Immunopurification and functional reconstitution of an amiloride
-sensitive na- channel complex from rat lymphocytes.
Bradford, Anne Lynn, Iskander I. Ismailov, Jean-Michel Achard, David
G. Warnock, James K. Bubien, and Dale J. Benos.
Departments of Physiology and Biophysics, Medicine, and the
Nephrology Research Training Center, University of Alabama at
Birmingham, Birmingham, AL 35294
APStracts 2:0177C, 1995.
Patch clamp experiments have demonstrated an amiloride-sensitive Na-
conductance in human B lymphoid cells. We measured whole-cell
currents in rat lymphocytes and observed a similar Na--specific
inward conductance. The presence of 400 M 8-CPT-cAMP in the bath
significantly increased the inward current, and this cAMP activation
was abolished by 2 M amiloride. We immunopurified a protein complex
from rat lymphocyte membranes using an anti - bovine kidney Na-
channel antibody. The complex consisted of five distinct polypeptides
with apparent Mr's of 110,000, 92,000, 59,000, 48,000, and 42,000.
This putative channel complex was incorporated into planar lipid
bilayers, where we observed single Na- channel activity that was
blocked by amiloride in a concentration-dependent manner. The
addition of protein kinase A (PKA) and ATP to the
"intracellular" solution elicited a two-fold increase in
channel activity. RT-PCR analysis was used to determine if the rat
lymphocytes express message for the recently cloned Na- channel of
the rat colon (rENaC). Primers for the -subunit of rENaC identified
no message in the lymphocyte RNA, while primers for the -subunit of
the clone produced low levels of the expected product. Thus, it
appears that a rENaC-like -subunit may be an essential component of
the lymphocyte Na- channel that was isolated. At the same time, this
channel is different from those recently cloned in that it does not
include an -subunit homologous to that of rENaC.
Received 12 January 1995; accepted in final form 19 April 1995.
APS Manuscript Number C30-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 2 May 1995.