ENaC Channels in Oocytes from <i>Xenopus laevis</i> and their Regulation by xShroom1 Protein

Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in X. oocytes and is required for the expression of amiloride sensitive sodium channels (ENaC). Oocytes were injected with a, b, and g mENaC and xShroom1 sense or antisense oligonucleotides. We used voltage clamp techniques to study the amiloride-sensitive Na(+) currents (INa((amil))). We observed a marked reduction in INa((amil)) in oocytes co-injected with xShroom1 antisense. Oocytes expressing a DEG mutant â-mENaC subunit (â-S518K) with an open probability of 1 had enhanced INa((amil)) although these currents were also reduced when co-injected with xShroom1 antisense. Addition of low concentration (20 ng/ml) of trypsin which activates the membrane-resident ENaC channels led to a slow increase in INa((amil)) in oocytes with xShroom1 sense but had no effect on the currents in oocytes coinjected with ENaC and xShroom1 antisense. The same results were obtained with higher concentrations of trypsin (2 ug/ml) exposed during 2.5 min. In addition, fluorescence positive staining of plasma membrane in the oocytes expressing a, b and g mENaC and xShroom1 sense were observed but not in oocytes coinjected with ENaC and xShroom1 antisense oligonucleotides. On this basis, we suggest that xShroom1-dependent ENaC inhibition may be through the number of channels inserted in the membrane.Fil: Assef, Yanina Andrea. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ozu, Marcelo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marino, Gabriela Inés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Galizia, Luciano. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kotsias, Basilio Aristides. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Salty Taste Deficits in CALHM1 Knockout Mice

Genetic ablation of calcium homeostasis modulator 1 (CALHM1), which releases adenosine triphosphate from Type 2 taste cells, severely compromises the behavioral and electrophysiological responses to tastes detected by G protein–coupled recep-tors, such as sweet and bitter. However, the contribution of CALHM1 to salty taste perception is less clear. Here, we evaluated several salty taste–related phenotypes of CALHM1 knockout (KO) mice and their wild-type (WT) controls: 1)  In a conditioned aversion test, CALHM1 WT and KO mice had similar NaCl avoidance thresholds. 2)  In two-bottle choice tests, CALHM1 WT mice showed the classic inverted U-shaped NaCl concentration-preference function but CALHM1 KO mice had a blunted peak response. 3)  In brief-access tests, CALHM1 KO mice showed less avoidance than did WT mice of high concentrations of NaCl, KCl, NH4Cl, and sodium lactate (NaLac). Amiloride further ameliorated the NaCl avoidance of CALHM1 KO mice, so that lick rates to a mixture of 1000 mM NaCl + 10 µM amiloride were statistically indistinguishable from those to water. 4)  Relative to WT mice, CALHM1 KO mice had reduced chorda tympani nerve activity elicited by oral application of NaCl, NaLac, and sucrose but normal responses to HCl and NH4Cl. Chorda tympani responses to NaCl and NaLac were amilorid

Epoxyeicosatrienoic Acids Attenuating Hypotonic-Induced Apoptosis of IMCD Cells via γ-ENaC Inhibition

Inner medulla collecting duct (IMCD) cells are the key part for urinary concentration. Hypotonic stress may trigger apoptosis of IMCD cells and induce renal injury. Epoxyeicosatrienoic acids (EETs) play an important role in anti-apoptosis, but their roles in hypotonic-induced apoptosis of IMCD cells are still unclear. Here we found increasing exogenous 11, 12-EET or endogenous EETs with Ad-CMV-CYP2C23-EGFP transfection decreased apoptosis of IMCD cells induced by hypotonic stress. Moreover, up-regulation of γ-ENaC induced by hypotonic stress was abolished by elevation of exogenous or endogenous EETs. Collectively, this study illustrated that EETs attenuated hypotonic-induced apoptosis of IMCD cells, and that regulation of γ-ENAC may be a possible mechanism contributing to the anti-apoptotic effect of EETs in response to hypotonic stress