Three broad classes of Ca2؉ -activated potassium channels are defined by their respective single channel conductances, i.e. the small, intermediate, and large conductance channels, often termed the SK, IK, and BK channels, respectively. SK channels are likely encoded by three genes, Kcnn1-3, whereas IK and most BK channels are most likely products of the Kcnn4 and Slo (Kcnma1) genes, respectively. IK channels are prominently expressed in cells of the hematopoietic system and in organs involved in salt and fluid transport, including the colon, lung, and salivary glands. IK channels likely underlie the K ؉ permeability in red blood cells that is associated with water loss, which is a contributing factor in the pathophysiology of sickle cell disease. IK channels are also involved in the activation of T lymphocytes. The fluid-secreting acinar cells of the parotid gland express both IK and BK channels, raising questions about their particular respective roles. To test the physiological roles of channels encoded by the Kcnn4 gene, we constructed a mouse deficient in its expression. Kcnn4 null mice were of normal appearance and fertility, their parotid acinar cells expressed no IK channels, and their red blood cells lost K ؉ permeability. The volume regulation of T lymphocytes and erythrocytes was severely impaired in Kcnn4 null mice but was normal in parotid acinar cells. Despite the loss of IK channels, activated fluid secretion from parotid glands was normal. These results confirm that IK channels in red blood cells, T lymphocytes, and parotid acinar cells are indeed encoded by the Kcnn4 gene. The role of these channels in water movement and the subsequent volume changes in red blood cells and T lymphocytes is also confirmed. Surprisingly, Kcnn4 channels appear to play no required role in fluid secretion and regulatory volume decrease in the parotid gland.It has become clear that multiple types of Ca 2ϩ -activated potassium channels underlie a wide range of distinct physiological processes. Physiological and pharmacological analyses have subdivided Ca 2ϩ -activated potassium channels into three groups, i.e. the small, intermediate, and large conductance channels, often termed SK, 1 IK, and BK channels, respectively. IK channels, as their designation implies, have a single channel conductance intermediate between the SK and BK channels. These three functional groups have rather distinct pharmacological profiles, and SK, IK, and BK channels can be specifically blocked by apamin, clotrimazole, and paxilline, respectively. SK and IK channels are encoded by four genes of the KCNN gene family. The three SK channels, KCNN1-3, share ϳ70 -75% amino acid identity. The IK channel KCNN4 is encoded by a protein sharing only ϳ40% amino acid identity with each of the three SK channels, KCNN1-3.

Death of the nematode Caenorhabditis elegans involves a conserved necrotic cell death cascade which generates endogenous blue anthranilate fluorescence, allowing death to be visualized.

Gel-forming mucins are major contributors to the viscoelastic properties of mucus secretion. Currently, four gel-forming mucin genes have been identified: MUC2, MUC5AC, MUC5B, and MUC6. All these genes have five major cysteine-rich domains (four von Willebrand factor [vWF] C or D domains and one Cystine-knot [CT] domain) as their distinctive features, in contrast to other non-gel-forming type of mucins. The CT domain is believed to be involved in the initial mucin dimer formation and have very succinct relationship between different gel-forming mucins across different species. Because of gene duplication and evolutional modification, it is very likely that other gel-forming mucin genes exist. To search for new gel-forming mucin candidate genes, a "Hidden Markov Model"(HMM) was built from the common features of the CT domains of those gel-forming mucins. By using this model to screen all protein databases as well as the six-frame translated expression sequence tag and translated human genomic databases, we identified a locus located at the peri-centromere region of human chromosome 12 and the corresponding homologous region of mouse chromosome 15. We cloned the 3' end of this gene and its mouse homolog. We found one vWF C domain, one CT domain, and various mucin-like threonine/serine-rich repeats. Phylogenetic analysis indicated the close relationship between this gene and the submaxillary mucin from porcine and bovine. A polydispersed signal was observed on the Northern blot, which indicates very large mRNA size. Further analysis of the upstream genomic sequences generated from human and mouse genome projects revealed three additional vWF D domains and many mucin-like threonine/serine-rich repeats. The expression of this gene is restricted to the mucous cells of various glandular tissues, including sublingual gland, submandibular gland, and submucosal gland of the trachea. Based on the chronological convention, we have given the name MUC19 to the human ortholog and Muc19 to the mouse.

