Worm Breeder's Gazette 13(1): 37 (October 1, 1993)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
mec-4 encodes a putative membrane protein required for the function of the six touch receptor neurons. Dominant gain-of-function mutations in mec-4 cause the swelling and degenerative death of these neurons. The capacity to mutate to forms that induce inappropriate neuronal death appears to be a general property of characterized members of the C. elegans degenerin gene family which includes mec-4 , deg-1 ,and mec-10 (Huang and Chalfie 1993 C.e. Meeting Abstracts, pg. 510.) We are interested in characterizing the cellular function of MEC-4 and describing how the normal activity of this protein is altered to induce neurodegeneration. A mammalian member of the degenerin family, rat alpha-rENaC, can induce an amiloride-sensitive Na+ current when introduced into Xenopus oocytes, suggesting that the encoded protein can form or activate an ion channel. Our working hypothesis is that MEC-4 is also a subunit of an ion channel, perhaps a mechanosensory channel.
We are investigating the role that the second hydrophobic domain, designated MSDII for membrane-spanning domain, plays in MEC-4 function. Several amino acids that project from one face of this putative transmembrane alpha helix are highly conserved in degenerins. Because of similarities between MEC-4 MSDII and transmembrane domains of well characterized ion channels thought to line the channel pore, we speculate that the conserved amino acids in MSDII of degenerins project into the channel lumen and influence conductance. Our analysis of EMS-induced mec-4 (r)mutations and site-directed mec-4 mutations indicates that amino acids on this face of the alpha helix are critical for function. We note three additional things about MSDII:
1) Amino acid substitutions on the putative lumen side of MSDII delay the onset of degeneration. Since we speculate that amino acid substitutions in MSDII slow or block ion flow, we wanted to ask whether changes in MSDII block degeneration induced by an amino acid substitution that we believe locks the channel in an open conformation. We constructed mec-4 alleles that encoded the death-inducing Val at mec-4 cDNA position 442 and a second amino acid substitution within or adjacent to MSDII. As we previously noted, MSDII amino acid substitutions that disrupt normal MEC-4 function appear to block degeneration when we assayed L1 animals (the mec-4 (d)clone induces a high proportion of touch cell degeneration in L1 s).However, when we examined touch neurons in the L4 stage in transgenic animals bearing the doubly substituted mec-4 alleles, degenerations were evident. Thus, most single amino acid substitutions delay, rather than block degeneration. Our observations are consistent with the idea that ion flow through the channel is important in the degeneration mechanism.
2) Certain alleles which encode single amino acid substitutions on the lumen side of MSDII can partially suppress degeneration when present in trans to a mec-4 (d)allele. We assessed the efficiency of cell death in heterozygotes which harbored one mec-4 (d)allele and a mec-4 (r)allele bearing a single amino acid substitution in MSDII. We used an integrated mec-7 /lacZfusiongene to assay cell viability in our heterozygotes since cells that have died no longer stain. Our experiments show that certain genes encoding single amino acid substitutions in MSDII, in trans to a gene encoding the degeneration-inducing amino acid substitution Val-442 ,can lead to partial suppression of cell death. The genetic data suggest that mec-4 monomers interact and thus we envision that the functional channel complex includes more than one MEC-4 protein.
3) Chimeric degenerins in which MEC-4 MSDII is replaced by DEG-1 MSDII or rat alpha-rENaC MSDII can rescue mec-4 (r)mutations. The MSDIIs of published degenerins are among the most highly conserved domains of this family, exhibiting 25% identity and 70% similarity. To test whether MSDII from DEG-1 and rat alpha-rENaC are sufficiently conserved to function within the context of the mec-4 protein, we performed a domain swap experiment, replacing MEC-4 MSDII with the corresponding 20 amino acids from either DEG-1 or rat alpha-rENaC. Chimeric genes were tested for the ability to confer touch sensitivity when introduced into mec-4 (r)animals. We find that these chimeras function in the touch receptor neurons.