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.

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  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.
Although 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 acidosis consequent to arterial occlusion (which models ischemia/ stroke), brain neurons die in large numbers, a channel toxicity that is on par with glutamate receptor channel excitotoxicity under the same circumstances (33). Disruption of ASIC1a dramatically ameliorates this neuronal death. ENaC channel hyperactivity can be accomplished by another molecular mechanism, assembly of excess functional channels at the plasma membrane. For human ␤and ␥-ENaC, disruption of an intracellular C-terminal motif limits channel retrieval from the plasma membrane, increasing overall conductance, the basis of the hyperten-sive disorder Liddle's syndrome (34 -36). Taken together, these findings underscore the profound therapeutic importance of understanding the molecular nature of both the normal and hyperactivated functions of DEG/ENaC channels.
One advantage of studying the nematode DEG/ENaCs is the capacity to conduct extensive genetic characterization of these channels within a physiological context. To identify molecular requirements for mec-4(d)-induced necrosis, we screened for genetic suppressors of neuronal death. One major suppressor class we isolated includes intragenic second site changes that maintain the channel hyperactivating mec-4(d) substitution (A713V) but have a second site mec-4 mutation that otherwise disrupts channel activity. Here we report on characterization of 22 intragenic mec-4 mutations. We focus on one distinctive mec-4 allele (mec-4(u231bz2)) in which the dramatic swelling associated with necrosis can occur, but some neurons can recover to survive. We show that the genetic change encoded by mec-4(u231bz2) alters an intracellular C-terminal residue that influences channel trafficking or stability. Moreover, this substitution (MEC-4(A745T)) renders necrosis strongly temperature-inducible. We discuss implications for structure/function/ regulation of neuronally expressed DEG/ENaCs and expand understanding of in vivo mechanisms of necrosis induction. (26)]. Strain ZB164 bzIs8 was constructed by co-injecting plasmid p mec-4 GFP 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). Integrated lines were outcrossed at least 3ϫ before further constructions. bzIs8 appeared X-linked because crossing bzIs8 males to N2 hermaphrodites yielded 70/70 males that were not fluorescent. Three factor mapping positioned bzIs8 at approximately ϩ18 on the X chromosome. Mutagenesis strain ZB1081 bzIs8 [p mec-4 GFP ϩpMJ23(lin-15)] mec-4(u231)] X was constructed by recombining in mec-4(u231) using standard genetic approaches.

