A cloned renal epithelial Na+ channel protein displays stretch activation in planar lipid bilayers.
We have previously cloned a bovine renal epithelial channel homologue (alpha-bENaC) belonging to the epithelial Na+ channel (ENaC) family. With the use of a rabbit nuclease-treated in vitro translation system, mRNA coding for alpha-bENaC was translated and the polypeptide products were reconstituted into liposomes. On incorporation into planar lipid bilayers, in vitro-translated alpha-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(i)amil approximately 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(i)amil > 25 microM) and a lack of monovalent cation selectivity in the presence of a hydrostatic pressure gradient. This observation of stretch activation (SA) of alpha-bENaC was confirmed in dual electrode recordings of heterologously expressed alpha-bENaC whole cell currents in Xenopus oocytes swelled by the injection of 15 nl of a 100 mM KCl solution. We conclude that alpha-bENaC, and by analogy other ENaCs, represent a novel family of cloned SA channels.
Awayda
M S
MS
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
Ismailov
I I
II
Berdiev
B K
BK
Benos
D J
DJ
D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D004848:Epithelium / Q000378:metabolism; D006874:Hydrostatic Pressure; D015640:Ion Channel Gating; D007679:Kidney Medulla / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000378:metabolism; D008051:Lipid Bilayers; D008564:Membrane Potentials / Q000187:drug effects; D011336:Probability; D014176:Protein Biosynthesis; D011510:Proteolipids; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D015222:Sodium Channels / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*
D008051:Lipid Bilayers; D011510:Proteolipids; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015222:Sodium Channels; C020485:proteoliposomes; D000584:Amiloride