Temperature-sensitive Mutant of the Caenorhabditis elegans Neurotoxic MEC-4(d) DEG/ENaC Channel Identifies a Site Required for Trafficking or Surface Maintenance
2005; Elsevier BV; Volume: 280; Issue: 51 Linguagem: Inglês
10.1074/jbc.m510732200
ISSN1083-351X
AutoresDewey C. Royal, Laura Bianchi, Mary Anne Royal, Michael A. Lizzio, Gargi Mukherjee, Yury O. Núnez, Monica Driscoll,
Tópico(s)Ion channel regulation and function
ResumoDEG/ENaC channel subunits are two transmembrane domain proteins that assemble into heteromeric complexes to perform diverse biological functions that include sensory perception, electrolyte balance, and synaptic plasticity. Hyperactivation of neuronally expressed DEG/ENaCs that conduct both Na+ and Ca2+, however, can potently induce necrotic neuronal death in vivo. For example, Caenorhabditis elegans DEG/ENaC MEC-4 comprises the core subunit of a touch-transducing ion channel critical for mechanosensation that when hyperactivated by a mec-4(d) mutation induces necrosis of the sensory neurons in which it is expressed. Thus, studies of the MEC-4 channel have provided insight into both normal channel biology and neurotoxicity mechanisms. Here we report on intragenic mec-4 mutations identified in a screen for suppressors of mec-4(d)-induced necrosis, with a focus on detailed characterization of allele bz2 that has the distinctive phenotype of inducing dramatic neuronal swelling without being fully penetrant for toxicity. The bz2 mutation encodes substitution A745T, which is situated in the intracellular C-terminal domain of MEC-4. We show that this substitution renders both MEC-4 and MEC-4(d) activity strongly temperature sensitive. In addition, we show that both in Xenopus oocytes and in vivo, substitution A745T disrupts channel trafficking or maintenance of the MEC-4 subunit at the cell surface. This is the first demonstration of a C-terminal domain that affects trafficking of a neuronally expressed DEG/ENaC. Moreover, this study reveals that neuronal swelling occurs prior to commitment to necrotic death and defines a powerful new tool for inducible necrosis initiation. DEG/ENaC channel subunits are two transmembrane domain proteins that assemble into heteromeric complexes to perform diverse biological functions that include sensory perception, electrolyte balance, and synaptic plasticity. Hyperactivation of neuronally expressed DEG/ENaCs that conduct both Na+ and Ca2+, however, can potently induce necrotic neuronal death in vivo. For example, Caenorhabditis elegans DEG/ENaC MEC-4 comprises the core subunit of a touch-transducing ion channel critical for mechanosensation that when hyperactivated by a mec-4(d) mutation induces necrosis of the sensory neurons in which it is expressed. Thus, studies of the MEC-4 channel have provided insight into both normal channel biology and neurotoxicity mechanisms. Here we report on intragenic mec-4 mutations identified in a screen for suppressors of mec-4(d)-induced necrosis, with a focus on detailed characterization of allele bz2 that has the distinctive phenotype of inducing dramatic neuronal swelling without being fully penetrant for toxicity. The bz2 mutation encodes substitution A745T, which is situated in the intracellular C-terminal domain of MEC-4. We show that this substitution renders both MEC-4 and MEC-4(d) activity strongly temperature sensitive. In addition, we show that both in Xenopus oocytes and in vivo, substitution A745T disrupts channel trafficking or maintenance of the MEC-4 subunit at the cell surface. This is the first demonstration of a C-terminal domain that affects trafficking of a neuronally expressed