a primer positioned 223 base pairs downstream from the stop codon (GTTAGTTCTTGGACAGTTC). After subcloning in pBluescript SK−vector (Stratagene), two independent clones were sequenced on both strands. All nucleic acid positions in the text refer to the rat nucleic acid sequence submitted to EMBL (accession numbers Y14635 for the rat clone and Y14634 for the mouse clone). MDEG2 rat cDNA in pBluescript vector was cut by BamHI, blunt ended, ligated with EcoRI linkers, and cut again byEcoRI and AccI. TheEcoRI/AccI part of the wild-type or mutated MDEG1 cDNAs in pBSK-SP6-globin vector (1Waldmann R. Champigny G. Voilley N. Lauritzen I. Lazdunski M. J. Biol. Chem. 1996; 271: 10433-10436Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar) was replaced by the previously prepared EcoRI/AccI fragment of the MDEG2 cDNAs. For expression in mammalian cells, the MDEG2 cDNA was excised from the pBluescript vector by XbaI andAccI. MDEG1 cDNA in pBSK-SP6-globin was cut byXhoI, blunt ended, and cut again by AccI. The fragment was subcloned with the MDEG2 XbaI/AccI fragment into the XbaI/SmaI-digested PCI expression vector (Promega). Human multi-tissue Northern blots containing about 2 μg of poly(A)+ RNA per lane (normalized for identical β-actin expression) were purchased fromCLONTECH. For the blots with RNAs from rat, total RNA was isolated as described (21Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). 10 μg of total RNA in each lane were separated on 1% agarose/formaldehyde gels and transferred onto nylon membranes. The probes were random primed 32P-labeled and correspond to bases 276 to 585 for the MDEG2 5′ probe, bases 154 to 612 for the MDEG1 5′ probe, bases 217 to 1363 for the rat, and bases 1 to 1308 for the human MDEG1 probe overlapping MDEG2. The human blots were hybridized overnight at 65 °C in 5 × SSC, 10 × Denhardt's solution, 0.1% SDS, 100 μg/ml fish sperm DNA, washed with 0.1 × SSC, 0.1% SDS at 70 °C and exposed to Kodak X-Omat AR film for 3–5 days at The rat blots were hybridized overnight at °C in 5 × SSC, 5 × Denhardt's solution, 0.1% SDS, 100 μg/ml fish sperm DNA, washed with × SSC, 0.1% SDS at 65 °C and with a to the were on using C. C. Lazdunski M. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). were in 0.1 sodium phosphate-buffered pH for and overnight at °C in a were cut on a at on and at °C to the rat cDNA sequence of MDEG2 and or MDEG1 and were used to MDEG2 and MDEG1 The of the used were the were with by to an specific of × were with 0.1 in for 10 for 5 μg/ml in 0.1 for at for 5 were 10 in PBS, for 10 in in and was carried overnight at °C in μg/ml sperm DNA, 1% μg/ml in 2 × SSC, and the probe with specific of × After were washed in 1 × at for before and to for were in with and exposed were with and The of was by in using of the probe with a of and by the of two specific probes to MDEG2 expression in dorsal root was obtained from in with the cDNA to bases with with and of with was carried the from The MDEG1 and DRASIC were amplified by PCR and subcloned in the PCI as described previously (13Waldmann R. Champigny G. Bassilana F. Heurteaux C. Lazdunski M. Nature. 1997; 386: 173-177Crossref PubMed Scopus (1155) Google Scholar, R. Bassilana F. de Weille J. Champigny G. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 20975-20978Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar). cells, at a of were with the ASIC and an expression vector containing the cDNA using the were used for days after cells were by their to G. 1994; 17: Google Scholar). currents were recorded using either the cell or and on for The 10 For the solution, was replaced by The in 10 in extracellular pH were by one of of a system in of the cell or a pH of than were with 10 rather than but were identical in were carried at 2 °C cDNA sequence from mouse to that of MDEG1 cDNA was found in the base of expressed sequence (GenBank accession number A fragment of this sequence was amplified by PCR from mouse brain cDNA and used to screen a mouse brain cDNA clones were isolated and the one base was It an open reading frame of 1689 base pairs preceded by stop and for a protein of amino acids. by PCR of the rat cDNA differences at amino with the mouse sequence and in the mouse were replaced by and in the The part of the sequence from amino acid 236 to the of the protein is identical to the rat MDEG1 sequence identity was also found at the nucleic acid from and including the The of between MDEG1 and MDEG2 cDNAs a splice This that from a single two for MDEG1 and the new protein MDEG2. The first acid sequence of MDEG2 by with MDEG1 identity and MDEG2 also two transmembrane one identical to MDEG1 and one different from MDEG1 this protein has the of the two hydrophobic flanking a large including a that was shown to be extracellular for the epithelial Na+ channel C.M. Rossier B.C. Am. J. Physiol. 