Genetic Screen for Suppressors of mec-4(d)-induced Cell Death-Strain
Our screen used nematode strain ZB1081, harboring the mec-4(d) mutation and expressing a GFP transgene exclusively in touch neurons (p mec-4 GFP). In this strain, the GFP signal is absent because of the death of the touch neurons. L4/young adult animals were mutagenized using ethane methyl sulfonate (EMS) according to standard protocols (38). F1 animals were distributed onto individual plates and allowed to selffertilize. Four days later, about 20 F2 animals from each plate were screened on a Nikon fluorescent microscope for fluorescent touch cells. Individuals with multiple fluorescent touch cells were cloned out so that stocks of candidate homozygous suppressor mutants with most animals harboring Ͼ3 fluorescent touch cells were generated for further study. Eighty-five mutants were isolated using a COPAS BIOSORT (Complex Object Parametric Analyzer and Sorter) from Union Biometrica. Mutations were mapped using standard procedures (38) and identified by sequence of PCR products. Primers used for sequencing were: 5Ј-GGC TGC TAC CGT TCT TGC TTC C-3Ј and 5Ј-GAG AAC GGA GCA ATG GTG GAA G-3Ј.
General Microscopy-For fluorescence microscopy, we immobilized L1, L4, and young adult nematodes with 20 mM NaN 3 and screened for fluorescent touch cells in L1 or L4 animal at ϫ20, ϫ40, ϫ60, or ϫ100 magnification. Because in some animals GFP signal persisted faintly even in dying neurons, we scored PLMs as alive when they expressed bright GFP, and neuronal processes were clearly visible. We scored for swollen necrotic-like PLM touch neurons by examining tails of L1 stage larvae with DIC microscopy as described (39). For the experiment in which individual animals were scored at L1 and then at the L4 stage, we immobilized L1 worms in drops of 17% ethanol in M9 placed on 8-well teflon-ringed slides. The L1 worms were then recovered to standard nematode growth medium (NGM) plates and scored at the L4 stage for glowing PLM tail cells. Photographs were taken by a digital camera mounted on a Zeiss Axioplan 2.
C. elegans Strains, Growth, and Touch Assay-Nematode strains were maintained at 20°C unless otherwise stated on NGM seeded with Escherichia coli strain OP50 as food source (38). For bz2 GFP expression studies, plasmids were injected into Bristol (N2) and mec-4(u253) (mec-4-null) strains. We performed gentle touch tests by stroking the body at anterior and posterior positions with an eyelash as described (40).
Oocyte Expression and Electrophysiology-Capped RNAs were synthesized using T7 mMESSAGE mMACHINE kit (Ambion), purified (Qiagen RNAeasy columns), and run on denaturating agarose gels to check for size and cRNA integrity. cRNA quantification was then performed spectroscopically. Stage V-VI oocytes were manually defolliculated after selecting them among multistaged oocytes dissected by a 2-h collagenase treatment (2 mg/ml in Ca 2ϩ -free OR2 solution) from Xenopus laevis ovaries (NASCO). Oocytes were incubated in OR2 media, which consists of 82.5 mM NaCl, 2.5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM Na 2 HPO 4 , 0.5 g/liter polyvinyl pyrrolidine, and 5 mM HEPES (pH 7.2), supplemented with penicillin and streptomycin (0.1 mg/ml) and 2 mM sodium pyruvate. Oocytes were then injected with 52 nl of cRNA mix for a final amount of 5 ng/oocyte of each cRNA except for MEC-6, which was injected at the concentration of 1 ng/oocyte. Oocytes were incubated in OR2 at 20°C for 4 days before recording.
Immunocytochemistry-Staining of oocytes was performed following previously reported procedures (31,43). Briefly, 5 days after injection, oocytes were fixed at 4°C overnight with 4% paraformaldehyde. The next day, oocytes were washed four times for 5 min each in PBS, imbedded in low melting point agarose (3% in PBS), and cut in 50-m thick slices using a vibrotome. Slices were incubated for 2 h at room temperature in 0.2% bovine serum albumin in PBS plus 0.1% Tween 20 and subsequently incubated with anti-MEC-4 antibody directed against amino acids 527-539 in the extracellular loop ((44)1:50 in 1% bovine serum albumin dissolved in PBS and 0.1% Tween 20) overnight at 4°C. Slices were washed three times for 5 min with PBS and incubated with Cy2-conjugated goat anti-rabbit antibody (1:2000; Jackson ImmunoResearch) for 1 h at room temperature. After slices were washed three times for 5 min in PBS, they were mounted with VECTOREX medium (Vector) and photographed using a Zeiss Axiplan 2 microscope equipped with digital camera. Images were analyzed and mounted with Adobe Photoshop.

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 expressed GFP exclusively in the six touch neurons using the mec-4 promoter (reporter bzIs8[p mec-4 GFP]) (Fig. 1A). We then 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[p mec-4 GFP] line such that 94% animals (nϾ200) 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). We confirmed that the extent of touch neuron degeneration, as scored by the presence of swollen PLM touch neurons in the L1 larval stage, was tightly correlated with the lack of fluorescent touch neurons in L4 or adult stage animals as we had previously documented (31,46) (TABLE ONE).
We reasoned that touch neurons genetically spared from death (but still able to express mec-4) would be easily identified by their In the small fraction in which one touch neuron survives (6%), PVM, which is a neuron that has structural features of other touch neurons but does not by itself mediate a touch response (45), is most often the sole survivor. Ͼ200 worms were scored for each measurement. D, strategy for identifying suppressors of mec-4(d)-induced cell death. Intragenic and extragenic suppressor mutations that block neurodegeneration should restore fluorescence in touch receptor neurons. restored fluorescence in the bzIs8[p mec-4 GFP];mec-4(d) background (Fig. 1D). We therefore treated this strain with EMS and screened ϳ76,000 mutagenized genomes for rare F2 progeny with multiple fluorescent touch neurons, initially scoring manually at high magnification, and later scoring with assistance of a COPAS BIOSORT, which can identify and sort animals based on differential fluorescence scores. We identified 110 strains that exhibit partial or complete suppression of necrosis.
Intragenic Death Suppressor Mutations Alter Multiple Amino Acids within and near the Channel Pore-We expected that second site intragenic mutations in mec-4(d) that disrupt function of the MEC-4 channel subunit would constitute one death suppressor class and that intragenic mutations should have genetic properties of X-linked recessive loss-offunction mec-4 alleles (i.e. mec-4(ϩ)/mec-4(u231bzx) trans-heterozygotes should be touch-sensitive, where x indicates the new intragenic mutation). In addition, we anticipated that most intragenic mec-4 loss-of-function alleles should express GFP strongly in all six touch receptor neurons. 80 necrosis suppressor alleles had these genetic properties, and we therefore considered them strong candidates for second site mutations in mec- 4(u231).
To determine if amino acid changes near or within the MEC-4 channel pore were encoded by any intragenic mec-4 alleles, we sequenced the mec-4 genomic sequence over an interval that included coding regions of part of the third extracellular Cys-rich domain, the transmembrane channel pore, and the cytosolic C-terminus (see Ref. 47 for details of domain structure). We identified single nucleotide changes in 22 mec-4(d) suppressor alleles, 14 of which encode single amino acid substitutions, 2 of which encode frame-shifts, 2 of which encode stop codons, and 4 of which alter splicing sites (TABLE TWO). Our data confirm that intragenic null mutations and other likely loss-of-function mutations constitute a significant death suppressor class isolated in our screen. In addition, we identify several residues previously unknown to be critical

