A Family of Acid-sensing Ion Channels from the Zebrafish
2004; Elsevier BV; Volume: 279; Issue: 18 Linguagem: Inglês
10.1074/jbc.m401477200
ISSN1083-351X
AutoresMartin Paukert, Samuel Sidi, Claire Russell, Maria Siba, Stephen W. Wilson, Teresa Nicolson, Stefan Gründer,
Tópico(s)Zebrafish Biomedical Research Applications
ResumoAcid-sensing ion channels (ASICs) are excitatory receptors for extracellular H+. Proposed functions include synaptic transmission, peripheral perception of pain, and mechanosensation. Despite the physiological importance of these functions, the precise role of ASICs has not yet been established. In order to increase our understanding of the physiological role and basic structure-function relationships of ASICs, we report here the cloning of six new ASICs from the zebrafish (zASICs). zASICs possess the basic functional properties of mammalian ASICs: activation by extracellular H+, Na+ selectivity, and block by micromolar concentrations of amiloride. The zasic genes are broadly expressed in the central nervous system, whereas expression in the peripheral nervous system is scarce. This pattern suggests a predominant role for zASICs in neuronal communication. Our results suggest a conserved function for receptors of extracellular H+ in the central nervous system of vertebrates. Acid-sensing ion channels (ASICs) are excitatory receptors for extracellular H+. Proposed functions include synaptic transmission, peripheral perception of pain, and mechanosensation. Despite the physiological importance of these functions, the precise role of ASICs has not yet been established. In order to increase our understanding of the physiological role and basic structure-function relationships of ASICs, we report here the cloning of six new ASICs from the zebrafish (zASICs). zASICs possess the basic functional properties of mammalian ASICs: activation by extracellular H+, Na+ selectivity, and block by micromolar concentrations of amiloride. The zasic genes are broadly expressed in the central nervous system, whereas expression in the peripheral nervous system is scarce. This pattern suggests a predominant role for zASICs in neuronal communication. Our results suggest a conserved function for receptors of extracellular H+ in the central nervous system of vertebrates. Acid-sensing ion channels (ASICs) 1The abbreviations used are: ASIC, acid-sensing ion channel; zASIC, zebrafish ASIC; MES, 2-(N-morpholino)ethanesulfonic acid; RACE, rapid amplification of cDNA ends; HA, hemagglutinin; VSV, vesicular stomatitis virus glycoprotein; hpf, hours postfertilization. 1The abbreviations used are: ASIC, acid-sensing ion channel; zASIC, zebrafish ASIC; MES, 2-(N-morpholino)ethanesulfonic acid; RACE, rapid amplification of cDNA ends; HA, hemagglutinin; VSV, vesicular stomatitis virus glycoprotein; hpf, hours postfertilization. are Na+ channels that are activated by extracellular H+ (1Waldmann R. Lazdunski M. Curr. Opin. Neurobiol. 1998; 8: 418-424Google Scholar). In the central nervous system, H+ is co-released with other transmitters, since synaptic vesicles are acidic (pH 5.7) (2Miesenböck G. De Angelis D.A. Rothman J.E. Nature. 1998; 394: 192-195Google Scholar). Thus, ASICs may act as excitatory receptors. During inflammation, the extracellular H+ concentration can also significantly increase. ASICs may therefore contribute to the perception of painful stimuli in the peripheral nervous system. In addition to the activation by H+, the homology of ASICs to mechanosensitive ion channels in Caenorhabditis elegans and the analysis of knock-out mice for individual ASIC subunits have suggested that ASICs are components of mechanically activated ion channels (3Welsh M.J. Price M.P. Xie J. J. Biol. Chem. 2002; 277: 2369-2372Google Scholar).There are four genes coding for ASICs in the human and mouse genome (asic1–asic4) (1Waldmann R. Lazdunski M. Curr. Opin. Neurobiol. 