Protons regulate electrogenic sodium absorption in a variety of epithelia, including the cortical collecting duct, frog skin, and urinary bladder. Recently, three subunits (α, β, γ) coding for the epithelial sodium channel (ENaC) were cloned. However, it is not known whether pH regulates Na+ channels directly by interacting with one of the three ENaC subunits or indirectly by interacting with a regulatory protein. As a first step to identifying the molecular mechanisms of proton-mediated regulation of apical membrane Na+ permeability in epithelia, we examined the effect of pH on the biophysical properties of ENaC. To this end, we expressed various combinations of α-, β-, and γ-subunits of ENaC in Xenopusoocytes and studied ENaC currents by the two-electrode voltage-clamp and patch-clamp techniques. In addition, the effect of pH on the α-ENaC subunit was examined in planar lipid bilayers. We report that α,β,γ-ENaC currents were regulated by changes in intracellular pH (pHi) but not by changes in extracellular pH (pHo). Acidification reduced and alkalization increased channel activity by a voltage-independent mechanism. Moreover, a reduction of pHi reduced single-channel open probability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited α,β-ENaC, α,γ-ENaC, and α-ENaC currents. We conclude that pHi but not pHo regulates ENaC and that the α-ENaC subunit is regulated directly by pHi.

The neurohypophysial peptide arginine vasopressin (AVP) increases Na+ absorption across A6 epithelia. In addition to the positive natriferic response, AVP increases net basolateral to apical Cl- flux. The time course of activation of electrogenic ion transport in A6 epithelia was examined by measuring transepithelial short-circuit current (ISC). Basolateral application of AVP (0.1 U/ml) or forskolin (10 microM) affects ISC in a biphasic manner. Shortly after addition of AVP, an early (transient) phase is observed in which ISC is rapidly stimulated, reaching a peak value at 1.4 +/- 0.1 min. A subsequent decrease in current is interrupted by a slower, late phase in which ISC reaches a peak 23 +/- 3 min after addition of AVP. The late increase in ISC is sustained over the remainder of the 40-min period of observation. The time course of ISC stimulation by forskolin is qualitatively similar. Replacement of external Cl- by aspartate lowers baseline transport nearly 40%, strongly blunts the early phase of ISC stimulation, and retains the late increase. Addition of amiloride (10 microM) to the apical bath before AVP or forskolin stimulation of ISC eliminates the late increase of ISC. Steady-state amiloride-insensitive ISC activated under these conditions was sensitive to apical application of the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (20 microM) and niflumic acid (100 microM). 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (1 mM) was not an effective inhibitor of this current. Basolateral bumetanide (100 microM) inhibited baseline ISC and reduced both the peak transient and steady-state amiloride-insensitive ISC.(ABSTRACT TRUNCATED AT 250 WORDS)

