TY - JOUR
T1 - Cholesterol depletion of the plasma membrane prevents activation of the epithelial sodium channel (ENaC) by SGK1
AU - Krueger, Bettina
AU - Haerteis, Silke
AU - Yang, Limin
AU - Hartner, Andrea
AU - Rauh, Robert
AU - Korbmacher, Christoph
AU - Diakov, Alexei
PY - 2009
Y1 - 2009
N2 - 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-β-cyclodextrin (MβCD) 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 MβCD shifted ENaC to non-raft fractions of higher density. Live cell imaging demonstrated that treatment with MβCD largely reduced filipin staining over time, confirming cholesterol depletion of the plasma membrane. For electrophysiological studies intact oocytes were exposed to 20 mM MβCD for three hours. MβCD 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 MβCD 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 MβCD. 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.
AB - 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-β-cyclodextrin (MβCD) 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 MβCD shifted ENaC to non-raft fractions of higher density. Live cell imaging demonstrated that treatment with MβCD largely reduced filipin staining over time, confirming cholesterol depletion of the plasma membrane. For electrophysiological studies intact oocytes were exposed to 20 mM MβCD for three hours. MβCD 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 MβCD 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 MβCD. 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.
KW - Cholesterol
KW - Epithelial sodium channel (ENaC)
KW - Lipid raft
KW - Methyl-β-cyclodextrin
KW - Serum-and glucocorticoid-induced kinase isoform 1
KW - Xenopus laevis oocytes
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U2 - 10.1159/000257516
DO - 10.1159/000257516
M3 - Article
C2 - 19910701
AN - SCOPUS:70450237212
SN - 1015-8987
VL - 24
SP - 605
EP - 618
JO - Cellular Physiology and Biochemistry
JF - Cellular Physiology and Biochemistry
IS - 5-6
ER -