1998; 8: 418-424Google Scholar, 4Gründer S. Geissler H.S. Bässler E.L. Ruppersberg J.P. Neuroreport. 2000; 11: 1607-1611Google Scholar, 5Akopian A.N. Chen C.C. Ding Y. Cesare P. Wood J.N. Neuroreport. 2000; 11: 2217-2222Google Scholar). Moreover, alternative splicing of the asic1 and asic2 genes generates the splice variants ASIC1a/ASIC1b and ASIC2a/ASIC2b, respectively, that differ in the N-terminal third of the protein (6Bässler E.L. Ngo-Anh T.J. Geisler H.S. Ruppersberg J.P. Gründer S. J. Biol. Chem. 2001; 276: 33782-33787Google Scholar, 7Lingueglia E. de Weille J.R. Bassilana F. Heurteaux C. Sakai H. Waldmann R. Lazdunski M. J. Biol. Chem. 1997; 272: 29778-29783Google Scholar). In rodents, ASICs show a broad expression pattern in the central and peripheral nervous system. All ASICs except ASIC4 are expressed in sensory neurons of the dorsal root and trigeminal ganglia. ASIC1a, -2a, -2b, and -4 are expressed in the central nervous system (1Waldmann R. Lazdunski M. Curr. Opin. Neurobiol. 1998; 8: 418-424Google Scholar, 4Gründer S. Geissler H.S. Bässler E.L. Ruppersberg J.P. Neuroreport. 2000; 11: 1607-1611Google Scholar, 5Akopian A.N. Chen C.C. Ding Y. Cesare P. Wood J.N. Neuroreport. 2000; 11: 2217-2222Google Scholar). ASICs activate rapidly upon H+ application (τact of about 10 ms for ASIC1) (6Bässler E.L. Ngo-Anh T.J. Geisler H.S. Ruppersberg J.P. Gründer S. J. Biol. Chem. 2001; 276: 33782-33787Google Scholar) and desensitize in the continuous presence of H+. ASIC1 and ASIC3 desensitize completely (τinact of about 1 s for ASIC1) (6Bässler E.L. Ngo-Anh T.J. Geisler H.S. Ruppersberg J.P. Gründer S. J. Biol. Chem. 2001; 276: 33782-33787Google Scholar), whereas ASIC2a desensitizes incompletely and more slowly (7Lingueglia E. de Weille J.R. Bassilana F. Heurteaux C. Sakai H. Waldmann R. Lazdunski M. J. Biol. Chem. 1997; 272: 29778-29783Google Scholar). ASIC2b and ASIC4 cannot be activated by H+ (4Gründer S. Geissler H.S. Bässler E.L. Ruppersberg J.P. Neuroreport. 2000; 11: 1607-1611Google Scholar, 5Akopian A.N. Chen C.C. Ding Y. Cesare P. Wood J.N. Neuroreport. 2000; 11: 2217-2222Google Scholar, 7Lingueglia E. de Weille J.R. Bassilana F. Heurteaux C. Sakai H. Waldmann R. Lazdunski M. J. Biol. Chem. 1997; 272: 29778-29783Google Scholar). However, functional ASICs are probably tetrameric proteins, and ASIC2b forms a heteromeric channel with ASIC3 (7Lingueglia E. de Weille J.R. Bassilana F. Heurteaux C. Sakai H. Waldmann R. Lazdunski M. J. Biol. Chem. 1997; 272: 29778-29783Google Scholar). Several other combinations of subunits form heteromeric ASICs (8Bassilana F. Champigny G. Waldmann R. de Weille J.R. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 28819-28822Google Scholar, 9Babinski K. Catarsi S. Biagini G. Séguéla P. J. Biol. Chem. 2000; 275: 28519-28525Google Scholar), increasing the variety of H+-gated Na+ channels in situ (10Baron A. Waldmann R. Lazdunski M. J. Physiol. (Lond.). 2002; 539: 485-494Google Scholar, 11Benson C.J. Xie J. Wemmie J.A. Price M.P. Henss J.M. Welsh M.J. Snyder P.