Reconstitution of immunopurified alveolar type II cell Na+ channel protein into planar lipid bilayers

Abstract

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.

Increased expression and activity of sodium channels in alveolar type II cells of hyperoxic rats.

“…Scatchard analysis of specific [3H]bromobenzamil binding to fetal rat ATII cell membranes was consistent with the presence of both H-and L-type conductive protein in these cells (21). Amiloride-inhibitable cation channels, activated by high cytoplasmic Ca2+ concentrations, are also present in cultured adult ATII cells (23 A putative Na+ channel protein has been recently isolated and immunopurified to homogeneity from ATII cells (24). This protein consists of two peptides of =130 and 70 kDa.…”

Cystic Fibrosis Transmembrane Conductance Regulator Inverts Protein Kinase A-mediated Regulation of Epithelial Sodium Channel Single Channel Kinetics

“…brane insertion and retrieval (12), but is similar to cAMP-dependent regulation of P o of partially purified renal (13) and lung alveolar type II cell Na ϩ channels (14). Studies of heterologously expressed ENaC in oocytes (15) and of reconstituted ENaC in lipid bilayers (16) detected no effect of PKA activation on single channel gating.…”

Hypoxia and β2-Agonists Regulate Cell Surface Expression of the Epithelial Sodium Channel in Native Alveolar Epithelial Cells

“…In normoxic ATII cells, only a mild and insignificant increase in rENaC subunit apical expression was evident following treatment with terbutaline, which was not surprising in view of the small magnitude of the I sc increase induced by terbutaline under normoxic conditions. This mild increase in rENaC subunit apical expression may be partly responsible for the terbutaline-induced stimulation of I sc , but we cannot exclude that additional effect(s) of ␤ 2 -agonists, such as augmented sodium channel open probability, might also be involved, at least under normoxic conditions (22,23).…”

“…Up to now, most studies trying to address the mechanism(s) whereby ␤ 2 -agonists increase sodium absorption in native epithelial cells have been limited to a functional approach, because the very small number of ENaC channels expressed at the plasma membrane has hampered the use of traditional biochemical techniques. In native ATII cells, functional studies have provided conflicting results, attributing the effect of ␤ 2 -agonists to either an increase in the number of highly selective sodium channels in the apical membrane (21), an increase in sodium channel open probability (22,23), or an indirect stimulation of transcellular sodium movement through the stimulation of apical chloride conductance (24). Recently, using a novel approach based on the covalent modification of cotransfected ENaC subunits at the cell surface of Fischer rat thyroid epithelial cells, Snyder (6) provided convincing evidence that the cAMP-mediated stimulation of I sc resulted from an increase in the ratio of translocation versus internalization of ENaC subunits to the cell surface.…”