CLH-3b is a Caenorhabditis elegans ClC anion channel that is expressed in the worm oocyte. The channel is activated during oocyte meiotic maturation and in response to cell swelling by serine/threonine dephosphorylation events mediated by the type 1 phosphatases GLC-7α and GLC-7β. We have now identified a new member of the Ste20 kinase superfamily, GCK-3, that interacts with the CLH-3b COOH terminus via a specific binding motif. GCK-3 inhibits CLH-3b in a phosphorylation-dependent manner when the two proteins are coexpressed in HEK293 cells. clh-3 and gck-3 are expressed predominantly in the C. elegans oocyte and the fluid-secreting excretory cell. Knockdown of gck-3 expression constitutively activates CLH-3b in nonmaturing worm oocytes. We conclude that GCK-3 functions in cell cycle– and cell volume–regulated signaling pathways that control CLH-3b activity. GCK-3 inactivates CLH-3b by phosphorylating the channel and/or associated regulatory proteins. Our studies provide new insight into physiologically relevant signaling pathways that control ClC channel activity and suggest novel mechanisms for coupling cell volume changes to cell cycle events and for coordinately regulating ion channels and transporters that control cellular Cl− content, cell volume, and epithelial fluid secretion.

2-Hydroxyglutarate (2-HG) is an important epigenetic regulator, with potential roles in cancer and stem cell biology. The D-(R)-enantiomer (D-2-HG) is an oncometabolite generated from ␣-ketoglutarate (␣-KG) by mutant isocitrate dehydrogenase, whereas L-(S)-2-HG is generated by lactate dehydrogenase and malate dehydrogenase in response to hypoxia. Because acidic pH is a common feature of hypoxia, as well as tumor and stem cell microenvironments, we hypothesized that pH may regulate cellular 2-HG levels. Herein we report that cytosolic acidification under normoxia moderately elevated 2-HG in cells, and boosting endogenous substrate ␣-KG levels further stimulated this elevation. Studies with isolated lactate dehydrogenase-1 and malate dehydrogenase-2 revealed that generation of 2-HG by both enzymes was stimulated severalfold at acidic pH, relative to normal physiologic pH. In addition, acidic pH was found to inhibit the activity of the mitochondrial L-2-HG removal enzyme L-2-HG dehydrogenase and to stimulate the reverse reaction of isocitrate dehydrogenase (carboxylation of ␣-KG to isocitrate). Furthermore, because acidic pH is known to stabilize hypoxia-inducible factor (HIF) and 2-HG is a known inhibitor of HIF prolyl hydroxylases, we hypothesized that 2-HG may be required for acid-induced HIF stabilization. Accordingly, cells stably overexpressing L-2-HG dehydrogenase exhibited a blunted HIF response to acid. Together, these results suggest that acidosis is an important and previously overlooked regulator of 2-HG accumulation and other oncometabolic events, with implications for HIF signaling.The field of cancer biology has long been rapt by the notion of a cancer-specific metabolic phenotype, perhaps most famously embodied in the "Warburg effect," wherein glycolytic metabolism predominates in cancer cells despite O 2 availability and largely intact mitochondrial respiratory function (1). A prominent feature of the cancer metabolic phenotype (reviewed in Ref. 2) is an elevated level of the small metabolic acid 2-hydroxyglutarate (2-HG), 2 derived from the TCA cycle intermediate ␣-ketoglutarate (␣-KG) (3). The D-(R)-enantiomer of 2-HG (D-2-HG) was shown to be generated by mutant forms of isocitrate dehydrogenase (ICDH) that are associated with a variety of cancers including aggressive gliomas (4). In addition, more recently the L-(S)-enantiomer (L-2-HG) was shown to be generated under hypoxic conditions by lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) (5, 6). We have also reported elevated D/L-2-HG levels in the heart following ischemic preconditioning (7).In addition to synthesis, 2-HG levels are regulated by a pair of dehydrogenases that convert 2-HG back to ␣-KG (i.e. L-2-HGDH and D-2-HGDH). Mutations in these enzymes manifest as the hydroxyglutaric acidurias, devastating inherited metabolic diseases with symptoms including epilepsy and cerebellar ataxia (8 -11). However, the importance of these dehydrogenases in regulating 2-HG levels in other settings is not clear.The downstream signaling roles ...

