The cytosolic termini of the beta- and gamma-ENaC subunits are involved in the functional interactions between cystic fibrosis transmembrane conductance regulator and epithelial sodium channel.
Epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) are co-localized in the apical membrane of many epithelia. These channels are essential for electrolyte and water secretion and/or reabsorption. In cystic fibrosis airway epithelia, a hyperactivated epithelial Na(+) conductance operates in parallel with defective Cl(-) secretion. Several groups have shown that CFTR down-regulates ENaC activity, but the mechanisms and the regulation of CFTR by ENaC are unknown. To test the hypothesis that ENaC and CFTR regulate each other, and to identify the region(s) of ENaC involved in the interaction between CFTR and ENaC, rENaC and its mutants were co-expressed with CFTR in Xenopus oocytes. Whole cell macroscopic sodium currents revealed that wild type (wt) alphabetagamma-rENaC-induced Na(+) current was inhibited by co-expression of CFTR, and further inhibited when CFTR was activated with a cAMP-raising mixture (CKT). Conversely, alphabetagamma-rENaC stimulated CFTR-mediated Cl(-) currents up to approximately 6-fold. Deletion mutations in the intracellular tails of the three rENaC subunits suggested that the carboxyl terminus of the beta subunit was required both for the down-regulation of ENaC by activated CFTR and the up-regulation of CFTR by ENaC. However, both the carboxyl terminus of the beta subunit and the amino terminus of the gamma subunit were essential for the down-regulation of rENaC by unstimulated CFTR. Interestingly, down-regulation of rENaC by activated CFTR was Cl(-)-dependent, while stimulation of CFTR by rENaC was not dependent on either cytoplasmic Na(+) or a depolarized membrane potential. In summary, there appear to be at least two different sites in ENaC involved in the intermolecular interaction between CFTR and ENaC.
Ji
H L
HL
Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA.
Chalfant
M L
ML
Jovov
B
B
Lockhart
J P
JP
Parker
S B
SB
Fuller
C M
CM
Stanton
B A
BA
Benos
D J
DJ
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D053503:Epithelial Sodium Channels; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D014176:Protein Biosynthesis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D014982:Xenopus laevis
D053503:Epithelial Sodium Channels; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins; D000584:Amiloride; D012964:Sodium