Maintenance of a constant cell volume in the face of osmotic stress is an evolutionarily ancient homeostatic process. Over the last two decades physiologists have gained an impressive understanding of the "volume-sensitive" channels, cotransporters, exchangers, metabolic pathways, and genes that are responsible for modulating intracellular solute content and cell volume. This review focuses on one part of this story, the characteristics and osmoregulatory functions of volume-sensitive anion channels. Three distinct types of swelling-activated anion channels have been observed and studied extensively in animal cells. These channels include 1) ClC-2, which is a member of the ClC family of voltage-gated anion channels, 2) an outwardly rectifying intermediate conductance channel, and 3) a large-conductance or "maxi" channel. In addition to these three channels, several other less well-characterized anion channels have been observed. This review discusses the electrophysiological and molecular biological characteristics and regulation of these channels. The possible roles different types of anion channels might play in cell volume homeostasis are also discussed.

The transcription factor SKN-1 protects Caenorhabditis elegans from stress and promotes longevity. SKN-1 is regulated by diverse signals that control metabolism, development, and stress responses, but the mechanisms of regulation and signal integration are unknown. We screened the C. elegans genome for regulators of cytoprotective gene expression and identified a new SKN-1 regulatory pathway. SKN-1 protein levels, nuclear accumulation, and activity are repressed by the WD40 repeat protein WDR-23, which interacts with the CUL-4/DDB-1 ubiquitin ligase to presumably target the transcription factor for proteasomal degradation. WDR-23 regulates SKN-1 target genes downstream from p38 mitogen-activated protein kinase, glycogen synthase kinase 3, and insulin-like receptor pathways, suggesting that phosphorylation of SKN-1 may function to modify its interaction with WDR-23 and/or CUL-4/DDB-1. These findings define the mechanism of SKN-1 accumulation in the cell nucleus and provide a new mechanistic framework for understanding how phosphorylation signals are integrated to regulate stress resistance and longevity.In response to xenobiotic and oxidative stress, eukaryotic cells activate conserved pathways that increase the expression of phase II detoxification enzymes that scavenge free radicals, synthesize glutathione, and catalyze conjugation reactions that increase xenobiotic solubility and excretion (20). Phase II detoxification plays a central role in preventing age-related diseases, such as cancer and neurodegeneration (34, 39), and in mediating the multidrug resistance of pathogenic fungi, helminthes, and tumor cells (30,44,57).Phase II detoxification in Caenorhabditis elegans is controlled by the transcription factor SKN-1 (1), which promotes stress resistance and longevity (1,2,31,55). In nonstressed animals, SKN-1 is constitutively localized in the nuclei of hypothalamus-like (ASI) neurons, where it is required for life span extension by dietary restriction (5). SKN-1 is absent from the nuclei of other cell types except during exposure to oxidative stress and xenobiotics, which induces its accumulation in intestinal-cell nuclei, where it activates the expression of phase II detoxification genes (1,2,15,27,55). Despite the central role of SKN-1 in stress resistance and longevity, the mechanisms that control nuclear accumulation of the transcription factor are unknown.Phosphorylation of SKN-1 by glycogen synthase kinase 3 (GSK-3) inhibits nuclear accumulation (2). Nuclear accumulation is also inhibited by phosphorylation via SGK-1, AKT-1, and AKT-2 kinases downstream from the insulin-like receptor DAF-2 (55). Conversely, accumulation of SKN-1 in the nucleus is promoted by phosphorylation by a p38 mitogen-activated protein kinase (MAPK) cascade (23) and the activities of at least four other protein kinases (31). Phosphorylation of SKN-1 by these diverse kinases allows C. elegans to integrate phase II gene expression with metabolism, development, stress, and aging (55). However, the mechanisms by which phosphorylation...

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The detection, stabilization, and repair of stress-induced damage are essential requirements for cellular life. All cells respond to osmotic stress-induced water loss with increased expression of genes that mediate accumulation of organic osmolytes, solutes that function as chemical chaperones and restore osmotic homeostasis. The signals and signaling mechanisms that regulate osmoprotective gene expression in animal cells are poorly understood. Here, we show that gpdh-1 and gpdh-2, genes that mediate the accumulation of the organic osmolyte glycerol, are essential for survival of the nematode Caenorhabditis elegans during osmotic stress. Expression of GFP driven by the gpdh-1 promoter (P gpdh-1::GFP) is detected only during hypertonic stress but is not induced by other stressors. Using Pgpdh-1::GFP expression as a phenotype, we screened Ϸ16,000 genes by RNAi feeding and identified 122 that cause constitutive activation of gpdh-1 expression and glycerol accumulation. Many of these genes function to regulate protein translation and cotranslational protein folding and to target and degrade denatured proteins, suggesting that the accumulation of misfolded proteins functions as a signal to activate osmoprotective gene expression and organic osmolyte accumulation in animal cells. Consistent with this hypothesis, 73% of these protein-homeostasis genes have been shown to slow age-dependent protein aggregation in C. elegans. Because diverse environmental stressors and numerous disease states result in protein misfolding, mechanisms must exist that discriminate between osmotically induced and other forms of stress-induced protein damage. Our findings provide a foundation for understanding how these damage-selectivity mechanisms function.Caenorhabditis elegans ͉ functional genomics ͉ organic osmolytes ͉ osmotic stress

C. elegans has provided important insights into neuromuscular system function and development. However, the animal's small size limits access to individual neurons and muscle cells for physiological, biochemical, and molecular study. We describe here primary culture methods that allow C. elegans embryonic cells to differentiate into neurons and muscle cells in vitro. Morphological, electrophysiological, and GFP reporter studies demonstrate that the differentiation and functional properties of cultured cells are similar to those observed in vivo. Enriched populations of cells expressing specific GFP reporters can be generated by fluorescence-activated cell sorting. Addition of double-stranded RNA to the culture medium induces dramatic knockdown of targeted gene expression. Primary nematode cell culture provides a new foundation for a wide variety of experimental opportunities heretofore unavailable in the field.