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J Biol Chem, Vol. 274, Issue 53, 37845-37854, December 31, 1999
From the The hypothesis that actin interactions account
for the signature biophysical properties of cloned epithelial
Na+ channels (ENaC) (conductance, ion selectivity,
and long mean open and closed times) was tested using planar lipid
bilayer reconstitution and patch clamp techniques. We found the
following. 1) In bilayers, actin produced a more than 2-fold decrease
in single channel conductance, a 5-fold increase in Na+
versus K+ permselectivity, and a substantial
increase in mean open and closed times of wild-type
Regulation of Epithelial Na+ Channels by Actin in
Planar Lipid Bilayers and in the Xenopus Oocyte Expression
System*
,,,¶,
,, and**
Department of Physiology and Biophysics and
the § Department of Cell Biology, University of Alabama at
Birmingham, Birmingham, Alabama 35294-0005
-rENaC but
had no effect on a mutant form of rENaC in which the majority of the C
terminus of the subunit was deleted
(R613X-rENaC). 2) When
R613X-rENaC was heterologously expressed
in oocytes and single channels examined by patch clamp, 12.5-pS
channels of relatively low cation permeability were recorded. These
characteristics were identical to those recorded in bilayers for either
R613X-rENaC or wild-type -rENaC in the absence of actin. Moreover, we show that rENaC subunits tightly
associate, forming either homo- or heteromeric complexes when prepared
by in vitro translation or when expressed in oocytes. Finally, we show that -rENaC is properly assembled but retained in
the endoplasmic reticulum compartment. We conclude that actin subserves
an important regulatory function for ENaC and that planar bilayers are
an appropriate system in which to study the biophysical and regulatory
properties of these cloned channels.
*
This work was supported by National Institutes of Health
Grant DK37206.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Dept. of Agricultural Sciences, University of
Bristol, Long Ashton, Bristol BS41 9AF, United Kingdom.
**
To whom correspondence should be addressed: Dept. of Physiology and
Biophysics, University of Alabama at Birmingham, 1918 University Blvd.,
MCLM 704, Birmingham, AL 35294-0005. Tel.: 205-934-6220; Fax:
205-934-2377; E-mail: benos@phybio.bhs.uab.edu.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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