Effects of mec-4(u231bz2) allele on cell death
In these experiments only PLM tail touch neurons were scored. All strains indicated here included also bzIs8͓p mec-4 GFP͔ so that touch neurons are fluorescent when alive. A necrotic neuron displays swelling of the cell body up to several times its original size (necrotic phenotype). To determine the percentage of PLMs that are dead at the later developmental stage of L4, the number of glowing PLMs with elongated shape typical of healthy neurons and neuronal processes was subtracted from the total number of scored PLM neurons. All experiments summarized in this table were performed at 20°C.

EMS-induced intragenic suppressor mec-4(d) mutations that alter the genome sequence in the vicinity of the MEC-4 pore-encoding region
Listed are nucleotide and amino acid changes (numbering according to Ref. 47) associated with intragenic mec-4(d) suppressor mutations that affect the 3Ј-half of the gene (nucleotides corresponding to ϳ2996 -3872, which encodes from amino acids 610 to the 3Ј-end untranslated region). All alleles include the mec-4(d) mutation u231 encoding A713V in addition to the change indicated. Bold indicates mutations not reported in previous screens. Genomic sequence encoding the N terminus was not determined for most alleles, and thus although unlikely, the alleles listed could include additional mutations. The mec-4(u231bz2) was fully sequenced from 221 bp prior the start codon to 316 bp after the stop codon.

Mutation site Base change Alteration Location
The underlined nucleotide is changed to A causing loss of the splice site. DECEMBER 23, 2005 • VOLUME 280 • NUMBER 51

C. elegans DEG/ENaC Channel MEC-4(d) Trafficking
for function of the hyperactivated mec-4(d) channel, including the first EMS-induced mutations affecting the MEC-4 C terminus. We note implications of specific changes for channel structure/function in greater detail in the "Discussion." In mec-4(u231bz2), Neurons Swell and Appear Necrotic but Some Can Then Recover-One X-linked death suppressor allele, mec-4(u231bz2), has distinctive properties. We noted that this mutation partially uncouples necrotic swelling and cell death. In the mec-4(u231) background, swollen PLM neurons are apparent in young L1 larvae and disappear by the L4 stage (46,48) (Fig. 2D). In mec-4(u231bz2) at 20°C, however, while 24% of PLM tail neurons still swell in the L1 larval stage, at the L4 stage only 11% appear to have died (TABLE ONE). These data suggested that some touch neurons might swell significantly but then recover. To address this possibility, we scored posterior touch neurons in individual animals at the L1 and then again at the L4 stage (Fig. 2). Indeed, when we followed 21 individuals (42 PLM neurons) from L1 to L4 we found that ϳone-fourth of PLMs became swollen but then recovered to appear as fluorescent neurons with normal morphology in the L4 stage; ϳone-third of PLM neurons appeared healthy at the L1 stage and then either remained healthy (n ϭ 9) or died (n ϭ 5). We conclude that in the mec-4(u231bz2) background, the toxic insult is sufficient to induce dramatic swelling but not strong enough to efficiently kill neurons. This uncoupling of swelling and death suggests that the swelling stage of necrosis occurs prior to the point of death commitment.
Another striking property of mec-4(u231bz2) that we noted is that this allele is strongly temperature sensitive for neurotoxicity (TABLE  ONE and Fig. 3, A and B). mec-4(u231) is a potent inducer of necrosis at either 15°C or 25°C. By contrast, in the mec-4(u231bz2) background, necrosis is potent at 15°C, intermediate at 20°C, and essentially absent at 25°C. The strong ts phenotype associated with this allele enables necrosis induction in specific cells late in development, a feature with highly advantageous potential for future necrosis analysis (see "Discussion").