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2338-2343Google Scholar).Despite the progress made in recent years, we are far from a clear understanding of the physiological role of ASICs. The zebrafish, Danio rerio, is a model organism for the study of vertebrate biology that offers some advantages over higher vertebrates. It has a translucent embryo and develops rapidly, allowing easy expression analysis of genes and detection of developmental abnormalities in mutant fish. In order to establish the zebrafish as a model to study the physiological role of ASICs, we report here the molecular cloning of cDNAs for six ASICs, their expression pattern during early zebrafish development, and their electrophysiological characterization in a heterologous expression system. Our results suggest a conserved function for receptors of extracellular H+ in the central nervous system of vertebrates.EXPERIMENTAL PROCEDURESCloning of ASIC cDNAs from Zebrafish—Partial cDNA clones for zASIC1.2, -1.3, and -4.1 were identified by homology cloning from embryonic zebrafish brain and eye. Degenerate oligonucleotides and conditions for PCR were as described elsewhere (4Gründer S. Geissler H.S. Bässler E.L. Ruppersberg J.P. Neuroreport. 2000; 11: 1607-1611Google Scholar). Partial sequences for zASIC1.1, -2, and -4.2 were identified by searching the online zebrafish genomic sequence trace data base (ENSEMBL) at the Sanger institute using the SSAHA program. Different rat ASIC subunits were used as a probe. These partial sequences were used to design primers for rapid amplification of 5′- and 3′-cDNA ends (RACE). Using the Smart RACE cDNA amplification kit (Clontech), RACE was performed with poly(A)+ RNA from different embryonic and adult zebrafish tissues. PCR products were subcloned with the TOPO-TA cloning kit (Invitrogen) and sequenced. Full-length zASICs were assembled from the longest 5′ and 3′ RACE products. cDNA sequences were deposited in the EMBL data base under the following accession numbers: zASIC1.1, AJ609615; zASIC1.2, AJ609616; zASIC1.3, AJ609617; zASIC2, AJ609618; zASIC4.1, AJ609619; zASIC4.2, AJ609620.For expression studies in Xenopus oocytes, the entire coding sequences of zASICs were amplified from cDNA of larval brain by PCR (ExpandHighFidelity PCR system; Roche Applied Science). Using terminal restriction endonuclease recognition sequences (BamHI and KpnI; zASIC1.1: XhoI and KpnI), the PCR product was ligated in the oocyte expression vector pRSSP containing the 5′-untranslated region from Xenopus β globin and a poly(A) tail. Several clones from two independent PCRs were sequenced to exclude PCR errors.Radiation Hybrid Mapping and Synteny Analyses—PCR primers designed to amplify single exons from zasic genes were used to screen pools of zebrafish/hamster radiation hybrid cell lines (Goodfellow radiation hybrid panel) (12Geisler R. Rauch G.J. Baier H. van Bebber F. Brobeta L. Dekens M.P. Finger K. Fricke C. Gates M.A. Geiger H. Geiger-Rudolph S. Gilmour D. Glaser S. Gnugge L. Habeck H. Hingst K. Holley S. Keenan J. Kirn A. Knaut H. Lashkari D. Maderspacher F. Martyn U. Neuhauss S. Haffter P. Nat. Genet. 1999; 23: 86-89Google Scholar). Two independent sets of primers were used per gene (primer sequences available upon request). The resulting 94-digit radiation hybrid scores were used to establish physical map positions using the Instant Mapping program at the Children's Hospital Zebrafish Genome Project Initiatives Web site (zfrhmaps.tch.harvard.edu/ZonRHmapper/instantMapping.htm). The same program was used to calculate Lod scores. Microsatellite markers (Zmarkers) and expressed sequence tags that flanked each locus were used to establish the putative genetic map positions of the different loci on the integrated genetic/physical Tübingen map (wwwmap.tuebingen.mpg.de/). zasic-containing regions were then screened for the presence of expressed sequence tags that had been used to establish the syntenic correspondence of the zebrafish and human genomes (13Barbazuk W.B. Korf I. Kadavi C. Heyen J. Tate S. Wun E. Bedell J.A. McPherson J.D. Johnson S.L. Genome Res. 2000; 10: 1351-1358Google Scholar) (zebrafish-human syntenic correspondence map available on the World Wide Web at zfish.wustl.edu/). Such expressed sequence