A cloned renal epithelial Na⁺ channel protein displays stretch activation in planar lipid bilayers

We have previously cloned a bovine renal epithelial channel homologue (α- bENaC) belonging to the epithelial Na⁺ channel (ENaC) family. With the use of a rabbit nuclease-treated in vitro translation system, mRNA coding for α- bENaC was translated and the polypeptide products were reconstituted into liposomes. On incorporation into planar lipid bilayers, in vitro-translated α-bENaC protein 1) displayed voltage-independent Na⁺ channel activity with a single-channel conductance of 40 pS, 2) was mechanosensitive in that the single-channel open probability was maximally activated with a hydrostatic pressure gradient of 0.26 mmHg across the bilayer, 3) was blocked by low concentrations of amiloride [apparent inhibitory constant of amiloride (K(amil)) ≃ 150 nM], and 4) was cation selective with a Li⁺:Na⁺:K⁺ permselectivity of 2:1:0.14 under nonstretched conditions. These pharmacological and selectivity characteristics were altered to a lower amiloride affinity (K(amil) > 25 μM) and a lack of monovalent cation selectivity in the presence of a hydrostatic pressure gradient. This observation of stretch activation (SA) of α-bENaC was confirmed in dual electrode recordings of heterologously expressed α-bENaC whole cell currents in Xenopus oocytes swelled by the injection of 15 nl of a 100 mM KCl solution. We conclude that α-bENaC, and by analogy other ENaCs, represent a novel family of cloned SA channels.