Temperature-sensitive mutant of the Caenorhabditis elegans neurotoxic MEC-4(d) DEG/ENaC channel identifies a site required for trafficking or surface maintenance.
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.
Royal
Dewey C
DC
Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
Bianchi
Laura
L
Royal
Mary Anne
MA
Lizzio
Michael
M
Mukherjee
Gargi
G
Nunez
Yury O
YO
Driscoll
Monica
M
D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D008565:Membrane Proteins; D009154:Mutation; D021381:Protein Transport; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014981:Xenopus
D029742:Caenorhabditis elegans Proteins; D017931:DNA Primers; C094927:Deg-1 protein, C elegans; D008565:Membrane Proteins; D015222:Sodium Channels; D049452:Green Fluorescent Proteins