mec-4(u231bz2) Encodes an A745T Change in the MEC-4 C Terminus That Causes Temperature-dependent Disruption of Both the MEC-4(d) and MEC-4(ϩ)
Channels-To determine the molecular basis of the mec-4(u231bz2) phenotype, we sequenced mec-4 genomic sequence throughout the coding region. We found that mec-4(u231bz2) specifies amino acid change A745T in the intracellular MEC-4 C terminus, the first identified EMS-induced mec-4 mutation to affect this domain. Although the C-terminal domains of DEG/ENaC family members are not highly conserved, ClustalW alignment of C termini indicates that the position corresponding to MEC-4 (745) is often a nonpolar residue in family members (supplemental Fig. 1).
We confirmed the A745T substitution to be causative for the mec-4(u231bz2) phenotype by engineering this second change into a mec-4(u231) transgene (which encodes substitution A713V), introducing it into wild-type animals, and assaying for toxicity.  induces necrosis efficiently at 15°C, but exerts only weak effects at 25°C, similar to the genomically encoded mec-4(u231bz2) (Fig. 3, A  and B).
Substitution of A745T affects the otherwise wild-type MEC-4 channel similarly to the MEC-4(d) channel. When we engineered the A745T substitution alone into a mec-4(ϩ) transgene, we found that the Pmec-4(bz2) transgene was able to complement the mec-4-null mutation u253 for touch sensitivity at the permissive temperature (15°C), but this complementation was not effective at 25°C (Fig. 3, C and D). We conclude that Ala 745 is critical for MEC-4 channel activity in vivo both for the wild-type protein and the hyperactivated MEC-4(d) mutant.

