The nematode degenerin UNC-105 forms ion channels that are activated by degeneration- or hypercontraction-causing mutations.

Nematode degenerins have been implicated in touch sensitivity and other forms of mechanosensation. Certain mutations in several degenerin genes cause the swelling, vacuolation, and death of neurons, and other mutations in the muscle degenerin gene unc-105 cause hypercontraction. Here, we confirm that unc-105 encodes an ion channel and show that it is constitutively active when mutated. These mutations disrupt different regions of the channel and have different effects on its gating. The UNC-105 channels are permeable to small monovalent cations but show voltage-dependent block by Ca2+ and Mg2+. Amiloride also produces voltage-dependent block, consistent with a single binding site 65% into the electric field. Mammalian cells expressing the mutant channels accumulate membranous whorls and multicompartment vacuoles, hallmarks of degenerin-induced cell death across species.

The C. elegans unc-104 gene encodes a putative kinesin heavy chain-like protein.

Mutations in the unc-104 gene of the nematode C. elegans result in uncoordinated and slow movement. Transposon insertions in three unc-104 alleles (e2184, rh1016, and rh1017) were used as physical markers to clone the unc-104 gene. DNA sequence analysis of unc-104 cDNAs revealed an open reading frame capable of encoding a 1584 amino acid protein with similarities to kinesin heavy chain. The similarities are greatest in the amino-terminal ATPase and microtubule-binding domains. Although the primary sequence relatedness to kinesin is weak in the remainder of the molecule, the predicted secondary structure and regional isoelectric points are similar to kinesin heavy chain.

Regulation of Caenorhabditis elegans degenerin proteins by a putative extracellular domain.

BACKGROUND: Rare, dominant mutations in the degenerin genes of Caenorhabditis elegans (deg-1, mec-4 and mec-10) cause neuronal degeneration. The extensive sequence similarity between degenerins and mammalian genes that encode subunits of the amiloride-sensitive sodium channel from kidney, colon and lung suggests that the C. elegans degenerins form ion channels. As mec-4 and mec-10 are needed for the reception of gentle touch stimuli, they may contribute to a mechanosensory ion channel. All the dominant degeneration-causing mutations in the C. elegans degenerin genes affect equivalent residues in a hydrophobic region that is structurally similar to the H5 domain of several ion channels, and so could form the channel lining. Increased channel activity may underlie the resulting degeneration, in which the affected cells vacuolate and swell. RESULTS: We now demonstrate that a missense change in a predicted extracellular region of the proteins encoded by deg-1 and mec-4 causes cell death similar to that caused by the dominant mutations. The missense mutation lies within a 22 amino-acid region found in all the C. elegans degenerins for which the sequences have been published, but not in the similar mammalian proteins. Deletion of nine amino acids surrounding the mutation site in mec-4 also causes neuronal degeneration. The degeneration-causing mutations in either the predicted pore-lining or the predicted extracellular regions of deg-1 are suppressed by additional, dominantly acting mutations that substitute larger for smaller residues within the channel lining. CONCLUSIONS: Our data suggest that the putative extracellular domain negatively regulates degenerin activity, perhaps by gating the channel. As this region is only found in the C. elegans proteins, it may allow more rapid regulation of the nematode channels, which may be needed for them to function in mechanosensation. The suppressor mutations, by adding larger amino acids to the putative pore lining, could prevent degeneration by blocking the pore of a multisubunit channel.

Touch at the molecular level. Mechanosensation.

The cloning of genes needed for gentle-touch sensitivity in the nematode Caenorhabditis elegans has provided new molecular details about a proposed mechanosensory ion channel complex.

Insect mechanoreception: what a long, strange TRP it's been.

Insect bristles are model mechanosensory organs. An ion channel of the TRP superfamily has recently been identified which is required for production of mechanoreceptor currents by insect bristles, and seems likely to represent a new kind of mechanically gated channel.

Chromosome organization of the protozoan Trypanosoma brucei.

The genome of the protozoan Trypanosoma brucei is known to be diploid. Karyotype analysis has, however, failed to identify homologous chromosomes. Having refined the technique for separating trypanosome chromosomes (L. H. T. Van der Ploeg, C. L. Smith, R. I. Polvere, and K. Gottesdiener, Nucleic Acids Res. 17:3217-3227, 1989), we can now provide evidence for the presence of homologous chromosomes. By determining the chromosomal location of different genetic markers, most of the chromosomes (14, excluding the minichromosomes), could be organized into seven chromosome pairs. In most instances, the putative homologs of a pair differed in size by about 20%. Restriction enzyme analysis of chromosome-sized DNA showed that these chromosome pairs contained large stretches of homologous DNA sequences. From these data, we infer that the chromosome pairs represent homologs. The identification of homologous chromosomes gives valuable insight into the organization of the trypanosome genome, will facilitate the genetic analysis of T. brucei, and suggests the presence of haploid gametes.

TRPA1.

