Background: Mechanisms that regulate plasma membrane expression of neuronally toxic DEG/ENaC channels are unclear. Results: Disruption of ER NRA-2, a Nicalin homolog, enhances C. elegans MEC-10(d) DEG/ENaC neurotoxicity. Immunocytochemistry, TIRF imaging, and electrophysiological assays support that NRA-2 controls relative MEC-10(d) distribution between ER and cell surface to regulate channel activity levels. Conclusion: NRA-2 regulates surface expression of a mutant DEG/ENaC channel. Significance: NRA-2 Nicalin can modulate DEG/ENaC pathophysiology.
Exposure of tissue to extreme temperature, corrosive chemicals, ischemia or other noxious stimuli can induce necrotic degeneration that is characterized by cellular swelling, loss of organellar integrity, accumulation of reactive oxygen species, activation of proteases and ultimately cell lysis. Necrosis can be genetically induced in C. elegans neurons by expression of mutant hyperactive ion channels. Mutant DEG/ENaC channels cause entry of excess calcium into the cell, triggering release of ER calcium stores and subsequent activation of calpain proteases. Due to the role of calcium in the induction and progression of this type of necrotic cell death we screened genes that encode calcium binding proteins and assayed for potential modulation of the cell death phenotype. RNAi and deletion mutant data indicates the involvement of EF hand containing proteins localized to ER and mitochondria. We will report on progress in deciphering their roles in neuronal necrosis.To extend mechanistic dissection, we have also been studying properties of hyperactivated ion channel‐induced necrosis in C. elegans embryonic cell culture and show that cultured neurons behave similarly to their counterparts in vivo. We have been pioneering the use of total internal reflection fluorescence microscopy to measure the MEC‐4/MEC‐10 channel at the neuron surface. We find changes in the surface expression and distribution of mechanotransduction complex proteins when necrosis is induced using a temperature‐sensitive mec‐4(d) allele. We are also studying the effects of mutant DEG/ENaC channels on mitochondrial morphology by imaging mitochondrially localized GFP in vitro and in vivo, as data suggest a relationship between maintenance of mitochondrial integrity and susceptibility to necrotic stimuli.
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