In Xenopus Oocytes, the MEC-4(A713V/A745T) Subunit Maintains Basic MEC-4(d) Electrophysiological Properties yet Whole Cell Currents
Are Reduced-To establish how mec-4(d) toxic function is impaired by second site substitution A745T, we heterologously expressed the double mutant channel in Xenopus oocytes and performed electrophysiological analysis. The best characterized MEC-4(d) channel includes DEG/ENaC subunits MEC-4(d) and MEC-10(d), stomatin-related protein MEC-2, and paraoxonase-like protein MEC-6 (29 -31). (We designate this channel arrangement as the MEC-4(d) channel complex.) We therefore co-expressed MEC-4(A713V/A745T) with MEC-10(d), MEC-2, and MEC-6 and compared current amplitude and properties to the L4 stage, three to five repetitions each, n Ն 300. Note that unlike the MEC-4(A713V) mutant, in the MEC-4(A713V/A745T) mutant necrosis is extensive at 15°C but is essentially eliminated at 25°C. This temperature-sensitive property is also observed for an engineered transgene bzEx11[P mec-4 (u231bz2::GFP)] introduced into the mec-4(ϩ) background, supporting that the temperature-sensitive property is conferred by the bz2 mutation. The bz11Ex[P mec-4 (u231bz2::GFP)] transgene array is expected to overexpress MEC-4(A713V/A745T)::GFP and thus will have some dominant-negative effects on endogenous channel activity as we have observed previously for MEC-4(ϩ) (47). C and D, MEC-4(A745T) channel function is temperature sensitive. The ability of engineered transgenic mec-4(bz2) (encoding MEC-4(A745T) to rescue the touch sensory defect of mec-4(u253)null mutants was assayed at 15°C (C) and 25°C (D). Transgenic animals were touched three times each on the tail, and the percent responses were recorded by an observer blind to genotype (two lines for mec-4(ϩ) and one line for mec-4(bz2) were tested; results obtained from one line each are shown here). Transmission rate of the extrachromosomal array was ϳ70% for all lines tested. Note that MEC-4(A745T) animals are markedly more touch-sensitive at 15°C as compared with 25°C. Data are expressed as mean Ϯ S.E., n ϭ 7-15. ** and * indicate p Ͻ 0.01 and p Ͻ 0.05 by comparison with mec-4(null), by Student's t test. NS indicates not statistically significant compared with mec-4(null). As we have noted previously, transgenic lines harboring mec-4(ϩ) do not exhibit full rescue because of occasional loss of the transgene and a partial dominant-negative effect of overexpression of one subunit of the channel complex (47).
We previously documented that MEC-4(d) is permeable to Ca 2ϩ ions (31), a Ca 2ϩ influx that may trigger catastrophic ER Ca 2ϩ release necessary for the completion of the necrotic program. In Xenopus oocytes Oocytes were maintained and assayed at 20°C. When oocytes were maintained at 25 or 15°C they were either leaky or unhealthy. A, example of sodium currents elicited by voltage steps from Ϫ160 to ϩ60 mV from a holding potential of Ϫ30 mV in an oocyte injected with mec-4(d), mec-10(d), mec-2, and mec-6 and exposed to a NaCl solution. B, same as in A for an oocyte injected with mec-4(u231bz2), mec-10(d), mec-2, and mec-6. Note the significant reduction in whole cell currents despite the injection of the same amount of RNA in both A and B (see "Experimental Procedures"). C, enlarged scale for current shown in B demonstrates that despite being strongly reduced, currents associated with expression of MEC-4(A713V/A745T) are still present. D, average Na ϩ current at Ϫ100 mV recorded from oocytes injected with mec-10(d), mec-2, and mec-6 ϩ mec-4(d) (n ϭ 14), or mec-4(u231bz2) (encoding MEC-4(A713V/A745T) (n ϭ 8). E, Ca 2ϩ -activated currents induced by mutant MEC-4 channels when extracellular Ca 2ϩ is present. The oocyte shown in A was exposed to a solution in which Na ϩ was substituted with Ca 2ϩ . This activates an endogenous Xenopus oocyte Ca 2ϩ -activated Cl Ϫ current, which we have previously shown to be activated by entry of Ca 2ϩ through the MEC-4(d) channel (31). F, MEC-4(A713V/ A745T) oocyte shown in B was exposed to the Ca 2ϩ solution resulting in the activation of the endogenous Ca 2ϩ -activated Cl Ϫ current. MEC-4(A713V/A745T) double mutant channels retain the Ca 2ϩ permeability property typical of toxic MEC-4(d), but the current is greatly reduced. G, same current in F is shown enlarged. H, average Ca 2ϩ -activated Cl Ϫ current at Ϫ160 mV. Note that for MEC-4(A713V/A745T), both Na ϩ currents and Ca 2ϩ -activated Cl Ϫ currents, which are a measure of the amount of Ca 2ϩ permeating through the degenerin channel, are reduced to ϳ30% of MEC-4(A713V). Thus, Ca 2ϩ permeability is not grossly affected by the second site mutation. MEC-4(A713V) n ϭ 6; MEC-4(A713V/A745T) n ϭ 8. Data are expressed as mean Ϯ S.E. ** indicates p Ͻ 0.01 by comparison with MEC-4(d)-expressing oocytes, by Student's t test.
bathed with CaCl 2 , entry of Ca 2ϩ through MEC-4(d) activates an endogenous Ca 2ϩ -activated Cl Ϫ current that we monitor as a measure of MEC-4(d) Ca 2ϩ permeability (31,49) (Fig. 4E). In oocytes expressing the MEC-4(A713V/A745T) channel complex, we detected activation of the endogenous Ca 2ϩ -activated Cl Ϫ current, but the amplitude of the current was reduced to ϳ30% of the MEC-4(d) channel complex (Fig. 4,  F-H). We conclude that although amiloride sensitivity, ion selectivity, and voltage/time characteristics are maintained in the mutant MEC-4(A713V/A745T) channel, a significant reduction in the amount of whole cell current is introduced by the A745T substitution.
The Number of Surface-expressed MEC-4(A713V/A745T) Subunits Is Reduced in Oocytes, Suggesting a Trafficking or Stability Defect-Reduced whole cell currents associated with MEC-4(A713V/A745T) could result from changes in single channel conductance and/or open probability or from reduced numbers of channels at the cell surface. To measure MEC-4(A713V/A745T) at the cell surface, we used anti-MEC-4 antibodies (polyclonal against epitope corresponding to extracellular amino acids 552-561 (44)) to visualize MEC-4 subunits in sectioned oocytes. Non-injected stained oocytes had no fluorescent signal at the plasma membrane, confirming specificity of the MEC-4 antibody that we employed (Fig. 5A). Oocytes expressing the MEC-4(d) subunit displayed clear membrane staining (Fig. 5B) (31). By contrast, 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). These results suggest that a significantly decreased number of MEC-4(A713V/A745T) channel subunits are present at the oocyte plasma membrane under conditions of equal cRNA injection for MEC-4(A713V) and MEC-4(A713V/A745T). We conclude that reduced ion fluxes associated with expression of MEC-4(A713V/A745T) in Xenopus oocytes result from diminished numbers of channel subunits at the cell surface.
MEC-4(A745T) Has a Temperature-sensitive Trafficking Defect in Vivo-A critical question regarding the mechanism of necrosis suppression for mec-4(u231bz2) is whether the MEC-4(A745T) subunit is disrupted for trafficking or protein stability in vivo in nematode neurons. To assess this possibility, we monitored full-length MEC-4::GFP fusion proteins in vivo. Wild-type MEC-4::GFP can functionally complement a mec-4-null mutation, suggesting that this protein traffics correctly in vivo (47). MEC-4::GFP is evident in the cell body and in puncta in the process that are thought to be sites of the MEC-4 channel complex (29,50,51). When we compared MEC-4(A745T)::GFP in transgenic animals at 15 and 25°C, we found that puncta and cell body fluorescence are present at low temperature (Fig. 6, A-C) when the protein is functional, but the GFP distribution to the process is markedly diminished at 25°C when the protein is non-functional (Fig. 6, D-F). We conclude that the MEC-4(A745T) substitution disrupts channel trafficking or stability in a temperature-dependent manner. Our data implicate a residue in the MEC-4 C terminus in the functional assembly of the channel complex. Necrosis suppression in the mec-4(u231bz2) mutant is most likely conferred by a significant reduction in channel activity that has a basis in defective trafficking and/or stability.

