Search

Epithelial [Na.sup.+] channels (ENaC) are inhibited by extracellular [Na.sup.+], a process referred to as [Na.sup.+] self-inhibition. We previously demonstrated that mutation of key residues within two furin cleavage consensus sites in [alpha], or one site in [gamma] blocked subunit proteolysis and inhibited channel activity when mutant channels were expressed in Xenopus laevis oocytes (Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD, and Kleyman TR. J Biol Chem 279: 18111-18114, 2004). Cleavage of subunits was also blocked by these mutations when expressed in Madin-Darby canine kidney cells, and both subunit cleavage and channel activity were blocked when wild-type subunits were expressed in furin-deficient Chinese hamster ovary cells. We now report that channels with mutant [alpha]-subunits lacking either one or both furin cleavage sites exhibited a marked enhancement of the [Na.sup.+] self-inhibition response, while channels with a mutant [gamma]/-subunit showed a modestly enhanced [Na.sup.+] self-inhibition response. Analysis of [Na.sup.+] self-inhibition at varying [Na.sup.+] indicates that channels containing mutant [alpha]-subunits exhibit an increased [Na.sup.+] affinity. At the single-channel level, channels with a mutant [alpha]-subunit had a low open probability ([P.sub.o]) in the presence of a high external [Na.sup.+] in the patch pipette. [P.sub.o] dramatically increased when trypsin was also present, or when a low external [Na.sup.+] was in the patch pipette. Our results suggest that furin cleavage of ENaC subunits activates the channels by relieving [Na.sup.+] self-inhibition and that activation requires that the [alpha]-subunit be cleaved twice. Moreover, we demonstrate for the first time a clear relationship between ENaC [P.sub.o] and extracellular [Na.sup.+], supporting the notion that [Na.sup.+] self-inhibition reflects a [P.sub.o] reduction due to high extracellular [Na.sup.+]. amiloride; open probability; voltage clamp; Xenopus laevis oocyte; mutagenesis

Epithelial sodium channels (ENaCs) are composed of three structurally related subunits that form a tetrameric channel. The Xenopus laevis oocyte expression system was used to identify regions within the ENaC [alpha]-subunit that confer a dominant negative phenotype on functional expression of [alpha][beta][gamma]-ENaC to define domains that have a role in subunit-subunit interactions. Coexpression of full-length mouse [alpha][beta][gamma]-ENaC with either 1) the [alpha]-subunit first membrane-spanning domain and short downstream hydrophobic domain ([alpha]-M1H1); 2) [alpha]-M1H1 and its downstream hydrophilic extracellular loop ([alpha]-M1H1-ECL); 3) the membrane-spanning domain of a control type 2 transmembrane protein (glutamyl transpeptidase; [gamma]-GT) fused to the [alpha]-ECL ([gamma]-GT[alpha]-ECL); 4) the extracellular domain of a control type 1 trans-membrane protein (Tac) fused to the [alpha]-subunit second membrane-spanning domain and short upstream hydrophobic domain (Tac-[alpha]-H2M2); or 5) the [alpha]-subunit cytoplasmic COOH terminus ([alpha]-Ct) significantly reduced amiloride-sensitive [Na.sup.+] currents in X. laevis oocytes. Functional expression of [Na.sup.+] channels was not inhibited when full-length [alpha][beta][gamma]-ENaC was coexpressed with either 1) the [alpha]-ECL lacking a signal-anchor sequence, 2) [alpha]-M1H1 and [alpha]-Ct expressed as a fusion protein, 3) full-length [gamma]-GT, or 4) full-length Tac. Furthermore, the expression of ROMK channels was not inhibited when full-length ROMK was coexpressed with either [alpha]-M1H1-ECL or [alpha]-Ct. Full-length FLAG-tagged [alpha], [beta], or [gamma]-ENaC coimmunoprecipitated with myc-tagged [alpha]-M1H1-ECL, whereas wild-type [gamma]-GT did not. These data suggest that multiple sites within the [alpha]-subunit participate in subunit-subunit interactions that are required for proper assembly of the heterooligomeric ENaC complex. amiloride-sensitive sodium channel; dominant negative mutants

[Na.sup.+] absorption in the renal cortical collecting duct (CCD) is mediated by apical epithelial [Na.sup.+] channels (ENaCs). The CCD is subject to continuous variations in intraluminal flow rate that we speculate alters hydrostatic pressure, membrane stretch, and shear stress. Although ENaCs share limited sequence homology with putative mechanosensitive ion channels in Caenorhabditis elegans, controversy exists as to whether ENaCs are regulated by biomechanical forces. We examined the effect of varying the rate of fluid flow on whole cell [Na.sup.+] currents ([I.sub.Na]) in oocytes expressing mouse [Alpha],[Beta],[Gamma]-ENaC (mENaC) and on net [Na.sup.+] absorption in microperfused rabbit CCDs. Oocytes injected with mENaC but not water responded to the initiation of superfusate flow (to 4-6 ml/min) with a reversible threefold stimulation of [I.sub.Na] without a change in reversal potential. The increase in [I.sub.Na] was variable among oocytes. CCDs responded to a threefold increase in rate of luminal flow with a twofold increase in the rate of net [Na.sup.+] absorption. An increase in luminal viscosity achieved by addition of 5% dextran to the luminal perfusate did not alter the rate of net [Na.sup.+] absorption, suggesting that shear stress does not influence [Na.sup.+] transport in the CCD. In sum, our data suggest that flow stimulation of ENaC activity and [Na.sup.+] absorption is mediated by an increase in hydrostatic pressure and/or membrane stretch. We propose that intraluminal flow rate may be an important regulator of channel activity in the CCD. epithelial sodium channel; ROMK; collecting duct; transepithelial transport; oocyte