DEG/ENaC. Moreover, this study reveals that neuronal swelling occurs prior to commitment to necrotic death and defines a powerful new tool for inducible necrosis initiation. DEG/ENaC 3The abbreviations used are: ENaCsepithelial amiloride-sensitive Na+ channelGFPgreen fluorescent proteinPBSphosphate-buffered salineEMSethane methyl sulfonatetstemperature sensitiveASICacid-sensing ion channelPLMposterior lateral microtubule cell. channels perform a broad range of critical biological functions across species (reviewed in Ref. 1Kellenberger S. Schild L. Physiol. Rev. 2002; 82: 735-767Crossref PubMed Scopus (864) Google Scholar). Mammalian DEG/ENaCs with predominant expression in epithelia are Na+-selective channels that mediate vectorial Na+ transport required for fluid clearance in lung (2Hummler E. Barker P. Gatzy J. Beermann F. Verdumo C. Schmidt A. Boucher R. Rossier B.C. Nat. Genet. 1996; 12: 325-328Crossref PubMed Scopus (778) Google Scholar, 3Barker P.M. Nguyen M.S. Gatzy J.T. Grubb B. Norman H. Hummler E. Rossier B. Boucher R.C. Koller B. J. Clin. Investig. 1998; 102: 1634-1640Crossref PubMed Scopus (230) Google Scholar, 4McDonald F.J. Yang B. 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Welsh M.J. Johnson W.A.U Curr. Biol. 2003; 13: 1557-1563Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). DEG/ENaC channel subunits have two transmembrane domains that are situated in the plasma membrane such that N and C termini face the intracellular milieu and a single large loop projects extracellularly (reviewed in Ref. 1Kellenberger S. Schild L. Physiol. Rev. 2002; 82: 735-767Crossref PubMed Scopus (864) Google Scholar). DEG/ENaC channels are heteromeric complexes that include multiple DEG/ENaC subunits (probably four). The functional channel complex also includes accessory subunits related to stomatin and at least in some cases, paraoxonases. epithelial amiloride-sensitive Na+ channel green fluorescent protein phosphate-buffered saline ethane methyl sulfonate temperature sensitive acid-sensing ion channel posterior lateral microtubule cell. One of the best studied of the invertebrate DEG/ENaCs is the C. elegans MEC channel that acts as the primary transducer of touch stimuli (24Suzuki H. Kerr R. Bianchi L. Frokjar-Jensen C. Slone D. Xue J. Gerstbrein B. Driscoll M. Schafer W.R. Neuron. 2003; 39: 1005-1017Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 25O'Hagan R. Chalfie M. Goodman M.B. Nat. Neurosci. 2005; 8: 43-50Crossref PubMed Scopus (375) Google Scholar). The MEC channel is assembled in six mechanosensory neurons that sense gentle touch delivered to the nematode body. The MEC touch-transducing channel complex includes DEG/ENaC subunits MEC-4 (26Driscoll M. Chalfie M. Nature. 1991; 349: 588-593Crossref PubMed Scopus (461) Google Scholar) and MEC-10 (27Huang M. Chalfie M. Nature. 1994; 367: 467-470Crossref PubMed Scopus (347) Google Scholar) as well as stomatin-related MEC-2 (28Huang M. Gu G. Ferguson E.L. Chalfie M. 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Molecular study of the MEC channel complex has outlined a premier model for mechanically gated channels and has provided mechanistic understanding of the physiological basis of the sense of touch. Although the normal physiological actions of DEG/ENaC channels are essential for diverse functions, exacerbated activation of these channels can be severely neurotoxic. In the case of the C. elegans MEC touch channel, large side chain amino acid substitutions near the MEC-4 channel pore enhance Na+ and Ca2+ conductance significantly (30Goodman M.B. Ernstrom G.G. Chelur D.S. O'Hagan R. Yao C.A. Chalfie M. Nature. 