DISCUSSION
We exploited C. elegans genetics to identify molecular changes that can block toxicity of the hyperactivated MEC-4(d) channel. Here we report several new amino acid changes within MEC-4 itself that disrupt D, quantitation of fluorescence at the plasma membrane. We used AdobePhotoshop (two pictures/oocyte, 6 oocytes/sample) to quantitate fluorescence at the plasma membrane by isolating pixels corresponding to the plasma membrane and determining average luminosity. Because of slightly different luminosity levels of the background, we subtracted background fluorescence from each measurement. Note that if we artificially manipulate photographs of MEC-4(d) expressing oocytes to reduce their luminosity level to 30% of its value (MEC-4(A713V/A745T)) currents are 30% of MEC-4(d)), we no longer detect a membraneassociated fluorescent signal. This suggests that lack of fluorescent signal in MEC-4(A713V/A745T) oocytes is because of reduction below detectable level. Data are expressed as mean Ϯ S.E. n is 12 for all samples. Pictures were taken with a Zeiss Axioplan 2 microscope equipped with a digital camera, 0.7 s exposure time for all photographs. channel function in vivo, including one that is strongly temperature sensitive for channel assembly but normal in channel functional properties. We examine "borderline" conditions that induce neuronal necrosis and probe the relationship of cell swelling to necrotic death. Our data provide novel information on MEC-4 structure/function and MEC channel complex formation at the same time they reveal mechanistic insight into necrosis.

New Insight into MEC-4 Structure/Function
Of the 80 mec-4 intragenic mutations identified in our large scale screen of 76,000 mutagenized genomes for suppressors of mec-4(d)induced death, five specify amino acid substitutions in the third extracellular cysteine-rich domain of MEC-4 (the cysteine-rich domain common to other DEG/ENaC family members), underscoring that this domain is critical for normal and toxic DEG/ENaC function (47). 14 mec-4 alleles encode single amino acid substitutions in and around MSDII (11 of these mutations are novel). Eight of these mutations affect residues in the pore region and highlight amino acids that have been implicated in mammalian DEG/ENaC amiloride blockage and ion selectivity (53)(54)(55)(56)(57), emphasizing the importance of pore integrity and ion transport for toxicity in vivo and supporting conservation of structure/ function throughout the channel class. Three novel sites in the region (Leu 728 , Thr 729 , and Leu 739 ) have not previously been implicated in channel function and might affect only the hyperactive configuration of the channel.

