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We report the synthesis of a radioactive, methylated analog of bromoamiloride which inhibits the amiloride-sensitive, epithelial Na+ channel reversibly and with high affinity. This synthesis was achieved by methylation of a nitrogen in the acylguanidinium moiety with tritiated methyliodide of high specific activity. This methylated bromoamiloride molecule (CH3BrA) was purified by both thin layer and high performance liquid chromatography. Proton nuclear magnetic resonance and mass spectroscopy techniques were used to determine the structure of this analog. This compound inhibited both short-circuit current of in vitro frog skin and 22Na+ influx into apical plasma membrane vesicles made from cultured toad kidney cells (line A6) with the same or lower apparent inhibitory dissociation constant as bromoamiloride. Irradiation with ultraviolet light rendered this inhibition irreversible in both A6 vesicles and frog skin. Preparation of radioactive CH3BrA yielded specific activities in excess of 1 Ci/mmol. We suggest that this compound will be useful in the isolation and purification of this ubiquitous Na+ channel.

Arginine vasopressin (antidiuretic hormone, ADH) stimulation of sodium transport in high electrical resistance epithelia is accompanied by adenylate cyclase stimulation and cAMP accumulation. The hypothesis of direct phosphorylation of the purified amiloride-blockable epithelial Na+ channel protein by cAMP-dependent protein kinase A after ADH treatment of cultured cells was investigated in this study. Phosphate-depleted A6 cells (a cell line derived from toad kidney) were exposed to 32PO4(3-) in the absence or presence of basolateral ADH (100 milliunits/ml). After 20 min (the time needed for ADH to increase maximally Na+ transport), the Na+ channels were extracted from the cells and purified. At every stage of purification, only one subunit of the Na+ channel, namely, the 315-kDa subunit, was specifically phosphorylated as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography or scintillation counting. In addition, a polyclonal antibody raised against purified epithelial Na+ channel protein was able to immunoprecipitate the phosphorylated channel protein from a detergent-solubilized fraction of vasopressin-treated A6 cells. This same subunit was also specifically phosphorylated in vitro when the purified Na+ channel protein was incubated with gamma-[32P]ATP and the purified catalytic subunit of the cAMP-dependent protein kinase. Thus, only a single component, the 315-kDa subunit, of the Na+ channel protein complex (which is composed of six subunits) can be phosphorylated both in vivo and in vitro. This subunit is selectively phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a level of 2-3 mol of 32P/mol of protein.

Net Na+ movement across the apical membrane of high-electrical resistance epithelia is driven by the electrochemical potential energy gradient. This entry pathway is rate limiting for transepithelial transport, occurs via a channel-type mechanism, and is specifically inhibited by the diuretic drug amiloride. This channel is selective for Na+, Li+, and H+, saturates with increasing extracellular Na+ concentration, and is not affected, at least in frog skin epithelium, by changes in apical membrane surface potential. There also appears to be multiple inhibitory regions associated with each Na+ channel. We discuss the possible implications of a voltage-dependent block by amiloride in terms of macroscopic inhibitory phenomena. We describe the use of cultured epithelial systems, in particular, the toad kidney-derived A6 cell line, and the preparation of apical plasma membrane vesicles to study the Na+ entry process. We discuss experiments in which single, amiloride-sensitive channel activity has been detected and summarize current experimental approaches directed at the biochemical identification of this ubiquitous Na+ transport system.

The amiloride-binding Na+ channel protein of high electrical resistance epithelia was solubilized and purified from cultured A6 toad kidney cells and bovine renal papilla. Purification was assessed by enrichment in [3H]methylbromoamiloride specific binding. Chromatography of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-solubilized plasma membrane vesicles on agarose-immobilized wheat-germ agglutinin provided a 130-fold enrichment of the amiloride-binding component compared to the cell homogenate. Further purification was achieved by either amiloride-affinity chromatography or size-exclusion HPLC. When the HPLC and amiloride affinity-purified material was injected into a second higher molecular weight exclusion HPLC column, only a single peak with Mr 800,000 was found. Further HPLC separation of the Mr 800,000 material at low ionic strength resolved two peaks with apparent Mrs 800,000 and 700,000. Only the 700-kDa component displayed specific [3H]methylbromoamiloride binding activity. The final binding specific activity achieved was 1300 pmol/mg of protein, corresponding to 91% homogeneity of the protein.

High resistance epithelia actively transport sodium from the luminal side to the blood. Aldosterone and vasopressin stimulate this sodium transport system; the diuretic drug amiloride inhibits it in a reversible fashion. The first step in the transepithelial transport of Na+ is the facilitated diffusion of Na+ across the apical membrane via Na+-specific, amiloride-sensitive channels. We report here the first direct measurements of single, amiloride-sensitive Na+ channel activity. The channel was isolated after incorporation of purified apical membrane vesicles from A6 cells into planar lipid bilayers. The channel had the following characteristics: single-channel conductance ranged from 4 to 80 pS at 200 mM NaCl; it was perfectly cation-selective; amiloride reduced the open-state conductance in a dose-dependent fashion when present in the cis compartment, and induced flickering when present in the trans chamber; channel conductance and gating were voltage-independent; and the Na+/K+ selectivity ratio of the channel was 2:1.

Single epithelial Na+ channels incorporated into planar lipid bilayers were studied to determine the effects of Na concentration on its own conductance. Amiloride-sensitive Na+ channels were obtained from apical membrane vesicles made from A6 cells, a continuous epithelial cell-line derived from amphibian kidney. Single-channel conductance was found to be a saturable function of Na+ concentration, with a Michaelis constant of approximately 17 or 47 mM, for a Gmax of approximately 4 or 44 pS, respectively.

Sodium dodecyl sulfate gel electrophoresis of the radioiodinated native amiloride-sensitive epithelial sodium channel protein isolated from bovine renal papilla and cultured amphibian A6 cells under denatured and nonreduced conditions revealed an 125I-labeled protein band of Mr approximately 730,000. Upon reduction, this protein was resolved into five major polypeptide bands with apparent average Mr values of 315,000, 149,000, 95,000, 71,000, and 55,000. The amiloride analog [3H]methylbromoamiloride has been used as a photoaffinity label to determine the location of the binding site for amiloride on the epithelial sodium channel protein. [3H]Methylbromoamiloride binds covalently to the sodium channel at high affinity binding sites with a half-maximal binding concentration of 0.2 microM. [3H]Methylbromoamiloride was specifically photoincorporated into the Mr approximately 150,000 polypeptide and this incorporation was blocked by addition of excess amiloride. These data suggest that the epithelial sodium channel protein is composed of at least five nonidentical polypeptide subunits, only one of which specifically binds amiloride.