Na؉ /H ؉ exchangers are involved in cell volume regulation, fluid secretion and absorption, and pH homeostasis. NHX-2 is a Caenorhabditis elegans Na ؉ /H ؉ exchanger expressed exclusively at the apical membrane of intestinal epithelial cells. The inactivation of various intestinal nutrient transport proteins has been shown previously to influence aging via metabolic potential and a mechanism resembling caloric restriction. We report here a functional coupling of NHX-2 activity with nutrient uptake that results in long lived worms. Gene inactivation of nhx-2 by RNAi led to a loss of fat stores in the intestine and a 40% increase in longevity. The NHX-2 protein was coincidentally expressed with OPT-2, an oligopeptide transporter that is driven by a transmembrane proton gradient and that is also known to be involved in fat accumulation. Gene inactivation of opt-2 led to a phenotype resembling that of nhx-2, although not as severe. In order to explore this potential functional interaction, we combined RNA interference with a genetically encoded, fluorescence-based reagent to measure intestinal intracellular pH (pH i ) in live worms under physiological conditions. Our results suggest first that OPT-2 is the main dipeptide uptake pathway in the nematode intestine, and second that dipeptide uptake results in intestinal cell acidification, and finally that recovery following dipeptide-induced acidification is normally a function of NHX-2. The loss of NHX-2 protein results in decreased steady-state intestinal cell pH i , and we hypothesize that this change perturbs proton-coupled nutrient uptake processes such as performed by OPT-2. Our data demonstrate a functional role for a Na ؉ /H ؉ exchanger in nutrient absorption in vivo and lays the groundwork for examining integrated acid-base physiology in a non-mammalian model organism.

No abstract

ClC-2 is localized to the apical membranes of secretory epithelia where it has been hypothesized to play a role in fluid secretion. Although ClC-2 is clearly the inwardly rectifying anion channel in several tissues, the molecular identity of the hyperpolarization-activated Cl ؊ current in other organs, including the salivary gland, is currently unknown. To determine the nature of the hyperpolarization-activated Cl ؊ current and to examine the role of ClC-2 in salivary gland function, a mouse line containing a targeted disruption of the Clcn2 gene was generated. The resulting homozygous Clcn2 ؊/؊ mice lacked detectable hyperpolarization-activated chloride currents in parotid acinar cells and, as described previously, displayed postnatal degeneration of the retina and testis. The magnitude and biophysical characteristics of the volume-and calcium-activated chloride currents in these cells were unaffected by the absence of ClC-2. Although ClC-2 appears to contribute to fluid secretion in some cell types, both the initial and sustained salivary flow rates were normal in Clcn2 ؊/؊ mice following in vivo stimulation with pilocarpine, a cholinergic agonist. In addition, the electrolytes and protein contents of the mature secretions were normal. Because ClC-2 has been postulated to contribute to cell volume control, we also examined regulatory volume decrease following cell swelling. However, parotid acinar cells from Clcn2 ؊/؊ mice recovered volume with similar efficiency to wild-type littermates. These data demonstrate that ClC-2 is the hyperpolarization-activated Cl ؊ channel in salivary acinar cells but is not essential for maximum chloride flux during stimulated secretion of saliva or acinar cell volume regulation.