Cloning and functional expression of the mouse epithelial sodium channel.
The epithelial sodium channel (ENaC) plays a major role in the transepithelial reabsorption of sodium in the renal cortical collecting duct, distal colon, and lung. ENaCs are formed by three structurally related subunits, termed alpha-, beta-, and gammaENaC. We previously isolated and sequenced cDNAs encoding a portion of mouse alpha-, beta-, and gammaENaC (alpha-, beta-, and gammamENaC). These cDNAs were used to screen an oligo-dT-primed mouse kidney cDNA library. Full-length betamENaC and partial-length alpha- and gammamENaC clones were isolated. Full-length alpha- and gammamENaC cDNAs were subsequently obtained by 5'-rapid amplification of cDNA ends (5'-RACE) PCR. Injection of mouse alpha-, beta-, and gammaENaC cRNAs into Xenopus oocytes led to expression of amiloride-sensitive (K(i) = 103 nM), Na(+)-selective currents with a single-channel conductance of 4.7 pS. Northern blots revealed that alpha-, beta-, and gammamENaC were expressed in lung and kidney. Interestingly, alphamENaC was detected in liver, although transcript sizes of 9.8 kb and 3.1 kb differed in size from the 3.2-kb message observed in other tissues. A partial cDNA clone was isolated from mouse liver by 5'-RACE PCR. Its sequence was found to be nearly identical to alphamENaC. To begin to identify regions within alphamENaC that might be important in assembly of the native heteroligomeric channel, a series of functional experiments were performed using a construct of alphamENaC encoding the predicted cytoplasmic NH(2) terminus. Coinjection of wild-type alpha-, beta-, and gammamENaC with the intracellular NH(2) terminus of alphamENaC abolished amiloride-sensitive currents in Xenopus oocytes, suggesting that the NH(2) terminus of alphamENaC is involved in subunit assembly, and when present in a 10-fold excess, plays a dominant negative role in functional ENaC expression.
Ahn
Y J
YJ
Departments of Medicine and Physiology, University of Pennsylvania, and Veterans Affairs Medical Center, Philadelphia, Pennsylvania 19104-6144, USA. yahn@mail.med.upenn.edu
Brooker
D R
DR
Kosari
F
F
Harte
B J
BJ
Li
J
J
Mackler
S A
SA
Kleyman
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004848:Epithelium / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism*; D014981:Xenopus
D015222:Sodium Channels