tags that turned out to be informative were fb82d01 (zasic1.1), fb36e06 and bact2 (zasic2), and fc17a11 (zasic4.1).In Situ Hybridization—PCR products comprising 360–520 bp from the N-terminal part of zASIC1.1 to zASIC4.2 cDNAs were subcloned in the TA cloning vector pCRII-TOPO (Invitrogen). Clones were sequenced to determine orientations relative to T3 and T7 sites and were used as templates for 50-μl PCRs (Advantage II PCR system; Clontech) using universal M13 forward and reverse primers. PCR products were precipitated and used as templates (∼1 μg/μl) for in vitro transcription of digoxygenin-labeled (Roche Applied Science) sense and antisense riboprobes. Probes were used for whole mount in situ hybridization as described previously (14Hauptmann G. Gerster T. Trends Genet. 1994; 10: 266Google Scholar). To obtain efficient labeling of internal tissues, hybridizations of larvae ≥3 days postfertilization were carried out using albino larvae that were treated 18 (3-day postfertilization larvae) to 22 min (4-day postfertilization larvae) in 10 μg/ml proteinase K.Electrophysiology—Using the mMessage mMachine kit (Ambion, Austin, TX), capped cRNA was synthesized by SP6 RNA polymerase from linearized cDNA. We injected 0.2–10 ng of cRNAs; currents ranged from 0.5 to 50 μA. cRNA was injected into stage V or VI oocytes of Xenopus laevis, and oocytes were kept in OR-2 medium (82.5 mm NaCl, 2.5 mm KCl, 1.0 mm Na2HPO4, 5.0 mm HEPES, 1.0 mm MgCl2, 1.0 mm CaCl2, and 0.5 g/liter polyvinylpyrrolidone) for 1–7 days. Whole cell currents were recorded with a TurboTec 03× amplifier (NPI Electronic, Tamm, Germany) using an automated, pump-driven solution exchange system together with the oocyte testing carousel controlled by the interface OTC-20 (NPI Electronic). Data acquisition and solution exchange were managed using the software CellWorks version 5.1.1 (NPI Electronic). Data were filtered at 20 Hz and acquired at 1 kHz. The bath solution for two-electrode voltage clamp contained 140 mm NaCl, 1.8 mm CaCl2, 1.0 mm MgCl2, 10 mm HEPES. For the acidic test solutions, HEPES was replaced by MES buffer. If not otherwise indicated, acidic application solutions were applied for 3.5 s, and neutral bath solution (pH 7.4) was applied for 30 s between channel activation. Holding potential was -60 mV.For patch clamp experiments, bath solution contained 140 mm NaCl, 1.8 mm CaCl2, 1.0 mm MgCl2, 10 mm HEPES, pH 7.4. Acidic test solutions were buffered with MES. Patch pipettes contained 140 mm KCl, 2.0 mm MgCl2, 5.0 mm EGTA, and 10 mm HEPES, pH 7.4. Rapid pH changes in the outside-out configuration were achieved by placing the patch in front of a piezo-driven double-barreled application pipette. Time constant for complete solution exchange is <2 ms (6Bässler E.L. Ngo-Anh T.J. Geisler H.S. Ruppersberg J.P. Gründer S. J. Biol. Chem. 2001; 276: 33782-33787Google Scholar). Holding potential was -70 mV. Currents were filtered at 5 kHz and acquired at 50 kHz.Data were analyzed using IgorPro software (WaveMetrics, Lake Oswego, OR). Dose-response curves were fitted to a Hill equation, and I-V curves were fitted to a polynomal function. Time constants of desensitization were determined by fitting current traces obtained in outsideout patch clamp experiments to a monoexponential function. The current rise time was evaluated by determining the rise time from 20 to 80% of the peak current (time to peak). All values reported represent the mean ± S.E. from n individual measurements, if not otherwise indicated. Statistical analysis was done with Student's t test.Immunoprecipitation and Western Blot—zASIC1.2 was epitope-tagged by inserting into the cDNA an oligonucleotide encoding the hemagglutinin (HA) epitope (YPYDVPDYA) of