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The inhibitory effect of G?? and G? isoform specificity on ENaC activity.

ABSTRACT: Epithelial Na(+) channel (ENaC) activity, which determines the rate of renal Na(+) reabsorption, can be regulated by G protein-coupled receptors. Regulation of ENaC by G?-mediated downstream effectors has been studied extensively, but the effect of G?? dimers on ENaC is unclear. A6 cells endogenously contain high levels of G?1 but low levels of G?3, G?4, and G?5 were detected by Q-PCR. We tested G?2 combined individually with G?1 through G?5 expressed in A6 cells, after which we recorded single-channel ENaC activity. Among the five ? and ?2 combinations, ?1?2 strongly inhibits ENaC activity by reducing both ENaC channel number (N) and open probability (Po) compared with control cells. In contrast, the other four ?-isoforms combined with ?2 have no significant effect on ENaC activity. By using various inhibitors to probe G?1?2 effects on ENaC regulation, we found that G?1?2-mediated ENaC inhibition involved activation of phospholipase C-? and its enzymatic products that induce protein kinase C and ERK1/2 signaling pathways.

SUBMITTER: Yu L

PROVIDER: S-EPMC3840225 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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The inhibitory effect of Gβγ and Gβ isoform specificity on ENaC activity.

Yu Ling L Al-Khalili Otor O Duke Billie Jeanne BJ Stockand James D JD Eaton Douglas C DC Bao Hui-Fang HF

American journal of physiology. Renal physiology 20130717 9

Epithelial Na(+) channel (ENaC) activity, which determines the rate of renal Na(+) reabsorption, can be regulated by G protein-coupled receptors. Regulation of ENaC by Gα-mediated downstream effectors has been studied extensively, but the effect of Gβγ dimers on ENaC is unclear. A6 cells endogenously contain high levels of Gβ1 but low levels of Gβ3, Gβ4, and Gβ5 were detected by Q-PCR. We tested Gγ2 combined individually with Gβ1 through Gβ5 expressed in A6 cells, after which we recorded single- ...[more]

PMID: 23863469

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Project description:The effects of small molecule ENaC activators N,N,N-trimethyl-2-((4-methyl-2-((4-methyl-1H-indol-3-yl)thio)pentanoyl)oxy)ethanaminium iodide (Compound 1) and N-(2-hydroxyethyl)-4-methyl-2-((4-methyl-1H-indol-3-yl)thio)pentanamide (Compound 2), were tested on the benzamil (Bz)-sensitive NaCl chorda tympani (CT) taste nerve response under open-circuit conditions and under ±60 mV applied lingual voltage-clamp, and compared with the effects of known physiological activators (8-CPT-cAMP, BAPTA-AM, and alkaline pH), and an inhibitor (ionomycin+Ca2+) of ENaC. The NaCl CT response was enhanced at -60 mV and suppressed at +60 mV. In every case the CT response (r) versus voltage (V) curve was linear. All ENaC activators increased the open-circuit response (ro) and the voltage sensitivity (?, negative of the slope of the r versus V curve) and ionomycin+Ca2+ decreased ro and ? to zero. Compound 1 and Compound 2 expressed a sigmoidal-saturating function of concentration (0.25-1 mM) with a half-maximal response concentration (k) of 0.49 and 1.05 mM, respectively. Following treatment with 1 mM Compound 1, 8-CPT-cAMP, BAPTA-AM and pH 10.3, the Bz-sensitive NaCl CT response to 100 mM NaCl was enhanced and was equivalent to the Bz-sensitive CT response to 300 mM NaCl. Plots of ? versus ro in the absence and presence of the activators or the inhibitor were linear, suggesting that changes in the affinity of Na+ for ENaC under different conditions are fully compensated by changes in the apical membrane potential difference, and that the observed changes in the Bz-sensitive NaCl CT response arise exclusively from changes in the maximum CT response (rm). The results further suggest that the agonists enhance and ionomycin+Ca2+ decreases ENaC function by increasing or decreasing the rate of release of Na+ from its ENaC binding site to the receptor cell cytosol, respectively. Irrespective of agonist type, the Bz-sensitive NaCl CT response demonstrated a maximum response enhancement limit of about 75% over control value.

Dataset Information

The inhibitory effect of G?? and G? isoform specificity on ENaC activity.

Publications

The inhibitory effect of Gβγ and Gβ isoform specificity on ENaC activity.

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