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, Thomas R. Kleyman

Research output: Contribution to journalArticle

49 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)F121-F129
JournalAmerican Journal of Physiology - Renal Physiology
Volume277
Issue number1 46-1
DOIs
StatePublished - Jul 1999

Keywords

  • Cloning
  • Structure-function relationship
  • Xenopus oocytes

ASJC Scopus subject areas

  • Physiology
  • Urology

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