A cloned renal epithelial na+ channel protein displays stretch activation
in planar lipid bilayers.
Awayda, Mouhamed S., Iskander I. Ismailov, Bakhram K. Berdiev and Dale J.
Benos.
Department of Physiology and Biophysics, The University of Alabama at
Birmingham, Birmingham, Alabama 35294.
APStracts 2:0049C, 1995.
We have previously cloned a bovine renal epithelial channel homolog
([alpha]bENaC) belonging to the ENaC (Epithelial Na+ Channel) family.
Utilizing a rabbit nuclease-treated in vitro translation system, mRNA coding
for [alpha]bENaC was translated and the polypeptide products were
reconstituted into liposomes. Upon 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 0.072 mmHg/Nm2 across the
bilayer; 3) was blocked by low concentrations of amiloride (Kiamil 150 nM);
and 4) was cation selective with a Li+:Na+:K+ permselectivity of 2:1:0.1 under
non-stretched conditions. These pharmacological and selectivity
characteristics were altered to a lower amiloride affinity (Kiamil>25 NM) 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 ENaC's, represent a novel family of cloned SA channels.
Received 7 September 1994; accepted in final form 7 December 1994
APS Manuscript Number C0518-4.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1994 The American Physiological Society.
Published in APStracts on 27 February 1995.