The TRPA1 protein has up to 18 N-terminal and presumed cytoplasmic ankyrin repeats followed by the six membrane spanning and single pore-loop domains characteristic of all TRPs. In mice, TRPA1 is almost exclusively expressed in nociceptive neurons of peripheral ganglia and in all the mechanosensory epithelia of inner ear. In nociceptive neurons, TRPA1 mediates the response to the proalgesic bradykinin as well as the response to pungent irritants found in mustards and garlic, and probably also to those found in cinnamon and tear gas. The channel properties of TRPA1 are discussed and compared to those of sensory transducers. TRPA1 is well conserved across the animal kingdom, with likely orthologs from human to nematode, which suggest an ancestral role for this channel, probably in sensation.

Transport and localization of the DEG/ENaC ion channel BNaC1alpha to peripheral mechanosensory terminals of dorsal root ganglia neurons.

Mammalian brain sodium channel (BNaC, also known as BNC/ASIC) proteins form acid-sensitive and amiloride-blockable sodium channels that are related to putative mechanosensory channels. Certain BNaC isoforms are expressed exclusively in dorsal root ganglia (DRG) and have been proposed to form the ion channels mediating tissue acidosis-induced pain. With antibody labeling, we find that the BNaC1alpha isoform is expressed by most large DRG neurons (low-threshold mechanosensors not involved in acid-induced nociception) and few small nociceptor neurons (which include high-threshold mechanoreceptors). BNaC1alpha is transported from DRG cell bodies to sensory terminals in the periphery, but not to the spinal cord, and is located specifically at specialized cutaneous mechanosensory terminals, including Meissner, Merkel, penicillate, reticular, lanceolate, and hair follicle palisades as well as some intraepidermal and free myelinated nerve endings. Accordingly, BNaC1alpha channels might participate in the transduction of touch and painful mechanical stimuli.

The molecules of mechanosensation.

Mechanosensation, the transduction of mechanical forces into a cellular electrochemical signal, enables living organisms to detect touch; vibrations, such as sound; accelerations, including gravity; body movements; and changes in cellular volume and shape. Ion channels directly activated by mechanical tension are thought to mediate mechanosensation in many systems. Only one channel has been cloned that is unequivocably mechanically gated: the MscL channel in bacteria. Genetic screens for touch-insensitive nematodes or flies promise to identify the proteins that constitute a mechanosensory apparatus in eukaryotes. In Caenorhabditis elegans, the mec genes thus identified encode molecules for a candidate structure, which includes a "degenerin" channel tethered to specialized extracellular and intracellular structural proteins. In hair cells of the inner ear, evidence suggests that an extracellular tip link pulls on a channel, which attached intracellularly to actin via a tension-regulating myosin 1beta. The channel and the tip link have not been cloned. Because degenerins and MscL homologs have not been found outside of nematodes and prokaryotes, respectively, and because intracellular and extracellular accessory structures apparently differ among organs and species, it may be that mechanosensory channel complexes evolved multiple times.

Neuropathology of degenerative cell death in Caenorhabditis elegans.

In Caenorhabditis elegans necrosis-like neuronal death is induced by gain-of-function (gf) mutations in two genes, mec-4 and deg-1, that encode proteins similar to subunits of the vertebrate amiloride-sensitive epithelial Na+ channel. We have determined the progress of cellular pathology in dying neurons via light and electron microscopy. The first detectable abnormality is an infolding of the plasma membrane and the production of small electron-dense whorls. Later, cytoplasmic vacuoles and larger membranous whorls form, and the cell swells. More slowly, chromatin aggregates and the nucleus invaginates. Mitochondria and Golgi are not dramatically affected until the final stages of cell death when organelles, and sometimes the cells themselves, lyse. Certain cells, including some muscle cells in deg-1 animals, express the abnormal gene products and display a few membrane abnormalities but do not die. These cells either express the mutant genes at lower levels, lack other proteins needed to form inappropriately functioning channels, or are better able to compensate for the toxic effects of the channels. Overall, the ultrastructural changes in these deaths suggest that enhanced membrane cycling precedes vacuolation and cell swelling. The pathology of mec-4(gf) and deg-1(gf) cells shares features with that of genetic disorders with alterations in channel subunits, such as hypokalemic periodic paralysis in humans and the weaver mouse, and with degenerative conditions, e.g., acute excitotoxic death. The initial pathology in all of these conditions may reflect attempts by affected cells to compensate for abnormal membrane proteins or functions.

BNaC1 and BNaC2 constitute a new family of human neuronal sodium channels related to degenerins and epithelial sodium channels.

The recently defined DEG/ENaC superfamily of sodium channels includes subunits of the amiloride-sensitive epithelial sodium channel (ENaC) of vertebrate colon, lung, kidney, and tongue, a molluscan FMRFamide-gated channel (FaNaC), and the nematode degenerins, which are suspected mechanosensory channels. We have identified two new members of this superfamily (BNaC1 and BNaC2) in a human brain cDNA library. Phylogenetic analysis indicates they are equally divergent from all other members of the DEG/ENaC superfamily and form a new branch or family. Human BNaC1 maps to 17q11.2-12 and hBNaC2 maps to 12q12. Northern blot and mouse brain in situ hybridizations indicate that both genes are coexpressed in most if not all brain neurons, although their patterns of expression vary slightly, and are expressed early in embryogenesis and throughout life. By analogy to the ENaCs and the degenerins, which form heteromultimeric channels, BNaC1 and BNaC2 may be subunits of the same channel.