2002; 415: 1039-1042Crossref PubMed Scopus (274) Google Scholar, 31Bianchi L. Gerstbrein B. Frokjaer-Jensen C. Royal D.C. Mukherjee G. Royal M.A. Xue J. Schafer W.R. Driscoll M. Nat. Neurosci. 2004; 7: 1337-1344Crossref PubMed Scopus (102) Google Scholar) and induce necrotic cell death by provoking a rise in intracellular [Ca2+] (31Bianchi L. Gerstbrein B. Frokjaer-Jensen C. Royal D.C. Mukherjee G. Royal M.A. Xue J. Schafer W.R. Driscoll M. Nat. Neurosci. 2004; 7: 1337-1344Crossref PubMed Scopus (102) Google Scholar, 32Xu K. Tavernarakis N. Driscoll M. Neuron. 2001; 31: 957-971Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). When mouse ASIC1a is hyperactivated in the brain by local acidosis consequent to arterial occlusion (which models ischemia/stroke), brain neurons die in large numbers, a channel toxicity that is on par with glutamate receptor channel excitotoxicity under the same circumstances (33Xiong Z.G. Zhu X.M. Chu X.P. Minami M. Hey J. Wei W.L. MacDonald J.F. Wemmie J.A. Price M.P. Welsh M.J. Simon R.P. Cell. 2004; 118: 687-698Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar). Disruption of ASIC1a dramatically ameliorates this neuronal death. ENaC channel hyperactivity can be accomplished by another molecular mechanism, assembly of excess functional channels at the plasma membrane. For human β- and γ-ENaC, disruption of an intracellular C-terminal motif limits channel retrieval from the plasma membrane, increasing overall conductance, the basis of the hypertensive disorder Liddle's syndrome (34Staub O. Sascha D. Henry P.C. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (741) Google Scholar, 35Goulet C.C. Volk K.A. Adams C.M. Prince L.S. Stokes J.B. Snyder P.M. J. Biol. Chem. 1998; 273: 30012-30017Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 36Snyder P.M. Endocr. Rev. 2002; 23: 258-275Crossref PubMed Scopus (189) Google Scholar). Taken together, these findings underscore the profound therapeutic importance of understanding the molecular nature of both the normal and hyperactivated functions of DEG/ENaC channels. One advantage of studying the nematode DEG/ENaCs is the capacity to conduct extensive genetic characterization of these channels within a physiological context. To identify molecular requirements for mec-4(d)-induced necrosis, we screened for genetic suppressors of neuronal death. One major suppressor class we isolated includes intragenic second site changes that maintain the channel hyperactivating mec-4(d) substitution (A713V) but have a second site mec-4 mutation that otherwise disrupts channel activity. Here we report on characterization of 22 intragenic mec-4 mutations. We focus on one distinctive mec-4 allele (mec-4(u231bz2)) in which the dramatic swelling associated with necrosis can occur, but some neurons can recover to survive. We show that the genetic change encoded by mec-4(u231bz2) alters an intracellular C-terminal residue that influences channel trafficking or stability. Moreover, this substitution (MEC-4(A745T)) renders necrosis strongly temperature-inducible. We discuss implications for structure/function/regulation of neuronally expressed DEG/ENaCs and expand understanding of in vivo mechanisms of necrosis induction. Genetic Screen for Suppressors of mec-4(d)-induced Cell Death—Strain ZB164 bzIs8 [pmec-4GFP+pMJ23(lin-15)]; lin-15(n765)ts X was used to generate mutagenesis strain ZB1081 bzIs8 [pmec-4GFP+pMJ23(lin-15)] mec-4(u231) X [TU231]; mec-4(u231) = mec-4(d) (26Driscoll M. Chalfie M. Nature. 