The ability to detect variations in humidity is critical for many animals. Birds, reptiles and insects all show preferences for specific humidities that influence their mating, reproduction and geographic distribution. Because of their large surface area to volume ratio, insects are particularly sensitive to humidity, and its detection can influence their survival. Two types of hygroreceptors exist in insects: one responds to an increase (moist receptor) and the other to a reduction (dry receptor) in humidity. Although previous data indicated that mechanosensation might contribute to hygrosensation, the cellular basis of hygrosensation and the genes involved in detecting humidity remain unknown. To understand better the molecular bases of humidity sensing, we investigated several genes encoding channels associated with mechanosensation, thermosensing or water transport. Here we identify two Drosophila melanogaster transient receptor potential channels needed for sensing humidity: CG31284, named by us water witch (wtrw), which is required to detect moist air, and nanchung (nan), which is involved in detecting dry air. Neurons associated with specialized sensory hairs in the third segment of the antenna express these channels, and neurons expressing wtrw and nan project to central nervous system regions associated with mechanosensation. Construction of the hygrosensing system with opposing receptors may allow an organism to very sensitively detect changes in environmental humidity.

G protein-coupled inward rectifier K ؉ (GIRK) channels regulate cellular excitability and neurotransmission. The GIRK channels are activated by a number of inhibitory neurotransmitters through the G protein ␤␥ subunit (G␤␥) after activation of G protein-coupled receptors and inhibited by several excitatory neurotransmitters through activation of phospholipase C. If the inhibition is produced by PKC, there should be PKC phosphorylation sites in GIRK channel proteins. To identify the PKC phosphorylation sites, we performed systematic mutagenesis analysis on GIRK4 and GIRK1 subunits expressed in Xenopus oocytes. Our data showed that the heteromeric GIRK1͞GIRK4 channels were inhibited by a PKC activator phorbol 12-myristate 13-acetate (PMA) through reduction of single channel open-state probability. Direct application of the catalytic subunit of PKC to excised patches had a similar inhibitory effect. This inhibition was greatly eliminated by mutation of Ser-185 in GIRK1 and Ser-191 in GIRK4 that remained G protein sensitive. The PKC-dependent phosphorylation seems to mediate the channel inhibition by the excitatory neurotransmitter substance P (SP) as specific PKC inhibitors and mutation of these PKC phosphorylation sites abolished the SP-induced inhibition of GIRK1͞GIRK4 channels. Thus, these results indicate that the PKC-dependent phosphorylation underscores the inhibition of GIRK channels by SP, and Ser-185 in GIRK1 and Ser-191 in GIRK4 are the PKC phosphorylation sites.play an important role in controlling membrane excitability and synaptic transmission (1, 2). Four members of GIRK channels have been cloned in mammals, i.e., GIRK1 through GIRK4 (Kir3.1 through Kir3.4). These channels are expressed in the heart, brain, and endocrine tissues (3, 4). Stoichiometric studies indicate that a functional GIRK channel consists of four homomeric subunits or two pairs of heteromeric subunits (3, 4). Coassembly of GIRK1 with GIRK4 forms muscarinic receptorcoupled K ϩ channels mainly in the heart (2, 5). The GIRK channels are activated by certain inhibitory transmitters and hormones. On activation of G i/o -coupled receptors by the neurotransmitters or hormones, the G protein ␤␥ subunit (G ␤␥ ) dissociated from the heterotrimeric G ␣␤␥ directly interacts with cytosolic domains of GIRK channel proteins (6-9), and activates the channels through a mechanism that involves movement of pore-lining helices (10-12). Critical domains and amino acids have been identified to be responsible for the basal G ␤␥ -dependent and agonist-induced channel activities (6,7,13,14). Furthermore, the GIRK channel activation may rely on local phosphatidylinositol-4,5-biphosphate (PIP 2 ) in membrane microdomains (15, 16) or cytosolic Na ϩ (16,17).In addition to direct activation by G ␤␥ , GIRK channels are inhibited by a number of excitatory neurotransmitters or hormones, such as acetylcholine (18-20), thyroid-stimulating hormone (TSH)-releasing hormones (21), bombesin (22), substance P (SP) (23), and glutamate (24). These transmitters or hormones sh...