influenza virus. zASIC1.3 was epitope-tagged by inserting an oligonucleotide encoding the vesicular stomatitis virus glycoprotein (VSV-G) epitope (YTDIEMNRLGK). Both epitopes were inserted at the C termini of the respective proteins. To have equal signal intensities in Western blots, half the amount of cRNA for zASIC1.3 compared with zASIC1.2 was injected in Xenopus oocytes. Microsomal membranes of oocytes injected with the tagged proteins were prepared 2 days after injection as described (15Gründer S. Firsov D. Chang S.S. Jaeger N.F. Gautschi I. Schild L. Lifton R.P. Rossier B.C. EMBO J. 1997; 16: 899-907Google Scholar). Proteins from part of the microsomal membranes (equal to two oocytes) were directly separated on a 9% polyacrylamide-SDS gel and transferred to a polyvinylidene difluoride membrane (PolyScreen; PerkinElmer Life Sciences). The polyvinylidene difluoride membrane was then incubated with either peroxidase-coupled anti-HA antibody (1:1000; Roche Applied Science) or anti-VSV-G antibody (1 μg/ml; Roche Applied Science) followed by peroxidase-coupled anti-mouse antibody (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (Lysate in Fig. 7). Bound antibodies were visualized using the ECL kit (Amersham Biosciences). The remaining part of the microsomal membranes (equal to 16 oocytes) was resuspended in immunoprecipitation buffer (20 mm Tris, pH 7.6, 100 mm NaCl, 2% bovine serum albumin, 0.5% digitonin, 2 mm phenylmethylsulfonyl fluoride, leupeptin, antipain, and pepstatin A) and incubated with an anti-VSV-G antibody coupled to agarose (1:10; Sigma) for 1 h at 4 °C. The immunoprecipitates were washed several times and then separated on a gel and subjected to Western blot using the peroxidase-coupled anti-HA antibody as described above.RESULTSMolecular Characterization of ASICs from the Zebrafish— We isolated six full-length cDNA clones coding for proteins with strong homology to ASICs from the zebrafish. The open reading frames of the respective cDNA clones code for proteins consisting of 501–558 amino acids with similar predicted molecular masses of ∼60 kDa. The predicted amino acid sequences for all six zebrafish clones are shown together with the sequence of rat ASICs in Fig. 1. The degree of amino acid identity between the zebrafish and rat ASICs ranges from 60 to 75%. All six ASICs from zebrafish are encoded by different genes (Table I). Since only four asic genes are present in the genome of mouse and humans, the zebrafish has a greater variety of ASICs, which may be compensated by, at least in part, alternative splicing of the asic1 and -2 genes in mammals. Phylogenetic analysis (Fig. 2) predicts that three of the zebrafish ASICs are orthologs of ASIC1, ASIC2, and ASIC4, respectively. We named these clones zASIC1.1, zASIC2, and zASIC4.1, respectively. Two other clones, zASIC1.2 and zASIC1.3, are paralogs of zASIC1.1, and one other clone, zASIC4.2, is a paralog of zASIC4.1. We did not identify an ortholog of ASIC3. However, there are genomic sequence data for additional zebrafish ASICs in the data base. Further studies will show if these genes are transcribed into mRNA coding for functional ASICs or if these genes are inactive pseudogenes.Fig. 1Sequence alignment of zASICs with rat ASIC1a, -2a, -3, and -4. Amino acids showing a high degree of identity are shown as white letters on a black background. The initiator methionine of each cDNA was assigned to the first ATG in frame. Transmembrane domains are indicated by bars, conserved cysteines by stars, and a conserved consensus site for N-linked glycosylation in the loop between transmembrane domains by a branched symbol. Accession numbers are as follows: U94403, ASIC1; U53211, ASIC2; AF013598, ASIC3; AJ271642, ASIC4.View