1991; 349: 588-593Crossref PubMed Scopus (461) Google Scholar)]. Strain ZB164 bzIs8 was constructed by co-injecting plasmid pmec-4GFP and pMJ23(lin-15(+)) into a lin-15(n765)ts mutant, selecting lin-15(+) transformants at the restrictive temperature of 20 °C, and γ-irradiating transgenics to identify stably transformed lines as described (37Rosenbluth R.E. Cuddeford C. Baillie D.L. Genetics. 1985; 109: 493-511Crossref PubMed Google Scholar). Integrated lines were outcrossed at least 3× before further constructions. bzIs8 appeared X-linked because crossing bzIs8 males to N2 hermaphrodites yielded 70/70 males that were not fluorescent. Three factor mapping positioned bzIs8 at approximately +18 on the X chromosome. Mutagenesis strain ZB1081 bzIs8 [pmec-4GFP +pMJ23(lin-15)] mec-4(u231)] X was constructed by recombining in mec-4(u231) using standard genetic approaches. Our screen used nematode strain ZB1081, harboring the mec-4(d) mutation and expressing a GFP transgene exclusively in touch neurons (pmec-4GFP). In this strain, the GFP signal is absent because of the death of the touch neurons. L4/young adult animals were mutagenized using ethane methyl sulfonate (EMS) according to standard protocols (38Brenner S. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar). F1 animals were distributed onto individual plates and allowed to self-fertilize. Four days later, about 20 F2 animals from each plate were screened on a Nikon fluorescent microscope for fluorescent touch cells. Individuals with multiple fluorescent touch cells were cloned out so that stocks of candidate homozygous suppressor mutants with most animals harboring >3 fluorescent touch cells were generated for further study. Eighty-five mutants were isolated using a COPAS BIOSORT (Complex Object Parametric Analyzer and Sorter) from Union Biometrica. Mutations were mapped using standard procedures (38Brenner S. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar) and identified by sequence of PCR products. Primers used for sequencing were: 5′-GGC TGC TAC CGT TCT TGC TTC C-3′ and 5′-GAG AAC GGA GCA ATG GTG GAA G-3′. General Microscopy—For fluorescence microscopy, we immobilized L1, L4, and young adult nematodes with 20 mm NaN3 and screened for fluorescent touch cells in L1 or L4 animal at ×20, ×40, ×60, or ×100 magnification. Because in some animals GFP signal persisted faintly even in dying neurons, we scored PLMs as alive when they expressed bright GFP, and neuronal processes were clearly visible. We scored for swollen necrotic-like PLM touch neurons by examining tails of L1 stage larvae with DIC microscopy as described (39Driscoll M. Methods Cell Biol. 1995; 46: 323-353Crossref PubMed Scopus (16) Google Scholar). For the experiment in which individual animals were scored at L1 and then at the L4 stage, we immobilized L1 worms in drops of 17% ethanol in M9 placed on 8-well teflon-ringed slides. The L1 worms were then recovered to standard nematode growth medium (NGM) plates and scored at the L4 stage for glowing PLM tail cells. Photographs were taken by a digital camera mounted on a Zeiss Axioplan 2. Temperature sensitivity assays of mec-4(u231) versus mec-4(u231bz2) genetic strains and bzEx10 [Pmec-4(u231)::GFP]inN2, as well as bzEx11[Pmec-4(u231bz2::GFP)] in null mutant mec-4(u253) were conducted as follows. Strains were grown at 15 and 25 °C. Extragenic transformants were maintained by picking animals that expressed the PMYO-2::GFP co-injection marker. After at least one generation, L4 worms were scored at 40× for glowing PLM tail cells. For puncta counts, processes from L4 bzEx12[PMEC-4::GFP]in mec-4(u253) and bzEx13[PMEC-4(bz2)::GFP] in null mec-4(u253) were photographed at ×40 and the puncta within 10 cell body lengths of the cell body were counted. C. elegans Strains, Growth, and Touch Assay—Nematode strains were maintained at 20 °C unless otherwise stated on NGM seeded with Escherichia coli strain OP50 as food source (38Brenner S. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar). For bz2 GFP expression studies, plasmids were injected into Bristol (N2) and mec-4(u253)(mec-4-null) strains. We performed gentle touch tests by stroking the body at anterior and posterior positions with an eyelash as described (40Chalfie M. Sulston J. Dev. Biol. 1981; 82: 358-370Crossref PubMed Scopus (505) Google Scholar). Molecular Biology—The pmec-4GFP vector was created by introducing a HindIII/BamHI fragment including the mec-4 promoter into vector pPD95.77, which includes enhanced GFP (constructed by Scott Clark, NYU). The BamHI fragment was introduced by site-directed mutagenesis at the mec-4 initiation codon. PMEC-4::GFP was constructed by subcloning a 4.7-kb HindIII-BamHI fragment from plasmid TU44 (41Mitani S. Du H. Hall D.H. Driscoll M. Chalfie M. Development. 1993; 119: 773-783Crossref PubMed Google Scholar), which includes mec-4 promoter and coding sequences except for those encoding the last 7 amino acids, into pPD95.77 (Fire lab vector kit, Ref. 42Mello C. Fire A. Methods Cell Biol. 1995; 48: 451-482Crossref PubMed Scopus (1161) Google Scholar). The PMEC-4(u231)::GFP and PMEC-4(u231bz2)::GFP were constructed by site-directed mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene) using PMEC-4::GFP as template. mec-2, mec-4(d), and mec-10(d) cDNAs subcloned into pGEM-HE or pSGEM, a gift from the Chalfie laboratory (30Goodman M.B. Ernstrom G.G. Chelur D.S. O'Hagan R. Yao C.A. Chalfie M. Nature. 2002; 415: 1039-1042Crossref PubMed Scopus (274) Google Scholar), were amplified using the SMC4 bacterial strain (30Goodman M.B. Ernstrom G.G. Chelur D.S. O'Hagan R. Yao C.A. Chalfie M. Nature. 2002; 415: 1039-1042Crossref PubMed Scopus (274) Google Scholar). The A745T mutation was introduced by site-directed mutagenesis (QuikChange site-directed mutagenesis kit). Oocyte Expression and Electrophysiology—Capped RNAs were synthesized using T7 mMESSAGE mMACHINE kit (Ambion), purified (Qiagen RNAeasy columns), and run on denaturating agarose gels to check for size and cRNA integrity. cRNA quantification was then performed spectroscopically. Stage V-VI oocytes were manually defolliculated after selecting them among multistaged oocytes dissected by a 2-h collagenase treatment (2 mg/ml in Ca2+-free OR2 solution) from Xenopus laevis ovaries (NASCO). Oocytes were incubated in OR2 media, which consists of 82.5 mm NaCl, 2.5 mm KCl, 1 mm CaCl2, 1 mm MgCl2, 1 mm Na2HPO4, 0.5 g/liter polyvinyl pyrrolidine, and 5 mm HEPES (pH 7.2), supplemented with penicillin and streptomycin (0.1 mg/ml) and 2 mm sodium pyruvate. Oocytes were then injected with 52 nl of cRNA mix for a final amount of 5 ng/oocyte of each cRNA except for MEC-6, which was injected at the concentration of 1 ng/oocyte. Oocytes were incubated in OR2 at 20 °C for 4 days before recording. Currents were measured 4–10 days after cRNA injection using a two-electrode voltage clamp amplifier (GeneClamp 500B, Axon Instruments) at room temperature. Electrodes (0.3–1 m) were filled with 3 m KCl, and oocytes were perfused with a NaCl solution containing (in mm): NaCl (100), KCl (2), CaCl2 (1), MgCl2 (2), HEPES (10), pH 7.2 or with a CaCl2 solution containing CaCl2 (73), KCl (2), HEPES (10), pH 7.2. Chemicals were obtained from Sigma and Calbiochem. We used the pCLAMP suite of programs (Axon Instruments) for data acquisition and analysis. Currents were filtered at 200 Hz and sampled at 1 kHz. Immunocytochemistry—Staining of oocytes was performed following previously reported procedures (31Bianchi L. Gerstbrein B. Frokjaer-Jensen C. Royal D.C. Mukherjee G. Royal M.A. Xue J. Schafer W.R. Driscoll M. Nat. Neurosci. 