The acid-sensing ion channel-1a (ASIC1a) is composed of 3 subunits and is activated by a decrease in extracellular pH. It plays an important role in diseases associated with a reduced pH and production of oxidants. Previous work showed that oxidants reduce ASIC1a currents. However, the effects on channel structure and composition are unknown. We found that ASIC1a formed inter-subunit disulfide bonds and the oxidant H 2O2 increased this link between subunits. Cys-495 in the ASIC1a C terminus was particularly important for inter-subunit disulfide bond formation, although other C-terminal cysteines contributed. Inter-subunit disulfide bonds also produced some ASIC1a complexes larger than trimers. Inter-subunit disulfide bond formation reduced the proportion of ASIC1a located on the cell surface and contributed to the H 2O2-induced decrease in H ؉ -gated current. These results indicate that channel function is controlled by disulfide bond formation between intracellular residues on distinct ASIC1a subunits. They also suggest a mechanism by which the redox state can dynamically regulate membrane protein activity by forming intracellular bridges.ACCN2 ͉ oxidation ͉ trimer ͉ acid-sensing ion channel A cid-sensing ion channels (ASICs) are members of the degenerin/epithelial Na ϩ channel family of non-voltagegated cation channels (1-3). There are 4 ASIC genes (ASIC1 to ASIC4) and 2 splice variants (a and b) for ASIC1 and ASIC2. ASIC subunits have intracellular N and C termini, 2 transmembrane domains, and a large extracellular loop. A trimer of subunits comprises the channel (4), which can be composed of homologous or heterologous subunits. ASIC1a, -1b, -2a, and -3 are activated by extracellular protons and conduct Na ϩ ; ASIC1a homo-multimers also conduct Ca 2ϩ (1-3). ASIC1a is widely expressed in the brain (5-7). Within individual neurons, it localizes to the cell soma and to dendritic spines, where it mediates an acid-activated increase in [Ca 2ϩ ] i and regulates spine number (8-10). ASIC1a is required for normal long-term potentiation (9), learning and memory (9), and both conditioned and innate fear-related behavior (7,11,12). In addition to its role in normal brain physiology, ASIC1a contributes to several pathophysiological conditions. Disrupting the ASIC1a gene or inhibiting ASIC1a protected animals from ischemia-induced brain damage (13,14), slowed disease progression in a mouse model of multiple sclerosis (15), and reduced disease in a mouse Parkinson model (16). ASIC1a also contributed to the termination of seizures (17). In all of these conditions, acidosis plays an important role (15,(17)(18)(19). In addition, these pathological conditions all generate free radicals, which in turn can contribute to the progression of disease (20)(21)(22)(23)(24).Because acidotic and oxidizing environments coexist in diseases involving ASIC1a, several groups have tested the effect of redox reagents on ASIC1a function. Reducing agents increased ASIC1a current amplitude (25-27). Conversely, extracellular modification with a ...

Abstract-ATP-sensitive K ϩ channels (K ATP ) couple intermediary metabolism to cellular activity, and may play a role in the autoregulation of vascular tones. Such a regulation requires cellular mechanisms for sensing O 2 , CO 2 , and pH. Our recent studies have shown that the pancreatic K ATP isoform (Kir6.2/SUR1) is regulated by CO 2 /pH. To identify the vascular K ATP isoform(s) and elucidate its response to hypercapnic acidosis, we performed these studies on vascular smooth myocytes (VSMs). Whole-cell and single-channel currents were studied on VSMs acutely dissociated from mesenteric arteries and HEK293 cells expressing Kir6.1/SUR2B. Hypercapnic acidosis activated an inward rectifier current that was K ϩ -selective and sensitive to levcromakalim and glibenclamide with unitary conductance of Ϸ35pS. The maximal activation occurred at pH 6.5 to 6.8, and the current was inhibited at pH 6.2 to 5.9. The cloned Kir6.1/SUR2B channel responded to hypercapnia and intracellular acidification in an almost identical pattern to the VSM current. In situ hybridization histochemistry revealed expression of Kir6.1/SUR2B mRNAs in mesenteric arteries. Hypercapnia produced vasodilation of the isolated and perfused mesenteric arteries. Pharmacological interference of the K ATP channels greatly eliminated the hypercapnic vasodilation. These results thus indicate that the Kir6.1/SUR2B channel is a critical player in the regulation of vascular tones during hypercapnic acidosis. Although these K ϩ channels are sensitive to ATP, studies have shown that they are also modulated by other nucleotides and phospholipids. [3][4][5][6][7][8][9] Previous studies have shown that K ATP channels in myocardium and insulin-secreting cell line are activated by intracellular acidification. 10,11 Similar activation was observed in the cloned pancreatic K ATP isoform (Kir6.2/ SUR1) during hypercapnic acidosis. [12][13][14][15] The regulation of K ATP by protons is particularly significant, because pH alterations occur in a large variety of physiological and pathophysiological conditions and are more frequently seen than sole energy depletion.The pH sensitivity may allow the K ATP channels to play a role in autoregulation of vascular tones. Experimental evidence suggests that the K ATP channels may be involved in reactive hyperemia, as sulfonylurea blocks hyperemic vasodilation. 16,17 The autoregulation occurs in most tissues including the heart and brain in which it underlies the cardioprotective effect of ischemic preconditioning and the activitydependent regulation of cerebral circulation. 18 -21 The reactive hyperemia is produced by hypoxia, hypercapnia, acidosis, and accumulation of other metabolic products in local tissues.Because under most physiological and pathophysiological conditions, ATP levels may not readily drop to submillimolar concentrations in cells to activate K ATP channels, 1,2 demonstration of the CO 2 /pH sensitivity of the K ATP channels becomes critical for understanding the molecular basis of the autoregulation of vascular to...