Temperature-sensitive Mutant of the Caenorhabditis elegans Neurotoxic MEC-4(d) DEG/ENaC Channel Identifies a Site Required for Trafficking or Surface Maintenance

Abstract

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 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. 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Neuronally expressed ASICs and C. elegans have in C termini and is and are the cell surface. the intracellular C-terminal domain are in the DEG/ENaC have or at the that to MEC-4 the at this This for is that the expression phenotype of the A745T subunit is to both Xenopus oocytes and One is that a in subunit is for the the and that it is for a mutation that with channel transport to the cell to have been for channel and channel as Anderson M.P. J. Welsh M.J. Nature. PubMed Scopus Google Scholar, E. D. C. M. S. A.M. J. Physiol. 2000; PubMed Google Scholar). In cases, the channel subunits also are into the plasma membrane and are in the E. D. C. M. S. A.M. J. Physiol. 2000; PubMed Google Scholar). without identified as a mutation that the of neuronal necrosis as with the We a for necrosis in the at death is at death is and at is to ion as functional channels are at the cell as the temperature is with the that a critical of Na+ is required for necrosis initiation. In mec-4(d) touch neurons to normal cell the S. Driscoll M. Nat. Biol. 2000; PubMed Scopus Google Scholar, J. L. M. Driscoll M. J. Neurosci. 1997; PubMed Google Scholar). the swelling is with of intracellular that in In we that some touch neurons and to and a normal in This that dramatic neuronal swelling can prior to the commitment to In addition, this that neurons have the to swelling to ion The of commitment to necrosis to for temperature of necrosis in the a for the in of necrosis by necrosis to by temperature For example, in C. elegans for that ion channel this have been by the that MEC-4(d) is in when are by the in the necrosis for the first for necrosis and Moreover, for necrosis of the of that We the C. elegans and for and and for the of the for We also and for critical of the and of the and Driscoll for a with