2004; 7: 1337-1344Crossref PubMed Scopus (102) Google Scholar, 43Bianchi L. Priori S.G. Napolitano C. Surewicz K.A. Dennis A.T. Memmi M. Schwartz P.J. Brown A.M. Am. J. Physiol. Heart Circ. Physiol. 2000; 279: H3003-H3011Crossref PubMed Google Scholar). Briefly, 5 days after injection, oocytes were fixed at 4 °C overnight with 4% paraformaldehyde. The next day, oocytes were washed four times for 5 min each in PBS, imbedded in low melting point agarose (3% in PBS), and cut in 50-μm thick slices using a vibrotome. Slices were incubated for 2 h at room temperature in 0.2% bovine serum albumin in PBS plus 0.1% Tween 20 and subsequently incubated with anti-MEC-4 antibody directed against amino acids 527–539 in the extracellular loop ((44Lai C.C. Hong K. Kinnell M. Chalfie M. Driscoll M. J. Cell Biol. 1996; 133: 1071-1081Crossref PubMed Scopus (97) Google Scholar)1:50 in 1% bovine serum albumin dissolved in PBS and 0.1% Tween 20) overnight at 4 °C. Slices were washed three times for 5 min with PBS and incubated with Cy2-conjugated goat anti-rabbit antibody (1:2000; Jackson ImmunoResearch) for 1 h at room temperature. After slices were washed three times for 5 min in PBS, they were mounted with VECTOREX medium (Vector) and photographed using a Zeiss Axiplan 2 microscope equipped with digital camera. Images were analyzed and mounted with Adobe Photoshop. Suppressors of mec-4(d)-induced Neurodegeneration Include Intragenic Mutations That Disrupt MEC-4 Function—With a goal of defining genes required for mec-4(d)-induced necrosis, we screened for novel mutations that block or delay the death of the touch receptor neurons in a mec-4(d) mutant background. We expressed GFP exclusively in the six touch neurons using the mec-4 promoter (reporter bzIs8[pmec-4GFP]) (Fig. 1A). We then introduced mec-4(d) (allele mec-4(u231)) into the bzIs8 background (Fig. 1B) and compared neuronal survival in the L4/young adult stage by counting fluorescent touch neurons. mec-4(d) induces necrosis efficiently in the bzIs8[pmec-4GFP] line such that 94% animals (n>200) lack any detectable fluorescent touch neurons and the remaining 6% have only one fluorescent touch cell (nearly always the PVM neuron that functionally differs from the other touch cells (45Chalfie M. Sulston J.E. White J.G. Southgate E. Thomson J.N. Brenner S. J. Neurosci. 1985; 5: 956-964Crossref PubMed Google Scholar)) (Fig. 1C). We confirmed that the extent of touch neuron degeneration, as scored by the presence of swollen PLM touch neurons in the L1 larval stage, was tightly correlated with the lack of fluorescent touch neurons in L4 or adult stage animals as we had previously documented (31Bianchi L. Gerstbrein B. Frokjaer-Jensen C. Royal D.C. Mukherjee G. Royal M.A. Xue J. Schafer W.R. Driscoll M. Nat. Neurosci. 2004; 7: 1337-1344Crossref PubMed Scopus (102) Google Scholar, 46Chung S. Gumienny T.L. Hengartner M.O. Driscoll M. Nat. Cell Biol. 2000; 2: 931-937Crossref PubMed Scopus (137) Google Scholar) (TABLE ONE).TABLE ONEEffects of mec-4(u231bz2) allele on cell deathmec-4(u231) mec-4(u231)mec-4(u231bz2) mec-4(u231bz2)mec-4(u231) +mec-4(u231bz2) +% Necrotic neurons in L1942478aRef. 61.0% Dead neurons in L49911880% Neurons that swell and survive013NDbND, not determined.0a Ref. 61Herman R.K. Genetics. 