Phosphatidylinositol phosphate-dependent regulation of Xenopus ENaC by MARCKS protein.
Phosphatidylinositol phosphates (PIPs) are known to regulate epithelial sodium channels (ENaC). Lipid binding assays and coimmunoprecipitation showed that the amino-terminal domain of the β- and γ-subunits of Xenopus ENaC can directly bind to phosphatidylinositol 4,5-bisphosphate (PIP(2)), phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), and phosphatidic acid (PA). Similar assays demonstrated various PIPs can bind strongly to a native myristoylated alanine-rich C-kinase substrate (MARCKS), but weakly or not at all to a mutant form of MARCKS. Confocal microscopy demonstrated colocalization between MARCKS and PIP(2). Confocal microscopy also showed that MARCKS redistributes from the apical membrane to the cytoplasm after PMA-induced MARCKS phosphorylation or ionomycin-induced intracellular calcium increases. Fluorescence resonance energy transfer studies revealed ENaC and MARCKS in close proximity in 2F3 cells when PKC activity and intracellular calcium concentrations are low. Transepithelial current measurements from Xenopus 2F3 cells treated with PMA and single-channel patch-clamp studies of Xenopus 2F3 cells treated with a PKC inhibitor altered Xenopus ENaC activity, which suggest an essential role for MARCKS in the regulation of Xenopus ENaC activity.
Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA. aalli@emory.edu
Abdel A
AA
Alli
Hui-Fang
HF
Bao
Alia A
AA
Alli
Yasir
Y
Aldrugh
John Z
JZ
Song
He-Ping
HP
Ma
Ling
L
Yu
Otor
O
Al-Khalili
Douglas C
DC
Eaton
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000032:analysis / Q000502:physiology; D061207:Calcium Ionophores / Q000494:pharmacology; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism*
D061207:Calcium Ionophores; D053503:Epithelial Sodium Channels; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D018129:Phosphatidylinositol Phosphates; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D015759:Ionomycin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium