RECONSTITUTION OF IMMUNOPURIFIED ALVEOLAR TYPE-II CELL NA+ CHANNEL PROTEIN INTO PLANAR LIPID BILAYERS

Low-amiloride-affinity (L-type) Na+ channels have been functionally an d immunologically localized to alveolar type II (ATII) cells. Purified rabbit ATII epithelial cells were isolated by elastase digestion and solubilized with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulf onate. The solubilized proteins were purified by ion-exchange chromato graphy, 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 immunoaffini ty column were assayed for specific binding of [H-3]Br-benzamil and re constituted into planar lipid bilayers. Sequential purification steps gave a final enrichment in specific [H-3]Br-benzamil binding of > 2,00 0 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 presu med cytoplasmic side of the bilayer resulted in a significant increase in the single-channel open probability (P-o), 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 P-o with no change in single-channel co nductance. Rabbit ATII Na+ channels in bilayers had an inhibitory cons tant for amiloride of 8 mu M and 1 mu M for EIPA. These data confirm t he presence of L-type Na+ channels in adult mammalian ATII cells.