Three dominant mutations of mec-4, a gene needed for mechanosensation, cause the touch-receptor neurons of Caenorhabditis elegans to degenerate. With deg-1, another C. elegans gene that can mutate to induce neuronal degeneration and that is similar in sequence, mec-4 defines a new gene family. Cross-hybridizing sequences are detectable in other species, raising the possibility that degenerative conditions in other organisms may be caused by mutations in similar genes. All three dominant mec-4 mutations affect the same amino acid. Effects of amino-acid substitutions at this position suggest that steric hindrance may induce the degenerative state (Driscoll, 1991).
Aberrant ion channel activity plays a causative role in several human disorders. Inappropriately regulated channel activity also appears to be the basis for neurodegeneration induced by dominant mutations of Caenorhabditis elegans mec-4, a member of the degenerin gene family postulated to encode a subunit of a mechanosensory channel. The degenerin gene family has been defined by two C. elegans genes, mec-4 and deg-1, which can mutate to gain-of-function alleles that induce degeneration of specific groups of neurons. A related mammalian gene, rat alpha-rENaC, induces an amiloride-sensitive Na+ current when introduced to Xenopus oocytes, strongly suggesting that degenerin genes encode ion channel proteins. Deduced amino-acid sequences of the degenerins include two predicted membrane-spanning domains. Conserved amino acids within the second membrane-spanning domain (MSDII) are critical for MEC-4 activity and specific substitutions within MSDII, whether encoded in cis or in trans to a mec-4(d) mutation, block or delay the onset of degeneration. Remarkably, MSDII from two other family members, C. elegans deg-1 and rat alpha-rENaC, can functionally substitute for MEC-4 MSDII in chimaeric proteins. These results support a structural model for a mechanosensory channels in which multiple MEC-4 subunits are oriented such that MSDII lines the channel pore, and a neurodegeneration model, in which aberrant ion flow through this channel is a key event (Hong, 1994).
The process by which mechanical stimuli are converted into cellular responses is poorly understood, in part because key molecules in this mode of signal transduction, the mechanically gated ion channels, have eluded cloning efforts. The Caenorhabditis elegans mec-4 gene encodes a subunit of a candidate mechanosensitive ion channel that plays a critical role in touch reception. Comparative sequence analysis of C. elegans and Caenorhabditis briggsae mec-4 genes was used to initiate molecular studies that establish MEC-4 as a 768-amino acid protein that includes two hydrophobic domains theoretically capable of spanning a lipid bilayer. Immunoprecipitation of in vitro translated Mec-4 protein with domain-specific anti-MEC-4 antibodies and in vivo characterization of a series of mec-4lacZ fusion proteins both support the hypothesis that MEC-4 crosses the membrane twice. The MEC-4 amino- and carboxy-terminal domains are situated in the cytoplasm and a large domain, which includes three Cys-rich regions, is extracellular. Definition of transmembrane topology defines regions that might interact with the extracellular matrix or cytoskeleton to mediate mechanical signaling (Lai, 1996).
Mechanosensory transduction in touch receptor neurons is believed to be mediated by DEG/ENaC (degenerin/epithelial Na+ channel) proteins in nematodes and mammals. In the nematode Caenorhabditis elegans, gain-of-function mutations in the degenerin genes mec-4 and mec-10 (denoted mec-4(d) and mec-10(d), respectively) cause degeneration of the touch cells. This phenotype is completely suppressed by mutation in a third gene, mec-6, that is needed for touch sensitivity. This last gene is also required for the function of other degenerins. mec-6 encodes a single-pass membrane-spanning protein with limited similarity to paraoxonases, which are implicated in human coronary heart disease. This gene is expressed in muscle cells and in many neurons, including the six touch receptor neurons. MEC-6 increases amiloride-sensitive Na+ currents produced by MEC-4(d)/MEC-10(d) by approximately 30-fold, and functions synergistically with MEC-2 (a stomatin-like protein that regulates MEC-4(d)/MEC-10(d) channel activity) to increase the currents by 200-fold. MEC-6 physically interacts with all three channel proteins. In vivo, MEC-6 co-localizes with MEC-4, and is required for punctate MEC-4 expression along touch-neuron processes. It is proposed that MEC-6 is a part of the degenerin channel complex that may mediate mechanotransduction in touch cells (Chelur, 2002).
In C. elegans, genes encoding components of a putative mechanotransducing channel complex have been identified in screens for light-touch-insensitive mutants. A long-standing question, however, is whether identified MEC proteins act directly in touch transduction or contribute indirectly by maintaining basic mechanoreceptor neuron physiology. In this study, the genetically encoded calcium indicator cameleon was used to record cellular responses of mechanosensory neurons to touch stimuli in intact, behaving nematodes. A gentle touch sensory modality is defined that adapts with a time course of approximately 500 ms and primarily senses motion rather than pressure. The DEG/ENaC channel subunit MEC-4 and channel-associated stomatin MEC-2 are specifically required for neural responses to gentle mechanical stimulation, but do not affect the basic physiology of touch neurons or their in vivo responses to harsh mechanical stimulation. These results distinguish a specific role for the MEC channel proteins in the process of gentle touch mechanosensation (Suzuki, 2003).
The molecular and functional characteristics of Ripped pocked (Rpk) were examined. Expression of an epitope-tagged Rpk construct in COS-7 cells generated a 73-kD glycoprotein that can be deglycosylated to its predicted molecular mass of 65 kD. On Northern analysis, an rpk probe detected transcripts in only embryonic and adult RNA, where a major 3.4-kb transcript was observed, as well as two smaller less-abundant messages (Adams, 1998).
