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.
Highlights
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 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 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 [45]) (Fig. 1C)
Oocytes expressing MEC-4(A713V/A745T) did not exhibit significant fluorescent signal at the plasma membrane (Fig. 5C), and quantitation of plasma membrane fluorescence intensity derived from several oocyte sections indicated that the signal in MEC-4(A713V/A745T) oocytes did not rise above background levels (Fig. 5D)
Summary
Genetic Screen for Suppressors of mec-4(d)-induced Cell Death—Strain ZB164 bzIs8 [pmec-4GFPϩpMJ23(lin-15)]; lin-15(n765)ts X was used to generate mutagenesis strain ZB1081 bzIs8 [pmec-4GFPϩpMJ23(lin-15)] mec-4(u231) X [TU231]; mec-4(u231) ϭ mec-4(d) [26]]. Strain ZB164 bzIs8 was constructed by co-injecting plasmid pmec-4GFP and pMJ23(lin-15(ϩ)) into a lin-15(n765)ts mutant, selecting lin-15(ϩ) transformants at the restrictive temperature of 20 °C, and ␥-irradiating transgenics to identify stably transformed lines as described [37]. Our screen used nematode strain ZB1081, harboring the mec-4(d) mutation and expressing a GFP transgene exclusively in touch neurons (pmec-4GFP). The L1 worms were recovered to standard nematode growth medium (NGM) plates and scored at the L4 stage for glowing PLM tail cells. PMEC-4::GFP was constructed by subcloning a 4.7-kb HindIII-BamHI fragment from plasmid TU44 [41], 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. 42).
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