Cholesterol depletion of the plasma membrane prevents activation of the epithelial sodium channel (ENaC) by SGK1.
The lipid environment of the epithelial sodium channel (ENaC) and its possible association with so-called lipid rafts may be relevant to its function. The aim of our study was to confirm the association of ENaC with lipid rafts and to analyze the effect of cholesterol depletion of the plasma membrane by methyl-beta-cyclodextrin (MbetaCD) on channel function and regulation. Using sucrose density gradient centrifugation we demonstrated that a significant portion of ENaC protein distributes to low density fractions thought to be typical lipid raft fractions. Importantly, cholesterol depletion of cell lysate by MbetaCD shifted ENaC to non-raft fractions of higher density. Live cell imaging demonstrated that treatment with MbetaCD largely reduced filipin staining over time, confirming cholesterol depletion of the plasma membrane. For electrophysiological studies intact oocytes were exposed to 20 mM MbetaCD for three hours. MbetaCD treatment had no consistent effect on baseline whole-cell ENaC currents. In addition to the typical single channel conductance of about 5 pS, subconductance states of ENaC were occasionally observed in patches from MbetaCD treated but not from control oocytes. Importantly, in outside-out patch clamp recordings the stimulatory effect of recombinant SGK1 in the pipette solution was essentially abolished in oocytes pretreated with MbetaCD. These results indicate that ENaC activation by cytosolic SGK1 is compromised by removing cholesterol from the plasma membrane. Thus, ENaC activation by SGK1 may require the presence of an intact lipid environment and/or of lipid rafts as signalling platform.
Krueger
Bettina
B
Institut fur Zellulare und Molekulare Physiologie, Erlangen-Nurnberg, Germany.
Haerteis
Silke
S
Yang
Limin
L
Hartner
Andrea
A
Rauh
Robert
R
Korbmacher
Christoph
C
Diakov
Alexei
A
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000378:metabolism*; D055724:Electrophysiological Phenomena; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D017874:Immediate-Early Proteins / Q000378:metabolism*; D021962:Membrane Microdomains; D009865:Oocytes / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D014982:Xenopus laevis; D047392:beta-Cyclodextrins / Q000494:pharmacology; D000099184:Serum-Glucocorticoid Regulated Kinases
D002784:Cholesterol; D053503:Epithelial Sodium Channels; D017874:Immediate-Early Proteins; D017346:Protein Serine-Threonine Kinases; D047392:beta-Cyclodextrins; D000099184:Serum-Glucocorticoid Regulated Kinases; C108732:methyl-beta-cyclodextrin