To determine the embryonic expression pattern of rpk
transcripts, in situ hybridization to whole
mount embryos was performed using an antisense rpk probe. In contrast
to ppk transcripts, rpk transcripts are detected in early
stage (0-3 h) embryos, but are not present in later stages
of embryogenesis. Furthermore, in early
stage embryos, rpk transcripts are not localized to a specific embryonic region or cell type. In Drosophila embryos, zygotic transcription does not initiate until the third
hour of development. Because rpk mRNA is detected in
embryos before the initiation of zygotic transcription, this result suggests that embryonic rpk message is of maternal
origin, and that Rpk may play a role in early development (Adams, 1998).
When expressed in Xenopus oocytes, Rpk generates small
whole cell Na+ currents that are reversibly blocked by
amiloride. Rpk is impermeable to K+, as
shown by the elimination of inward current when external Na+ is replaced with K+. Thus, in contrast to
Ppk, Rpk forms functional ion channels by itself (Adams, 1998).
In several C. elegans degenerins, mutation of a specific
residue near M2, the 'Deg' mutation, causes a dominant
form of neurodegeneration suggestive of constitutive ion
channel activity. Similarly, BNC1 containing a
Deg mutation (BNC1G430V) is activated, producing much
larger currents in Xenopus oocytes. To learn whether
Rpk could also be activated by the Deg mutation, a valine residue was incorporated at the appropriate position
(residue 524). Like wild-type Rpk, RpkA524V generates
Na+-selective currents that are reversibly inhibited by
amiloride. However, RpkA524V currents
are 20-50 times larger than wild-type Rpk currents. This indicates that the Deg mutation activates Rpk.
RpkA524V is slightly more permeable to Li+ than Na+ but was impermeable to K+. RpkA524V is significantly more sensitive to amiloride than wild-type Rpk. Gadolinium, an inhibitor of mechanosensation and some stretch-activated channels, also reversibly inhibits RpkA524V current (Adams, 1998).
Individual DEG/ENaC proteins are subunits that form
homo- or hetero-multimeric ion channels. Because DEG/
ENaC proteins with Deg mutations produce a genetically
dominant phenotype in C. elegans, it is thought that the Deg
mutation in one or a few subunits might activate the channel complex, producing larger currents. The hypothesis that the Deg mutation is dominant at the molecular level was tested by asking if channels composed of both wild-type
Rpk and RpkA524V would generate small or large Na+ currents. Coexpression of Rpk and RpkA524V generate
large Na+ currents that are similar in size to those generated by RpkA524V alone. However, the amiloride sensitivity of the current, was similar to that generated by wild-type
Rpk alone. These observations indicate that
the increase in current amplitude depends on RpkA524V, and the low amiloride sensitivity depends on wild-type
Rpk. Thus, the data suggest that at least two subunits
combine to produce multimeric channels, that the A524V
mutation dominantly activates the channel, and that Ala524
dominantly determines amiloride sensitivity. Gadolinium
also inhibits wild-type Rpk current, and gadolinium sensitivity is not significantly altered by the presence of
wild-type Rpk in a complex with RpkA524V.
Coexpression of Ppk with Rpk or RpkA524V does not significantly alter the amount, ionic selectivity, or amiloride
sensitivity of Rpk or RpkA524V current. Thus,
it appears that Ppk and Rpk are not subunits of the
same ion channel but likely have distinct physiological roles (Adams, 1998).
The Drosophila tracheal system and mammalian airways are branching networks of tubular epithelia that deliver oxygen to the organism. In mammals, the epithelial Na+ channel (ENaC) helps clear liquid from airways at the time of birth and removes liquid from the airspaces in adults. The hypothesis was tested that related Drosophila degenerin (DEG)/ENaC family members might play a similar role in the fly. Among 16 Drosophila DEG/ENaC genes, called pickpocket (PPK) genes, 9 were found expressed in the tracheal system. By in situ hybridization, expression appeared in late-stage embryos after tracheal tube formation, with individual PPK genes showing distinct temporal and spatial expression patterns as development progressed. Promoters for several PPK genes drove reporter gene expression in the larval and adult tracheal systems. Adding the DEG/ENaC channel blocker amiloride to the medium inhibits liquid clearance from the trachea of first instar larvae. Moreover, when RNA interference is used to silence PPK4 and PPK11, larvae fail to clear tracheal liquid. These data suggest substantial molecular diversity of DEG/ENaC channel expression in the Drosophila tracheal system where the PPK proteins likely play a role in Na+ absorption. Extensive similarities between Drosophila and mammalian airways offer opportunities for genetic studies that may decipher further the structure and function of DEG/ENaC proteins and development of the airways (Liu, 2003a).
The ability to detect salt is critical for the survival of terrestrial animals. Based on amiloride-dependent inhibition, the receptors that detect salt have been postulated to be DEG/ENaC channels. Drosophila DEG/ENaC genes Pickpocket11 (ppk11) and Pickpocket19 (ppk19) are expressed in the larval taste-sensing terminal organ and in adults on the taste bristles of the labelum, the legs, and the wing margins. When Ppk11 or Ppk19 function is disrupted, larvae lose their ability to discriminate low concentrations of Na+ or K+ from water, and the electrophysiologic responses to low salt concentrations are attenuated. In both larvae and adults, disrupting Ppk11 or Ppk19 affects the behavioral response to high salt concentrations. In contrast, the response of larvae to sucrose, pH 3, and several odors remains intact. These results indicate that the DEG/ENaC channels Ppk11 and Ppk19 play a key role in detecting Na+ and K+ salts (Liu, 2003b).
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