Large Image Figure ViewerDownload (PPT)Table IIntegrated physical and genetic map positions of zasic lociGeneLGaLinkage groupNearest markerDistanceLODbLogarithm of the odds scoreMinimal genetic interval (p-d)cProximal to distalGenetic Position (from top of LG)Human chromosomedThe physical position of human orthologs; the syntenic region for zasic4.2 was equivocalSyntenycReCentirayscMfCentimorganszasic1.122Wz12404.11110.78Z11379-Z6507∼1512q12Yeszasic1.28Zkp113g7ya435.81Z7819-Z4323∼3312q12Nozasic1.324unp2650gConsidered as duplicate markers by InstantMapperMaximumZ9603-Z6438∼5612q12Nozasic23Wz12338.1715.86Z9664-Z11227∼68.517q11.2-12Yeszasic4.19bz31c2716.54Z7319-Z25375∼432q35-36Yeszasic4.26fj66a042411.62Z17248-Z1209439.5-492q35-36?a Linkage groupb Logarithm of the oddsc Proximal to distald The physical position of human orthologs; the syntenic region for zasic4.2 was equivocale Centiraysf Centimorgansg Considered as duplicate markers by InstantMapper Open table in a new tab Fig. 2Evolutionary relationship of zASIC subunits and other family members. Other family members are ASICs and intestinal Na+ channels (INaCs) from human (h) and rat (r); ripped pocket (RPK) and pickpocket (PPK) from Drosophila; DEG-1, MEC-4, MEC-10, and UNC-8, degenerins from Caenorhabditis; subunits of the epithelial Na+ channel (ENaC) from rat; and the FMRFamid receptor (FaNaCh) from the snail Helix aspersa. Highly divergent sequences at the N and C termini as well as in the proximal part of the extracellular loop had been deleted, and the alignment and the tree for the phylogram have been established by Neighbor-Joining with ClustalX. The tree was then imported into TreeView and rooted with FMRFamid receptor as an outgroup. Maximum likelihood analysis using TreePuzzle gave similar results.View Large Image Figure ViewerDownload (PPT)All six zASICs show the hallmarks of the degenerin/epithelial Na+ channel gene family: two hydrophobic domains, rather short N and C termini, and a large loop containing conserved cysteines between the two hydrophobic domains. There is one consensus site for N-linked glycosylation that is predicted to have an extracellular location and that is conserved in all zASICs (Fig. 1). Homology between different ASICs is particularly high in the second transmembrane domain and the region preceding this domain (Fig. 1). The C-terminal motif ((E/D)(F/I)(A/T)C) of rat ASIC1 and -2 that mediates the interaction of these ASICs with the PDZ domain-containing protein PICK1 (16Hruska-Hageman A.M. Wemmie J.A. Price M.P. Welsh M.J. Biochem. J. 2002; 361: 443-450Google Scholar, 17Duggan A. García-Añoveros J. Corey D.P. J. Biol. Chem. 2002; 277: 5203-5208Google Scholar) is conserved in rat ASIC4 and in zASIC1.1, -2, -4.1, and -4.2 but not in zASIC1.2 and -1.3 (Fig. 1).Expression Pattern of asic Genes in Zebrafish Embryos and Larvae—The expression pattern of the six zasic genes in different zebrafish tissues was analyzed by in situ hybridization at 24, 30, 48, 72, and 96 h postfertilization (hpf). Expression of the asic genes was restricted mainly to neurons and maybe some glial cells. In general, each zasic showed spatially localized expression in specific neuronal tissues up until 48 hpf (Fig. 3) and then more widespread expression throughout the central nervous system from 72 hpf. Although overlapping in several cases, the expression patterns of zasic genes were distinguishable, indicating that each pattern was unique. Hybridization of sense strand cRNAs, which was performed as controls, did not give hybridization signals above background.Fig. 3zasic genes are widely expressed in the developing central nervous system. Lateral (A, C, E, G, I, and K) and ventral (B, D, F, H, J, and L) views of whole brains