1987; 116: 377-388Crossref PubMed Google Scholar.b ND, not determined. Open table in a new tab We reasoned that touch neurons genetically spared from death (but still able to express mec-4) would be easily identified by their restored fluorescence in the bzIs8[pmec-4GFP];mec-4(d) background (Fig. 1D). We therefore treated this strain with EMS and screened ∼76,000 mutagenized genomes for rare F2 progeny with multiple fluorescent touch neurons, initially scoring manually at high magnification, and later scoring with assistance of a COPAS BIOSORT, which can identify and sort animals based on differential fluorescence scores. We identified 110 strains that exhibit partial or complete suppression of necrosis. Intragenic Death Suppressor Mutations Alter Multiple Amino Acids within and near the Channel Pore—We expected that second site intragenic mutations in mec-4(d) that disrupt function of the MEC-4 channel subunit would constitute one death suppressor class and that intragenic mutations should have genetic properties of X-linked recessive loss-of-function mec-4 alleles (i.e. mec-4(+)/mec-4(u231bzx) trans-heterozygotes should be touch-sensitive, where x indicates the new intragenic mutation). In addition, we anticipated that most intragenic mec-4 loss-of-function alleles should express GFP strongly in all six touch receptor neurons. 80 necrosis suppressor alleles had these genetic properties, and we therefore considered them strong candidates for second site mutations in mec-4(u231). To determine if amino acid changes near or within the MEC-4 channel pore were encoded by any intragenic mec-4 alleles, we sequenced the mec-4 genomic sequence over an interval that included coding regions of part of the third extracellular Cys-rich domain, the transmembrane channel pore, and the cytosolic C-terminus (see Ref. 47Hong K. Mano I. Driscoll M. J. Neurosci. 2000; 20: 2575-2588Crossref PubMed Google Scholar for details of domain structure). We identified single nucleotide changes in 22 mec-4(d) suppressor alleles, 14 of which encode single amino acid substitutions, 2 of which encode frame-shifts, 2 of which encode stop codons, and 4 of which alter splicing sites (TABLE TWO). Our data confirm that intragenic null mutations and other likely loss-of-function mutations constitute a significant death suppressor class isolated in our screen. In addition, we identify several residues previously unknown to be critical for function of the hyperactivated mec-4(d) channel, including the first EMS-induced mutations affecting the MEC-4 C terminus. We note implications of specific changes for channel structure/function in greater detail in the “Discussion.”TABLE TWOEMS-induced intragenic suppressor mec-4(d) mutations that alter the genome sequence in the vicinity of the MEC-4 pore-encoding regionAlleleMutation siteBase changeAlterationLocationbz1492956C-TR601CExtracellularbz1833055G-AR619KExtracellularbz1953103ΔCS635F frameshiftExtracellularbz1333116G-A5′-AGGTRAGExtracellularbz1343116G-A5′-AGGTRAGExtracellularbz1433116G-A5′-AGGTRAGExtracellularbz943194C-TP643LExtracellularbz1043220C-TP655LExtracellularbz1123230G-AW658StopExtracellularbz1503246C-TQ664StopExtracellularbz1223349G-A3′-TTTCAGRExtracellularbz1593406C-TT701IExtracellularbz1733590G-AG716SPorebz113593G-AG717EPorebz1653602G-AG720DPorebz1393611G-AC723YPorebz453620C-TS726FPorebz1013625C-TL728FPorebz1843629C-TT729IPorebz83658G-AL739EPorebz1793748T-Astop769R, 7 AA extensionC terminusbz23676G-AA745TC terminusa The underlined nucleotide is changed to A causing loss of the splice site. Open table in a new tab a The underlined nucleotide is changed to A causing loss of the splice site. In mec-4(u231bz2), Neurons Swell and Appear Necrotic but Some Can Then Recover—O
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