Intersubunit interactions between mutant DEG/ENaCs induce synthetic neurotoxicity

Abstract

Ion channel hyperactivation can result in neuronal loss in injury, stroke and neurodegenerative disease. Acidosis-associated hyperactivation of the Degenerin/epithelial amiloride-sensitive Na þ channel (DEG/ENaC) acid-sensing ion channel 1a (ASIC1a), a proton-gated channel expressed in the mammalian brain, contributes significantly to neuronal loss in ischemia. Analogously, in invertebrates, genetic hyperactivation of the Caenorhabditis elegans mechanosensory (MEC) channel (MEC-4(d)) of the DEG/ ENaC ion channel superfamily induces neuronal necrosis. Similarly substituted MEC-10(d) mutant subunits of the same MEC channel are only marginally neurotoxic, and we therefore exploited the weak necrosis phenotype of mec-10(d) lines to screen for novel extragenic mutations that enhance neuronal death. Here, we report on one mec-10(d) necrosis enhancer, which we show is MEC-4 variant MEC-4(A149V). MEC-4(A149V) executes normal MEC-4 function in touch sensation and does not induce necrosis on its own, but rather combines with MEC-10(d) to create a strongly neurotoxic channel. The MEC-4(A149V) þ MEC-10(d) channel conducts elevated Na þ and Ca 2 þ currents (with a disproportionate increase in Ca 2 þ current) in the Xenopus oocyte expression system, and exhibits altered binding of the channel inhibitor amiloride. Our data document the first example of synergistically toxic intersubunit interactions in the DEG/ENaC channel class and provide evidence that Ca 2 þ current levels may be decisive factors in tipping the balance between neuronal survival and necrosis. Ion channel dysfunction can result in the neuronal death that underlies the devastating consequences of stroke and nervous system injury. Primary determinants of mammalian neuronal loss associated with ion channel hyperactivation include the glutamate-gated channels 1 and the less extensively studied ASIC (acid-sensing ion channels) 2 of the DEG/ ENaC superfamily (named after founding members Caenorhabditis elegans degenerins and the mammalian epithelial amiloride-sensitive Na þ channels). DEG/ENaC channel complexes include three DEG/ENaC subunits, 3 with each subunit having a large extracellular domain, two transmembrane domains that contribute to the channel pore, and intracellular N and C termini (reviewed in Kellenberger and Schild 4 ). Structure/activity studies in the DEG/ENaC channel class have provided insights into function, but the relationship between toxicity and subunit interactions is largely unexplored.Of DEG/ENaCs studied to date, the C. elegans MEC channel, which transduces gentle touch stimuli in six mechanosensory (MEC) neurons 5,6 has been analyzed in most genetic detail. The core of the MEC channel is formed by DEG/ENaC subunits MEC-4 7,8 and MEC-10, 9 which associate with paraoxonase-like transmembrane protein MEC-6 10 and stomatin-like protein MEC-2.7,11,12 Dominant gain-offunction mutations alter MEC-4(A713) (the d position) to introduce large amino acids (AAs) near the channel pore, causing channel hyperactivity that induces necrotic-like neurod...

SRGP 1 regulation, targets, and contribution to cell killing inC. elegans

“…Intriguingly, significantly fewer cells survived when engulfment signaling was over-activated, as in srgp-1 mutants. Similar results were observed in mutants with neurotoxic or cytotoxic cell death, 17,18 where fewer cells survived in srgp-1 mutants. Thus, engulfment signaling does more than remove apoptotic debris, it can also promote the killing of unhealthy or less 'fit' cells within a tissue.…”

Insight into DEG/ENaC Channel Gating from Genetics and Structure

“…Classical genetic approaches including unbiased chemical screens have uncovered more than 50 missense mutations in the DEG/ENaC proteins that mediate C. elegans touch sensation (12, 20, 24, 27, 31-33, 51, 57, 66, 72). Such mutations disrupt behavioral responses to mechanical stimuli either because of a loss of DEG channel function or because of neuronal cell death caused by inappropriate activation of DEG channels.…”

NRA-2, a Nicalin Homolog, Regulates Neuronal Death by Controlling Surface Localization of Toxic Caenorhabditis elegans DEG/ENaC Channels

“…1A). At 15°C we observed baseline PLM tail touch neuron degeneration of ϳ2%, as described previously (15). We then fed this strain either empty vector control (L4440) or dsRNA EF hand gene clones from the L1 to L4 larval stages and evaluated the extent of neuronal death by counting surviving fluorescent PLM tail TRNs.…”

“…Introduction of an engineered mec-10 transgene encoding the mec-4(d)-analogous toxic amino acid substitution (namely A693V) to create mec-10(d) induces a low level of necrosis associated with neuronal swelling and featuring a similar toxicity mechanism to mec-4(d) (15). The low level of toxicity associated with the mec-10(d) transgene presents an opportunity to screen for genes that can mutate, or be knocked down with RNAi approaches, to enhance necrosis.…”