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 |
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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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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. 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Renal physiology</title><addtitle>Am J Physiol Renal Physiol</addtitle><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.</description><subject>Amiloride - pharmacology</subject><subject>Animals</subject><subject>Biological Transport - drug effects</subject><subject>Biological Transport - physiology</subject><subject>Cell Compartmentation - physiology</subject><subject>Cell Fractionation</subject><subject>Cells, Cultured</subject><subject>Detergents</subject><subject>Diuretics - pharmacology</subject><subject>Epithelial Cells - metabolism</subject><subject>Kidney - cytology</subject><subject>Kidney - metabolism</subject><subject>Lipid Bilayers - metabolism</subject><subject>Octoxynol</subject><subject>Sodium - metabolism</subject><subject>Sodium Channels - metabolism</subject><subject>Xenopus laevis</subject><issn>1931-857X</issn><issn>1522-1466</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2003</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpFkM1Lw0AQxRdRbK3-BYLk5C11P5rs5iilfkChFwVvyySZ2C3JJu5uhP73bm3F0xuY994MP0JuGZ0zlvEH2A0OLbRzSjnL5zzKGZnGDU_ZIs_P41wIlqpMfkzIlfc7SiljnF2SCeNCKVGwKdmsBhO22BpoE9_XZuySagvWYptAFcy3CfvE2KTGgO4TbUgdeuMD2JB02JUOLCadqVxf9x0Y66_JRQOtx5uTzsj70-pt-ZKuN8-vy8d1WsWzIRWcZqgyhAVISflCFg1tKEhFBcuhzqUAlYmSFjLLoSg4LTKhSqY45CVWpRIzcn_sHVz_NaIPujO-wraND_Wj15IrWeRCRqM4GuOP3jts9OBMB26vGdUHjvqPo_7lqA8cY-ruVD-WHdb_mRM48QMngnD1</recordid><startdate>200301</startdate><enddate>200301</enddate><creator>Shlyonsky, Vadim G</creator><creator>Mies, Frederique</creator><creator>Sariban-Sohraby, Sarah</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>200301</creationdate><title>Epithelial sodium channel activity in detergent-resistant membrane microdomains</title><author>Shlyonsky, Vadim G ; Mies, Frederique ; Sariban-Sohraby, Sarah</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c391t-3205e85ea4a7702479f0f0a780316ad673a853b09756a99209538b182a6becb83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2003</creationdate><topic>Amiloride - pharmacology</topic><topic>Animals</topic><topic>Biological Transport - drug effects</topic><topic>Biological Transport - physiology</topic><topic>Cell Compartmentation - physiology</topic><topic>Cell Fractionation</topic><topic>Cells, Cultured</topic><topic>Detergents</topic><topic>Diuretics - pharmacology</topic><topic>Epithelial Cells - metabolism</topic><topic>Kidney - cytology</topic><topic>Kidney - metabolism</topic><topic>Lipid Bilayers - metabolism</topic><topic>Octoxynol</topic><topic>Sodium - metabolism</topic><topic>Sodium Channels - metabolism</topic><topic>Xenopus laevis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Shlyonsky, Vadim G</creatorcontrib><creatorcontrib>Mies, Frederique</creatorcontrib><creatorcontrib>Sariban-Sohraby, Sarah</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>American journal of physiology. Renal physiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Shlyonsky, Vadim G</au><au>Mies, Frederique</au><au>Sariban-Sohraby, Sarah</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Epithelial sodium channel activity in detergent-resistant membrane microdomains</atitle><jtitle>American journal of physiology. Renal physiology</jtitle><addtitle>Am J Physiol Renal Physiol</addtitle><date>2003-01</date><risdate>2003</risdate><volume>284</volume><issue>1</issue><spage>F182</spage><epage>F188</epage><pages>F182-F188</pages><issn>1931-857X</issn><eissn>1522-1466</eissn><abstract>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.</abstract><cop>United States</cop><pmid>12388391</pmid><doi>10.1152/ajprenal.00216.2002</doi></addata></record> |
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source | MEDLINE; American Physiological Society; EZB-FREE-00999 freely available EZB journals |
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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