1994; PubMed Google Scholar, S. E. Voilley N. Lazdunski M. Barbry P. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, P.M. Welsh M.J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) and for the C. M. Chalfie M. Driscoll M. J. Biol. 1996; PubMed Scopus Google Scholar). Northern was on human and on rat brain with a probe overlapping the of MDEG1 and MDEG2 with two probes specific of each splice variant The two of and by the probe in human and rat brain shown by correspond to each splice the to the MDEG1 form and the to the MDEG2 form Of the by Northern both splice variant are expressed in brain 2 A more of MDEG1 and MDEG2 in the nervous system was by in of the of MDEG1 and MDEG2 was on film and of rat cut in and The of MDEG2 or MDEG1 identical MDEG2 or MDEG1 and of the MDEG2 were found in the of the neuronal but were not in cells. expression in the and and In the system cells MDEG2 were in the cell the and the and cell the were to the cells. In contrast, was observed in the and the In the the large neurons were in as well as in the small cells to or cells. In the MDEG2 were expressed in cells and neurons, as well as in cells of In addition, were observed in large in and of the the and of MDEG2 In the cells and cells of MDEG2 The molecular was in small neurons with a and to and cells. expression was in the large neurons of A expression was observed most including and of the and MDEG2 were expressed at low in MDEG2 was in the same brain as MDEG1 and the that the two can be present in the same neurons and can with each High MDEG2 expression was also observed in sensory neurons of the dorsal root and MDEG1 was not that cause of in the C. elegansdegenerins and neurodegeneration are to activate MDEG1 (1Waldmann R. Champigny G. Voilley N. Lauritzen I. Lazdunski M. J. Biol. Chem. 1996; 271: 10433-10436Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). These mutations are just before the second hydrophobic in a MDEG1 and MDEG2 are the same 1 and 2 The two mutations and were introduced in MDEG2. these mutations are to activate MDEG1 (1Waldmann R. Champigny G. Voilley N. Lauritzen I. Lazdunski M. J. Biol. Chem. 1996; 271: 10433-10436Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar), MDEG2 were not active after expression in Xenopus oocytes differences in the part of MDEG1 and MDEG2 which includes the first 236 amino and the first transmembrane have differences between the properties of the two MDEG The of sequence identity between MDEG1 or MDEG2, and ASIC and the that are proton-gated ion of COS cells with MDEG1 a proton-gated Na+ current with properties different from both ASIC and DRASIC A and MDEG1 requires more acidic pH values for activation and to open at pH a pH ASIC and DRASIC are activated (13Waldmann R. Champigny G. Bassilana F. Heurteaux C. Lazdunski M. Nature. 1997; 386: 173-177Crossref PubMed Scopus (1155) Google R. Bassilana F. de Weille J. Champigny G. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 20975-20978Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar). The inactivation kinetics of MDEG1 are slower than for ASIC of COS cells with MDEG2 did not a proton-gated current, at the low pH of MDEG2 is by MDEG2 and ASIC are present in the same and dorsal root ganglion cells, of the two in COS cells was It H+-gated currents that were not different from those recorded with ASIC of MDEG2 and MDEG1 to a new current that activated the pH was from to 5 The current by the acid to pH 5 a slowly inactivating component that was not on expression of MDEG1 after a of to the of pH, a small but current be recorded from most cells the pH currents and at pH the current not This is in with the MDEG1 current which at a pH between and These that MDEG2 with MDEG1 to form with new properties. the of the current at pH is of the MDEG1 current activated at the same pH it is that if not channels are expressed in the form in cells with both MDEG1 and MDEG2. in inactivation of the current was between MDEG1 and currents currents were recorded from excised from cells and