of embryos at 48 hpf show the distribution of various ASIC mRNAs (blue labeling). Black and red dashes indicate the positions of the anterior commissure and optic chiasm and tracts, respectively. a, anterior; d, dorsal; Hb, hindbrain; h, hypothalamus; LL, lateral line; Mb, midbrain; OB, olfactory bulb; OSN, otic sensory neurons; p, posterior; PG, pituitary gland; POA, preoptic area; RGC, retinal ganglion cells; T, telencephalon; Tg, trigeminal; Th, thalamus; v, ventral; ♦, ventral midbrain.View Large Image Figure ViewerDownload (PPT)zasic1.1 was first expressed by 30 hpf when its expression was restricted to the anterior and posterior lateral line ganglia and the otic sensory neurons (aLL, pLL, and OSN in Fig. 4, A and B). At 48 hpf, expression also became evident in the trigeminal ganglia (Tg in Fig. 3, A and B). At 72 and 96 hpf, expression could be seen throughout most of the central nervous system except for the eyes (Fig. 4K). It was excluded from the dorsal forebrain except for the habenula nuclei. Like asic1.3 (see below), asic1.1 expression was stronger in the left habenula in comparison with the right habenula (Fig. 4K).Fig. 4Localized expression of zasic genes in specific central nervous system neurons and cranial ganglia. Shown are lateral (A, C, E, and I), ventral (B, D, G, H, and J), and dorsal (F, K, and L) views of ASIC mRNA expression (blue) in brains of embryos at the various stages indicated. The solid line in K represents the midline. Black and red dashes indicate the position of the anterior commisure and of the tract of the postoptic commisure, respectively. AC, anterior commissure; aLL, anterior lateral line; Cb, cerebellum; dT, dorsal telencephalon; OB, olfactory bulb; OSN, otic sensory neurons; pLL, posterior lateral line; RGC, retinal ganglion cells; Te, tectum; Tg, trigeminal; TPOC, tract of the postoptic commissure.View Large Image Figure ViewerDownload (PPT)Expression of zasic1.2 was first evident at 48 hpf, at which stage weak expression could be observed in the ventral thalamus, ventral midbrain, ventral cerebellum, and ventral hindbrain and in the dorsal thalamus and hypothalamus (Fig. 3, C and D). There was also expression in the telencephalon, along the tract of the anterior commissure (black dashes in Fig. 3, C and D). zasic1.2 was additionally weakly expressed in the dorsal midbrain (dMb) and olfactory bulb (OB) at 48 hpf (Fig. 3, C and D). The level of staining increased by 96 hpf (Fig. 4, C and D) when zasic1.2 was also expressed in the tectum (Te in Fig. 4C). Unlike zasic1.1 and -1.3, zasic1.2 was not expressed asymmetrically in the habenulae at 96 h. zasic1.2 was the only zasic to be faintly expressed in the trunk of the fish (Fig. 4L, 96 hpf). Presumably, these cell bodies represent dorsal root ganglia. However, labeling was so faint that we could not unequivocally attribute it to a certain cell or tissue type.zasic1.3 was first expressed at 30 hpf in the anterior and posterior lateral line ganglia (aLL and pLL in Fig. 4, E and F), where it persisted until at least 48 hpf (Fig. 3, E and F). It was also expressed between 30 and 72 hpf in the telencephalon, along the tract of the anterior commissure (black dashes in Fig. 3E and data not shown). There was expression in the ventral thalamus, ventral midbrain, ventral cerebellum, and ventral hindbrain from 30 hpf. By 48 hpf, expression was also evident in the dorsal thalamus and hypothalamus (Fig. 3, E and F). At 72 hpf, weak expression was apparent in the habenulae, but by 96 hpf expression was stronger in the left habenula (LH) compared with the right habenula (RH) (not shown). This lateralized expression of zasic1.3 in the habenula has previously been published under the name habenular expressed sequence tag 1 (hest1) (18Concha M.L. Russell C. Regan J.C. Tawk M. Sidi S. Gilmour D.T. Kapsimali M. Sumoy L. Goldstone K. Amaya E. Kimelman D. Nicolson T. Gründer S. Gomperts M. Clarke J.D. Wilson S.W. Neuron. 