Delta-subunit confers novel biophysical features to alpha beta gamma-human epithelial sodium channel (ENaC) via a physical interaction.
Native amiloride-sensitive Na+ channels exhibit a variety of biophysical properties, including variable sensitivities to amiloride, different ion selectivities, and diverse unitary conductances. The molecular basis of these differences has not been elucidated. We tested the hypothesis that co-expression of delta-epithelial sodium channel (ENaC) underlies, at least in part, the multiplicity of amiloride-sensitive Na+ conductances in epithelial cells. For example, the delta-subunit may form multimeric channels with alpha beta gamma-ENaC. Reverse transcription-PCR revealed that delta-ENaC is co-expressed with alpha beta gamma-subunits in cultured human lung (H441 and A549), pancreatic (CFPAC), and colonic epithelial cells (Caco-2). Indirect immunofluorescence microscopy revealed that delta-ENaC is co-expressed with alpha-, beta-, and gamma-ENaC in H441 cells at the protein level. Measurement of current-voltage that cation selectivity ratios for the revealed relationships Na+/Li+/K+/Cs+/Ca2+/Mg2+, the apparent dissociation constant (Ki) for amiloride, and unitary conductances for delta alpha beta gamma-ENaC differed from those of both alpha beta gamma- and delta beta gamma-ENaC (n = 6). The contribution of the delta subunit to P(Li)/P(Na) ratio and unitary Na+ conductance under bi-ionic conditions depended on the injected cRNA concentration. In addition, the EC50 for proton activation, mean open and closed times, and the self-inhibition time of delta alpha beta gamma-ENaC differed from those of alpha beta gamma- and delta beta gamma-ENaC. Co-immunoprecipitation of delta-ENaC with alpha- and gamma-subunits in H441 and transfected COS-7 cells suggests an interaction among these proteins. We, therefore, concluded that the interactions of delta-ENaC with other subunits could account for heterogeneity of native epithelial channels.
Ji
Hong-Long
HL
Department of Anesthesiology, University of Alabama at Birmingham, Alabama 35205, USA.
Su
Xue-Feng
XF
Kedar
Shrestha
S
Li
Jie
J
Barbry
Pascal
P
Smith
Peter R
PR
Matalon
Sadis
S
Benos
Dale J
DJ
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001703:Biophysics / Q000379:methods*; D019556:COS Cells; D018938:Caco-2 Cells; D002412:Cations; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D053503:Epithelial Sodium Channels; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011522:Protons; D018075:RNA, Complementary / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000494:pharmacology; D015222:Sodium Channels / Q000737:chemistry*; D013997:Time Factors; D014162:Transfection
D002412:Cations; D053503:Epithelial Sodium Channels; D011522:Protons; D018075:RNA, Complementary; D012333:RNA, Messenger; D015222:Sodium Channels; D000584:Amiloride; D012964:Sodium