Reconstitution of immunopurified alveolar type II cell Na+ channel protein into planar lipid bilayers.
Low-amiloride-affinity (L-type) Na+ channels have been functionally and immunologically localized to alveolar type II (ATII) cells. Purified rabbit ATII epithelial cells were isolated by elastase digestion and solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate. 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 > 2,000 compared with 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 ethylisopropyl amiloride (EIPA) to the side opposite that in 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 microM and 1 microM for EIPA. These data confirm the presence of L-type Na+ channels in adult mammalian ATII cells.
Senyk
O
O
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
Ismailov
I
I
Bradford
A L
AL
Baker
R R
RR
Matalon
S
S
Benos
D J
DJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology; D004848:Epithelium / Q000378:metabolism; D007158:Immunologic Techniques; D008051:Lipid Bilayers / Q000378:metabolism*; D008297:Male; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism*; D011817:Rabbits; D015222:Sodium Channels / Q000378:metabolism*
D008051:Lipid Bilayers; D015222:Sodium Channels; D000584:Amiloride; D000255:Adenosine Triphosphate; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; C039614:ethylisopropylamiloride