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Identification of epithelial Na+ channel (ENaC) intersubunit Cl- inhibitory residues suggests a trimeric alpha gamma beta channel architecture.

ABSTRACT: The extracellular domain of the epithelial Na(+) channel (ENaC) is exposed to a wide range of anion concentrations in the kidney. We have previously demonstrated that extracellular Cl(-) inhibits ENaC activity. To identify sites involved in Cl(-) inhibition, we mutated residues in the extracellular domain of ?-, ?-, and ?ENaC that are homologous to the Cl(-) binding site in acid-sensing ion channel 1a and tested the effect of Cl(-) on the activity of ENaC expressed in Xenopus oocytes. We identified two Cl(-) inhibitory sites in ENaC. One is formed by residues in the thumb domain of ?ENaC and the palm domain of ?ENaC. Mutation of residues at this interface decreased Cl(-) inhibition and decreased Na(+) self-inhibition. The second site is formed by residues at the interface of the thumb domain of ?ENaC and the palm domain of ?ENaC. Mutation of these residues also decreased Cl(-) inhibition yet had no effect on Na(+) self-inhibition. In contrast, mutations in the thumb domain of ?ENaC and palm of ?ENaC had little or no effect on Cl(-) inhibition or Na(+) self-inhibition. The data demonstrate that Cl(-) inhibits ENaC activity by two distinct Na(+)-dependent and Na(+)-independent mechanisms that correspond to the two functional Cl(-) inhibitory sites. Furthermore, based on the effects of mutagenesis on Cl(-) inhibition, the additive nature of mutations, and on differences in the mechanisms of Cl(-) inhibition, the data support a model in which ENaC subunits assemble in an ??? orientation (listed clockwise when viewed from the top).

SUBMITTER: Collier DM

PROVIDER: S-EPMC3057804 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Identification of epithelial Na+ channel (ENaC) intersubunit Cl- inhibitory residues suggests a trimeric alpha gamma beta channel architecture.

Collier Daniel M DM Snyder Peter M PM

The Journal of biological chemistry 20101213 8

The extracellular domain of the epithelial Na(+) channel (ENaC) is exposed to a wide range of anion concentrations in the kidney. We have previously demonstrated that extracellular Cl(-) inhibits ENaC activity. To identify sites involved in Cl(-) inhibition, we mutated residues in the extracellular domain of α-, β-, and γENaC that are homologous to the Cl(-) binding site in acid-sensing ion channel 1a and tested the effect of Cl(-) on the activity of ENaC expressed in Xenopus oocytes. We identif ...[more]

PMID: 21149458

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Project description:BACKGROUND: In rodents, dietary Na+ deprivation reduces gustatory responses of primary taste fibers and central taste neurons to lingual Na+ stimulation. However, in the rat taste bud cells Na+ deprivation increases the number of amiloride sensitive epithelial Na+ channels (ENaC), which are considered as the "receptor" of the Na+ component of salt taste. To explore the mechanisms, the expression of the three ENaC subunits (alpha, beta and gamma) in taste buds were observed from rats fed with diets containing either 0.03% (Na+ deprivation) or 1% (control) NaCl for 15 days, by using in situ hybridization and real-time quantitative RT-PCR (qRT-PCR). Since BDNF/TrkB signaling is involved in the neural innervation of taste buds, the effects of Na+ deprivation on BDNF and its receptor TrkB expression in the rat taste buds were also examined. RESULTS: In situ hybridization analysis showed that all three ENaC subunit mRNAs were found in the rat fungiform taste buds and lingual epithelia, but in the vallate and foliate taste buds, only alpha ENaC mRNA was easily detected, while beta and gamma ENaC mRNAs were much less than those in the fungiform taste buds. Between control and low Na+ fed animals, the numbers of taste bud cells expressing alpha, beta and gamma ENaC subunits were not significantly different in the fungiform, vallate and foliate taste buds, respectively. Similarly, qRT-PCR also indicated that Na+ deprivation had no effect on any ENaC subunit expression in the three types of taste buds. However, Na+ deprivation reduced BDNF mRNA expression by 50% in the fungiform taste buds, but not in the vallate and foliate taste buds. The expression of TrkB was not different between control and Na+ deprived rats, irrespective of the taste papillae type. CONCLUSION: The findings demonstrate that dietary Na+ deprivation does not change ENaC mRNA expression in rat taste buds, but reduces BDNF mRNA expression in the fungiform taste buds. Given the roles of BDNF in survival of cells and target innervation, our results suggest that dietary Na+ deprivation might lead to a loss of gustatory innervation in the mouse fungiform taste buds.

Project description:We have previously reported that Dot1a is located in the cytoplasm and nucleus (Reisenauer MR, Anderson M, Huang L, Zhang Z, Zhou Q, Kone BC, Morris AP, Lesage GD, Dryer SE, Zhang W. J Biol Chem 284: 35659-35669, 2009), widely expressed in the kidney as detected by its histone H3K79 methyltransferase activity (Zhang W, Hayashizaki Y, Kone BC. Biochem J 377: 641-651, 2004), and involved in transcriptional control of the epithelial Na(+) channel subunit-alpha gene (alphaENaC) (Zhang W, Xia X, Jalal DI, Kuncewicz T, Xu W, Lesage GD, Kone BC. Am J Physiol Cell Physiol 290: C936-C946, 2006). Aldosterone releases repression of alphaENaC by reducing expression of Dot1a and its partner AF9 (Zhang W, Xia X, Reisenauer MR, Hemenway CS, Kone BC. J Biol Chem 281: 18059-18068, 2006) and by impairing Dot1a-AF9 interaction via Sgk1-mediated AF9 phosphorylation (Zhang W, Xia X, Reisenauer MR, Rieg T, Lang F, Kuhl D, Vallon V, Kone BC. J Clin Invest 117: 773-783, 2007). This network also appears to regulate transcription of several other aldosterone target genes. Here, we provide evidence showing that Dot1a contains at least three potential nuclear localization signals (NLSs). Deletion of these NLSs causes green fluorescent protein-fused Dot1a fusions to localize almost exclusively in the cytoplasm of 293T cells as revealed by confocal microscopy. Deletion of NLSs abolished Dot1a-mediated repression of alphaENaC-promoter luciferase construct in M1 cells. AF9 is widely expressed in mouse kidney. Similar to alphaENaC, the mRNA levels of betaENaC, gammaENaC, and Sgk1 are also downregulated by Dot1a and AF9 overexpression. Small interference RNA-mediated knockdown of Dot1a and AF9 or aldosterone treatment leads to an opposite effect. Using single-cell fluorescence imaging or equivalent short-circuit current in IMCD3 and M1 cells, we show that observed transcriptional alterations correspond to changes in ENaC and Sgk1 protein levels as well as benzamil-sensitive Na(+) transport. In brief, Dot1a and AF9 downregulate Na(+) transport, most likely by regulating ENaC mRNA and subsequent protein expression and ENaC activity.

Dataset Information

Identification of epithelial Na+ channel (ENaC) intersubunit Cl- inhibitory residues suggests a trimeric alpha gamma beta channel architecture.

Publications

Identification of epithelial Na+ channel (ENaC) intersubunit Cl- inhibitory residues suggests a trimeric alpha gamma beta channel architecture.

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