Worm Breeder's Gazette 15(2): 18 (February 1, 1998)
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.
|1||Howard Hughes Medical Institute, Department of Neurobiology, Harvard Medical School Massachusetts General Hospital Boston, MA 02114|
|2||Department of Genetics, Washington University School of Medicine, St. Louis, MO|
|3||These authors contributed equally to this work|
The degenerins are a family of proteins of Caenorhabditis elegans that have been implicated in touch sensitivity and other forms of mechanosensation. Gain-of-function mutations in several degenerin genes cause the swelling, vacuolation and eventual death of neurons that express them, and similar mutations in the muscle degenerin gene unc-105 cause hypercontraction. Degenerins bear sequence similarity with several amiloride-sensitive ion channels from vertebrate kidney and brain, so it has been suggested that the nematode genes encode mechanically gated ion channels, and that these gain-of-function mutations create constituitively open ion channels. We expressed wild type unc-105 in human embryonic kidney cells (HEK 293) and in Xenopus oocytes, and detected no currents, in keeping with the prediction that these channels would remain closed unless activated by the proper (i.e., mechanical) stimulus. However, expression of unc-105 harboring degeneration- (A692V) or hypercontraction-causing mutations (P134S) results in constitutive cationic currents in both cell types. The UNC-105 channels are permeable to small monovalent cations, but comparatively impermeable to Ca++ and Mg++, which instead act as voltage-dependent blockers that may bind as deep as 40% into the pore. Single channel records indicate that the mutant channels have very long open times of hundreds to thousands of milliseconds, and fluctuate constantly between open and close states. Thus we consider these channels to be constitutively active, rather than constitutively open. Amiloride also produces voltage-dependent block, consistent with a single binding site 65% into the electric field of the pore. These properties distinguish degenerins from the mechanosensitive channels of vertebrate hair cells and of Xenopus oocytes, and suggest that these channels are not degenerins. Genetic evidence has suggested that other degenerins (e.g., MEC-4 and MEC-10) form heteromultimers that also require a third gene, mec-6. In contrast, because expression of unc-105 alone produced channels in two different cell types, UNC-105 channels are most likely homomultimers. Expression of mutant channels in cultured human and frog cells causes their death. The degeneration phenotype may be the result of sodium poisoning. Observations of dying HEK cells with the electron microscope reveal the presence of vacuoles and whorls (concentric rings of membrane) that also characterize the degeneration of nematode neurons, and therefore represent a hallmark of degenerin-induced cell death across species. In addition we observed in some cells a clear cytoplasm and mitochondria with a condensed matrix. The constitutive activity of the mutated UNC-105 channels is apparently the basis for the constitutive currents observed in whole cells, and provides an explanation of the muscle hypercontraction phenotype observed in unc-105 mutant animals.