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Am J Physiol Renal Physiol 277: F121-F129, 1999;
0363-6127/99 $5.00
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Vol. 277, Issue 1, F121-F129, July 1999

Cloning and functional expression of the mouse epithelial sodium channel

Yoon J. Ahn, David R. Brooker, Farhad Kosari, Brian J. Harte, Jinqing Li, Scott A. Mackler, and Thomas R. Kleyman

Departments of Medicine and Physiology, University of Pennsylvania, and Veterans Affairs Medical Center, Philadelphia, Pennsylvania 19104-6144

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 gamma ENaC. We previously isolated and sequenced cDNAs encoding a portion of mouse alpha -, beta -, and gamma ENaC (alpha -, beta -, and gamma mENaC). These cDNAs were used to screen an oligo-dT-primed mouse kidney cDNA library. Full-length beta mENaC and partial-length alpha - and gamma mENaC clones were isolated. Full-length alpha - and gamma mENaC cDNAs were subsequently obtained by 5'-rapid amplification of cDNA ends (5'-RACE) PCR. Injection of mouse alpha -, beta -, and gamma ENaC cRNAs into Xenopus oocytes led to expression of amiloride-sensitive (Ki = 103 nM), Na+-selective currents with a single-channel conductance of 4.7 pS. Northern blots revealed that alpha -, beta -, and gamma mENaC were expressed in lung and kidney. Interestingly, alpha mENaC 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 alpha mENaC. To begin to identify regions within alpha mENaC that might be important in assembly of the native heteroligomeric channel, a series of functional experiments were performed using a construct of alpha mENaC encoding the predicted cytoplasmic NH2 terminus. Coinjection of wild-type alpha -, beta -, and gamma mENaC with the intracellular NH2 terminus of alpha mENaC abolished amiloride-sensitive currents in Xenopus oocytes, suggesting that the NH2 terminus of alpha mENaC is involved in subunit assembly, and when present in a 10-fold excess, plays a dominant negative role in functional ENaC expression.

cloning; Xenopus oocytes; structure-function relationship


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