Reconstitution of immunopurified alveolar type ii cell na+ channel protein
into planar lipid bilayers.
Senyk, Oksana, Iskander Ismailov, Anne Lynn Bradford, R. Randall Baker, Sadis
Matalon, and Dale J. Benos.
Departments of Physiology and Biophysics, Pulmonary Medicine, and
Anesthesiology, The University of Alabama at Birmingham, Birmingham, Alabama
35294
APStracts 2:0025C, 1995.
Low amiloride affinity (L-type) Na+ channels have been functionally and
immunologically localized to alveolar type II cells (ATII). Purified rabbit
ATII epithelial cells were isolated by elastase digestion and solubilized
with CHAPS. The solubilized proteins were purified by ion exchange
chromatography, followed by immunoaffinity purification over a column to
which rabbit polyclonal antibodies raised against purified bovine renal Na+
channel protein were bound. The proteins eluted from the immunoaffinity
column were assayed for specific binding of [3H]Br-benzamil and
reconstituted into planar lipid bilayers. Sequential purification steps gave
a final enrichment in specific [3H]Br-benzamil binding of >2000
compared to the homogenate. Single channel currents of 25 pS were recorded
from the immunopurified rabbit ATII cell protein. Addition of the catalytic
subunit of protein kinase A (PKA) plus ATP to the presumed cytoplasmic side
of the bilayer resulted in a significant increase in the single channel open
probability (PO), from 0.40 0.14 to 0.8 0.12, without altering single channel
conductance. The addition of amiloride or ethylisopropylamiloride (EIPA) to
the side opposite that which PKA acts reduced PO with no change in single
channel conductance. Rabbit ATII Na+ channels in bilayers had an inhibitory
constant for amiloride of 8 _M, and 1 _M for EIPA. These data confirm the
presence of L-type Na+ channels in adult mammalian ATII cells.
Received 1 August 1994; accepted in final form 9 November 1994
APS Manuscript Number C448-4.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1994 The American Physiological Society.
Published in APStracts on 27 February 1995.