Hyperactivation of the Caenorhabditis elegans MEC-4 Na(+) channel of the DEG/ENaC superfamily (MEC-4(d)) induces neuronal necrosis through an increase in intracellular Ca(2+) and calpain activation. How exacerbated Na(+) channel activity elicits a toxic rise in cytoplasmic Ca(2+), however, has remained unclear. We tested the hypothesis that MEC-4(d)-induced membrane depolarization activates voltage-gated Ca(2+) channels (VGCCs) to initiate a toxic Ca(2+) influx, and ruled out a critical requirement for VGCCs. Instead, we found that MEC-4(d) itself conducts Ca(2+) both when heterologously expressed in Xenopus oocytes and in vivo in C. elegans touch neurons. Data generated using the Ca(2+) sensor cameleon suggest that an induced release of endoplasmic reticulum (ER) Ca(2+) is crucial for progression through necrosis. We propose a refined molecular model of necrosis initiation in which Ca(2+) influx through the MEC-4(d) channel activates Ca(2+)-induced Ca(2+) release from the ER to promote neuronal death, a mechanism that may apply to neurotoxicity associated with activation of the ASIC1a channel in mammalian ischemia.

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 Ca 2؉ , 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. 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 (24, 25). 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 (26) and MEC-10 (27) as well as stomatin-related MEC-2 (28) and paraoxonase-related MEC-6 (29). Additional proteins are thought to associate with the channel complex to assemble a macromolecular structure in which physical force gates the channel. The MEC channel conducts both Na ϩ and Ca 2ϩ (PCa/PNa ϳ0.2; Refs. 30 and 31). 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. DEG/ENaCAlthough 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 ϩ and Ca 2ϩ conductance significantly (30, 31) and induce necrotic cell death by provoking a rise in intracellular [Ca 2ϩ ] (31, 32). When mouse ASIC1a is hyperactivated in the brain by local acidos...

The sperm of Caenorhabditis elegans translocate in a fashion similar to sperm of Ascaris suum even though their pseudopods are longer, more plastic in shape, and form multiple expansions zones around their perimeter. Mutants in spe-11 form primary spermatocytes with a defective perinuclear region, but the resulting spermatozoa can still crawl and fertilize eggs. However, the resultant zygotes die due to the absence of sperm-supplied spe-11. Computer-assisted analysis of translocating spe-11 sperm reveals a novel defect in the dynamic morphology of their pseudopods. A similar analysis of the C. elegans mutant unc-54, which lacks the most abundant isoform of myosin II, reveals no defect in sperm motility, as expected, since C. elegans sperm have substituted the protein MSP for actin in the process of pseudopod expansion. These results reveal an unexpected defect in the dynamic morphology of pseudopods of spe-11 sperm. This defect, however, does not significantly affect crawling velocity, and it demonstrates how computer-assisted motion analysis systems can reveal subtle behavioral phenotypes in C. elegans mutant spermatozoa.

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