2003; 39: 423-438Google Scholar).zasic2 expression was first detected at 30 hpf along the tract of the anterior commissure, where it persisted until 48 hpf (data not shown and dashed line in Fig. 3, G and H). At 48 hpf, zasic2 was also expressed in the preoptic area, ventral thalamus, and ventral midbrain and weakly in the ventral hindbrain (Fig. 3, G and H). At 72 and 96 hpf, expression was seen throughout most of the brain except for the dorsal forebrain. It was also expressed in the retinal ganglion cells (RGC in Fig. 4G).Expression of zasic4.1 was first evident at 48 hpf, at which stage the expression pattern was very similar to but stronger than that of zasic1.2. Of note, zasic4.1 (Fig. 3, I and J) was expressed in an extra domain in the dorsal midbrain (dMb) and in the retinal ganglion cells (data not shown).zasic4.2 was the earliest zasic to be expressed, being evident along the tract of the anterior commissure between 24 and 30 hpf (black dashes in Fig. 4, I and J, and data not shown). At 30 hpf, cells along the tract of the postoptic commissure also expressed zasic4.2 (red dashes in Fig. 4, I and J). At 48 hpf, very localized expression was evident in the preoptic area (POA in Fig. 3, K and L), which was maintained until at least 96 hpf. The posterior hypothalamus, ventral midbrain, hindbrain (Fig. 3, K and L), and retinal ganglion cells (Fig. 4H) all expressed zasic4.2 by 48 hpf. These domains of expression persisted and strengthened in older embryos. Notably, zasic4.2 was the only zasic almost devoid of expression in the telencephalon at 96 hpf (not shown).Functional Properties of zASICs—The functional properties of zASICs were investigated in X. laevis oocytes using the two-electrode voltage clamp technique. Oocytes expressing zASIC1.1, -1.2, -1.3, or -4.1 showed rapidly desensitizing inward currents when acidic solutions were applied, whereas zASIC2 and zASIC4.2 could not be activated by acidic test solutions. All zASIC currents had a similar overall shape (Fig. 5A). They rapidly activated and desensitized completely within less than 1 s. Occasionally, a small transient current increase appeared during desensitization of the channels that was highly variable among oocytes. Prolonged activation of zASIC4.1 led to the appearance of a sustained current component following the transient component (Fig. 5A, inset). This behavior is similar to rat ASIC3 (19Waldmann R. Bassilana F. de Weille J. Champigny G. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 20975-20978Google Scholar). In this study, we analyzed only the transient current component of zASIC4.1 in detail. Recovery from desensitization of zASICs was complete within less than 30 s in pH 7.4 (not shown). The pH at which half-maximal activation of zASICs was reached varied between pH 5.0 and 6.6 (Fig. 5 and Table II). This corresponds to an almost 100-fold difference in agonist concentration. However, these large differences in agonist affinity are comparable with the mammalian ASICs, for which pH of half-maximal activation varies between pH 4.3 (ASIC2a) (20Champigny G. Voilley N. Waldmann R. Lazdunski M. J. Biol. Chem. 1998; 273: 15418-15422Google Scholar) and 6.7 (ASIC3) (21Sutherland S.P. Benson C.J. Adelman J.P. McCleskey E.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 711-716Google Scholar).Fig. 5H+ sensitivity of zASICs. A, representative current traces of zASIC expressing oocytes elicited by varying acidic pH. Acidic application solution w
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