Functions of the MEC-4 C-terminal Domain
Genetic screens for touch-insensitive mutants (40,58), and our screen for specific suppressors of mec-4(d)-induced neuronal death have been extensive and yet a striking paucity of point mutations affecting the intracellular C-terminal MEC-4 domain (amino acids 740 -768) has been identified (47). In fact, of the 136 (50 (47) ϩ 86 (this study)) sequenced mutant mec-4 alleles, only two (both newly reported here) affect the C-terminal domain. One implication of this finding is that the intracellular C-terminal domain may not provide functions essential for channel activity. However, since genetically engineered mutations that eliminate the C terminus or that substitute Ala for a stretch of four lysines (amino acids 753-756) disrupt both normal MEC-4 and hyperactive MEC-4(d) activity (47), it appears more likely that the C terminus is critical to MEC-4 function but that multiple changes may be often required to disrupt biological activity.
One of the substitutions at the C terminus (encoded by bz179) eliminates the MEC-4 stop codon and would extend the C terminus by 7 amino acids if translated. Because the last 12 MEC-4 amino acids appear dispensable (as determined by analysis of genetically engineered mec-4 mutants capacity for functional complementation) and because GFP can be added at the end of MEC-4 to create a MEC-4 protein fusion that can restore function in a null mutant background (47), it may be that the mutation bz179 disrupts transcription termination sites or confers a novel function to disrupt channel activity.

A Mutation That Confers a Temperature-sensitive Defect in Channel Surface Expression
Another change in the MEC-4 C terminus, the A745T substitution, confers several interesting properties to the channel subunit. The A745T substitution introduces additional amino acid volume and polarity at this site in the MEC-4 C-terminal region. If the amphipathic transmembrane pore ␣-helix extends into the C-terminal domain, res-  (47). Evident are the puncta structures along the neuronal processes that are thought to be sites of the MEC-4 channel (29,50,51). C, number of puncta from the cell body up to ten times the cell body length (ϳ20 m) in the two strains reared at 15°C. The number of neuronal processes scored was 29 and 25, respectively. D and E, same as in A and B, but the worms were reared at 25°C. Note the dramatically reduced number of puncta in PMEC-4(A745T)::GFP neuron. Puncta become less evident and are substituted by a more diffuse staining about halfway through the scored length. F, scoring of the number of puncta in the two strains reared at 25°C confirms the strong reduction in PMEC-4(A745T)::GFP neurons. Note the slight reduction in the number of puncta for PMEC-4::GFP worms when reared at 25°C (compare first bar on the left in C and F, p Ͻ 0.05 by Student's t test) suggesting that the wildtype channel trafficking has a moderate temperature sensitivity. Neuronal processes scored were 12 and 14, respectively. ** indicates p Ͻ 0.01 by comparison with PMEC-4::GFP neurons, by Student's t test.
idue 745 would be situated near the hydrophilic-hydrophobic boundary of the amphipathic structure, so the polar Thr substitution could disrupt polarity distribution. Interestingly, when we aligned all neuronally expressed members of the DEG/ENaC superfamily (characterized C. elegans degenerins and the mammalian ASICs) we find that all members have small or non-polar residues at this site, possibly implying a functional constraint.
The A745T substitution confers strong temperature sensitivity for function of the hyperactivated MEC-4(d) variant channel. Although the A745T mutant was originally identified for its capacity to disrupt MEC-4(d) toxicity, this amino acid change appears to exert equivalent effects on the otherwise MEC-4(ϩ) subunit in terms of temperature-sensitive functional activity. Thus, A745T affects both the wild-type and hyperactivated forms of the MEC-4 channel complex.
How does the A745T substitution disrupt MEC-4 function? Our electrophysiological study of the MEC-4(A713V/A745T) channel indicates that amiloride sensitivity, ion permeation selectivity, and voltage/time characteristics are maintained when the mutant channel subunit is assayed in Xenopus oocytes. Thus, although we cannot rule out changes in single channel properties by experiments we conducted here, the whole cell electrophysiological properties of the heterologously expressed channel appear normal. Still, current amplitudes from whole cell recordings are markedly reduced, and our data support that this is likely because of the fact that the mutant channel is not efficiently trafficked to, or maintained at, the oocyte cell surface. We find strong evidence for a similar defect in vivo in nematode neurons when we examined how a MEC-4(A745T)::GFP fusion protein was situated in nematode neurons, we found significantly fewer fluorescent MEC-4::GFP puncta (likely sites of channel assembly (29,50,51)) distributed out to processes at the restrictive temperature as compared with the permissive temperature. We favor a model in which the MEC-4 channel distribution defect reflects a trafficking disruption rather than gross instability of the MEC-4 subunit because the biophysical properties of MEC-4(A713V/A745T) in whole cell recordings are indistinguishable from MEC-4(d) when reconstituted in Xenopus oocytes, even though current amplitudes are greatly reduced, suggesting that the mutant channel can function correctly once assembled. Given that the A745T substitution disrupts channel distribution but not gross stability or channel subunit function, we suggest that the A745T substitution disrupts efficient channel trafficking or maintenance at the cell surface.

