Cloning and functional expression of the mouse epithelial sodium channel

Abstract

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 α-, β-, and γENaC. We previously isolated and sequenced cDNAs encoding a portion of mouse α-, β-, and γENaC (α-, β-, and γmENaC). These cDNAs were used to screen an oligo-dT-primed mouse kidney cDNA library. Full-length βmENaC and partial-length α- and γmENaC clones were isolated. Full-length α- and γmENaC cDNAs were subsequently obtained by 5′-rapid amplification of cDNA ends (5′-RACE) PCR. Injection of mouse α-, β-, and γENaC 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 α-, β-, and γmENaC were expressed in lung and kidney. Interestingly, αmENaC 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 αmENaC. To begin to identify regions within αmENaC that might be important in assembly of the native heteroligomeric channel, a series of functional experiments were performed using a construct of αmENaC encoding the predicted cytoplasmic NH2 terminus. Coinjection of wild-type α-, β-, and γmENaC with the intracellular NH2 terminus of αmENaC abolished amiloride-sensitive currents in Xenopus oocytes, suggesting that the NH2 terminus of αmENaC is involved in subunit assembly, and when present in a 10-fold excess, plays a dominant negative role in functional ENaC expression.