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.

The Nematode Degenerin UNC-105 Forms Ion Channels that are Activated by Degeneration- or Hypercontraction-Causing Mutations.

Jaime Garcia-Ailoveros[l]3, Jesus A. Garcia[l]3, Jing-Dong Liu2, David P. Corey[l]

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.