Implications for Trafficking in the Channel Class
The A745T MEC-4 substitution constitutes the first change in a degenerin C terminus to be specifically implicated in channel trafficking or cell surface maintenance in a neuronally expressed DEG/ENaC family member. Interestingly, in the ENaC branch of the DEG/ENaC superfamily, channel stability at the surface is mediated by conserved PY motifs (consensus PPXY) situated in subunit C termini. The PY motif interacts with Nedd-4 ubiquitin ligase, which mediates channel turnover (34,35). The PY motif is missing or disrupted in ENaC mutants associated with enhanced channel distribution at the surface and increases overall Na ϩ transport, constituting the basis of human Liddle's syndrome, a hypertensive disorder (reviewed in Ref. 36). Neuronally expressed ASICs and C. elegans degenerins do not have PY motifs in their C termini and virtually nothing is understood about how they get to, and are maintained at, the cell surface. Although the intracellular C-terminal domain sequences are not well conserved in the DEG/ENaC family, all do have hydrophobic or neutral amino acids at the residue that corresponds to MEC-4 A745, so the trafficking/maintenance function operative at this site might be conserved among family members.
This possibility could easily be tested for mammalian family members using site-directed mutagenesis techniques.
It is interesting that the low efficiency surface expression phenotype of the A745T subunit is common to both Xenopus oocytes and nematode neurons. One possibility is that a change in subunit structure is causative for the defect; alternatively, the trafficking/maintenance machinery might be conserved between nematodes and vertebrates. Note that it is not unusual for a mutation that interferes with efficient channel transport to the cell surface to be temperature-sensitive (ts); ts mutants have been characterized for other channel types including CFTR and cardiac K ϩ channel KCNH2 (also known as HERG) (59,60). In these cases, the channel subunits also function normally but are less efficiently inserted into the plasma membrane and are massively retained in the endoplasmic reticulum (60).

Implications for the in Vivo Study of Necrosis Mechanisms
Necrotic Swelling Can Occur without Death-We initially identified mec-4(u231bz2) as a mutation that limited the extent of neuronal necrosis as compared with the mec-4(u231) background. We later found a strong temperature-dependence for necrosis induction in the mec-4(u231bz2) mutant: at 15°C death is extensive, at 20°C death is reduced, and at 25°C is nearly eliminated. Necrosis suppression appears likely attributed to limited ion influx as fewer functional channels are at the cell surface as the temperature rises. Such an explanation is consistent with the working hypothesis that a critical threshold level of Na ϩ and/or Ca 2ϩ influx is required for necrosis initiation.
In mec-4(d) mutants, touch receptor neurons swell to several times their normal cell diameter before they disappear from the animals (46,48). At the ultrastructural level, swelling is associated with expansion of intracellular electron dense whorls that appear membranous in nature. In mec-4(u231bz2) mutants, we observed that some touch receptor neurons swell and appear necrotic, but these same cells then recover to survive and adopt a normal morphology later in adulthood. This finding indicates that dramatic neuronal swelling can occur prior to the commitment to death. In addition, this result suggests that neurons have the capacity to recover after extensive swelling to restore ion homeostasis. The point of commitment to necrosis must occur consequent to swelling.
New Capacity for Inducible Necrosis Induction-The strong temperature dependence of necrosis induction in the mec-4(u231bz2) mutant constitutes a long sought breakthrough for the in vivo analysis of necrosis mechanisms by enabling necrosis to be induced by temperature shift. For example, in C. elegans screens for pharmacological reagents that disrupt ion channel toxicity using this model have been thus far hampered by the fact that MEC-4(d) toxicity is induced in late embryogenesis when embryos are still protected by the egg shell from drugs in the external environment. Delaying necrosis until adulthood should for the first time render high throughput screens for novel necrosis inhibitors feasible and efficient. Moreover, options for temperature-sensitive necrosis induction will facilitate elaboration of the temporal sequence of events that transpire during necrosis.