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 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.
Shlyonsky
Vadim G
VG
Laboratory of Physiology and Physiopathology, Université Libre de Bruxelles, Belgium.
Mies
Frederique
F
Sariban-Sohraby
Sarah
S
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002451:Cell Compartmentation / Q000502:physiology*; D002458:Cell Fractionation; D002478:Cells, Cultured; D003902:Detergents; D004232:Diuretics / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism*; D017830:Octoxynol; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000378:metabolism*; D014982:Xenopus laevis
D003902:Detergents; D004232:Diuretics; D008051:Lipid Bilayers; D015222:Sodium Channels; D000584:Amiloride; D017830:Octoxynol; D012964:Sodium