Epithelial sodium channel activity in detergent-resistant membrane microdomains

The activity of epithelial Na(+) selective channels is modulated by various factors, with growing evidence that membrane lipids also participate in the regulation. In the present study, Triton X-100 extracts of whole cells and of apical membrane-enriched preparations from cultured A6 renal epithelia...

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Veröffentlicht in:American journal of physiology. Renal physiology 2003-01, Vol.284 (1), p.F182-F188
Hauptverfasser: Shlyonsky, Vadim G, Mies, Frederique, Sariban-Sohraby, Sarah
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container_title American journal of physiology. Renal physiology
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creator Shlyonsky, Vadim G
Mies, Frederique
Sariban-Sohraby, Sarah
description The activity of epithelial Na(+) selective channels is modulated by various factors, with growing evidence that membrane lipids also participate in the regulation. In the present study, Triton X-100 extracts of whole cells and of apical membrane-enriched preparations from cultured A6 renal epithelial cells were floated on continuous-sucrose-density gradients. Na(+) channel protein, probed by immunostaining of Western blots, was detected in the high-density fractions of the gradients (between 18 and 30% sucrose), which contain the detergent-soluble material but also in the lighter, detergent-resistant 16% sucrose fraction. Single amiloride-sensitive Na(+) channel activity, recorded after incorporation of reconstituted proteoliposomes into lipid bilayers, was exclusively localized in the 16% sucrose fraction. In accordance with other studies, high- and low-density fractions of sucrose gradients likely represent membrane domains with different lipid contents. However, exposure of the cells to cholesterol-depleting or sphingomyelin-depleting agents did not affect transepithelial Na(+) current, single-Na(+) channel activity, or the expression of Na(+) channel protein. This is the first reconstitution study of native epithelial Na(+) channels, which suggests that functional channels are compartmentalized in discrete domains within the plane of the apical cell membrane.
doi_str_mv 10.1152/ajprenal.00216.2002
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subjects Amiloride - pharmacology
Animals
Biological Transport - drug effects
Biological Transport - physiology
Cell Compartmentation - physiology
Cell Fractionation
Cells, Cultured
Detergents
Diuretics - pharmacology
Epithelial Cells - metabolism
Kidney - cytology
Kidney - metabolism
Lipid Bilayers - metabolism
Octoxynol
Sodium - metabolism
Sodium Channels - metabolism
Xenopus laevis
title Epithelial sodium channel activity in detergent-resistant membrane microdomains
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