from cells. In both it was found that a be recorded The currents at 2 for both MDEG1 and that the MDEG1 and are to The between and is 2 for MDEG1 and 2 for excised from COS cells were exposed to a drop in pH from the pH of to pH Na+ was from the and were at and the MDEG1 current inward the pH the current This that channel of the transient component and of the component are Both current components expressed by the are by of the epithelial Na+ channel It has been shown previously that the DRASIC current, like the native proton-gated current in dorsal root sensory neurons, consists of two a rapidly inactivating current followed by a sustained current (14Waldmann R. Bassilana F. de Weille J. Champigny G. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 20975-20978Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar). However, the native current, the two DRASIC components at the same membrane of and MDEG2 is present in dorsal root ganglion cells, we the that of DRASIC and MDEG2 a late non-selective current, an transient sodium-selective current. show that of the two different a current which at first like a current. The transient current by activation of at with the it at a well in the of Na+ and the transient current is mostly carried by The current, to in the or of that it is not a Na+-selective current. It has been shown previously that the pH for activation of the DRASIC current is at (14Waldmann R. Bassilana F. de Weille J. Champigny G. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 20975-20978Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar). It was found that with MDEG2 did not the pH of the current The that the sustained current to independent of the Na+ that the sustained non-selective current to the was not by the of the DRASIC sustained current. It that the transient Na+ current recorded from cells be a current from the of a as in the of the current, both the and the non-selective current A a single channel protein displays two different ion is not It has been described previously for the A. F. E. Buell G. 1996; 272: PubMed Scopus Google Scholar). is the molecular for the of the and the to 2 different of A first is that a single channel selectivity with the of the different to different different of one and one be that the 2 different of channel expression be to differences in if the non-selective channel be from Na+-selective by we for the well as a of membrane the of that be separated into two independent channel it that in the between and both a Na+-selective and a non-selective be These not a However, it was shown that the is for it likely that the non-selective current is with a channel of In the DRASIC be found between the of the and sustained current. In cells the sustained current was of cells, the sustained component was than of the in of the sustained component was at of the current. This in of the the of 2 different for ion channels Trends Sci. 1996; 17: Full Text PDF PubMed Google Scholar), in the H+-gated channel family can be by and MDEG2 is present in sensory neurons it the expression of DRASIC. of the two yields a H+-gated current that now a non-selective sustained component. it that these two at part of the native proton-gated channel of nociceptive neurons (18Bevan S. Yeats J. J. Physiol. 1991; 433: 145-161Crossref PubMed Scopus (304) Google Scholar). The expression of acid sensing ion channels is not to sensory neurons. ASIC (14Waldmann R. Bassilana F. de Weille J. Champigny G. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 20975-20978Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar), both MDEG1 and MDEG2 are well expressed in the brain and MDEG1 is in present in the in extracellular pH have been shown to be with neuronal M. Trends Neurosci. 1992; 15: Full Text PDF PubMed Scopus (477) Google Scholar), the that brain ion channels are involved in or However, the acid for activation of MDEG1 by pH not to be with a as an acid The also is at acidic pH. It is not MDEG1 channels or in their native can associate with or can be by such as to bring their pH to pH the of MDEG1 channels in the brain remain Both MDEG1 and channels can be activated in such as and which large of the extracellular C. J. 49: PubMed Scopus Google Scholar). In such the of these particular channels and the non-selective cation current by the have to neuronal cell We are to G. Champigny and P. Barbry for We C. G. M. and N. for their for and F. for with the