An epithelial sodium channel (ENaC) is composed of three homologous subunits: ␣, ␤, and ␥. To elucidate the function of the cytoplasmic, NH 2 terminus of rat ENaC (rENaC) subunits, a series of mutant cDNAs was constructed and the cRNAs for all three subunits were expressed in Xenopus oocytes. Amiloride-sensitive Na ؉ currents (I Na ) were measured by the two-electrode voltage clamp technique. Deletion of the cytoplasmic, NH 2 terminus of ␣ (⌬2-109), ␤ (⌬2-49), or ␥-rENaC (⌬2-53) dramatically reduced I Na . A series of progressive, NH 2 -terminal deletions of ␣-rENaC were constructed to identify motifs that regulate I Na . Deletion of amino acids 2-46 had no effect on I Na : however, deletion of amino acids 2-51, 2-55, 2-58, and 2-67 increased I Na by ϳ4-fold. By contrast, deletion of amino acids 2-79, 2-89, 2-100, and 2-109 eliminated I Na . To evaluate the mechanism whereby ⌬2-67-␣-rENaC increased I Na , single channels were evaluated by patch clamp. The single-channel conductance and open probability of ␣,␤,␥-rENaC and ⌬2-67-␣,␤,␥-rENaC were similar. However, the number of active channels in the membrane increased from 6 ؎ 1 channels per patch with ␣,␤,␥-rENaC to 11 ؎ 1 channels per patch with ⌬2-67-␣,␤,␥-rENaC. Laser scanning confocal microscopy confirmed that there were more ⌬2-67-␣,␤,␥-rENaC channels in the plasma membrane than ␣,␤,␥-rENaC channels. Deletion of amino acids 2-67 in ␣-rENaC reduced the endocytic retrieval of channels from the plasma membrane and increased the half-life of the channel in the membrane from 1.1 ؎ 0.2 to 3.5 ؎ 1.1 h. We conclude that the cytoplasmic, NH 2 terminus of ␣-, ␤-, and ␥-rENaC is required for channel activity. The cytoplasmic, NH 2 terminus of ␣-rENaC contains two key motifs. One motif regulates the endocytic retrieval of the channel from the plasma membrane. The second motif is required for channel activity.An amiloride-sensitive, epithelial sodium channel (ENaC) 1 mediates Na ϩ transport across the apical membrane of a variety of epithelia including the kidney, lung, and intestine and, thereby, plays a vital role in maintaining Na ϩ and fluid homeostasis (1-4). ENaC is composed of three subunits: ␣, ␤, and ␥ (5, 6). The expression of the ␣ subunit in Xenopus oocytes produces very small currents, and the expression of ␤ and/or ␥ subunits generates no current (5, 6). However, coexpression of ␣-, ␤-, and ␥-rENaC produces large Na ϩ currents in oocytes (5, 6). The ENaC subunits are members of a growing family of ion channels that include the FMRFamide-gated Na ϩ channel, Na ϩ channels in brain (BNC1 and BNC2), and the degenerins of Caenorhabditis elegans that encode mechanosensitive channels (e.g. DEG-1, MEC-4, and MEC-10) (4, 7).Amino acid sequence analysis and biochemical studies suggest that the ENaC subunits have cytoplasmic NH 2 and COOH termini, two hydrophobic transmembrane domains (M1 and M2) and a large extracellular domain (4 -8). Several lines of evidence suggest that the ␣-, ␤-, and ␥-subunits interact to form a heteromultimeric channel complex and that this c...

The M-1 cell line is derived from the mouse cortical collecting duct and displays the low-conductance, highly Na(+)-selective channel activity of the alpha,beta, gamma-heterotrimeric epithelial Na+ channel (ENaC). The short-circuit current (Isc) across M-1 monolayers was 89 +/- 4 microA/cm2, and the transepithelial conductance was 2.1 +/- 0.2 mS/cm2. Isc was abolished by blocking the Na+ pump with ouabain. Both Isc and transepithelial conductance (gT) were inhibited by benzamil > amiloride >> dimethylamiloride. Under our experimental conditions, vasopressin, vasopressin, forskolin, and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) had no detectable effects on Isc or gT. Increasing apical Na+ entry with nystatin increased Isc. The possible regulation of the M-1 Na+ channel by cAMP-activated protein kinase A (PKA) was further examined with excised inside-out patches. The open-time probability (Po) was not fixed, displaying substantial variance. Perfusion with ATP itself, with the catalytic subunit of PKA with ATP, or with alkaline phosphatase had no consistent effect on Po, the unitary current, or the kinetics of the M-1 Na+ channel. The data are consistent with the concept that PKA stimulates ENaCs by phosphorylating a site with access to but not within the apical membrane patch during cell-attached and excised-patch studies.

Epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) are colocalized in the apical membrane of many epithelia. These channels are essential for electrolyte and water secretion and/or reabsorption. In cystic fibrosis airway epithelia, a hyperactivated epithelial Na ؉ conductance operates in parallel with defective Cl ؊ secretion. Several groups have shown that CFTR down-regulates ENaC activity, but the mechanisms and the regulation of CFTR by ENaC are unknown. To test the hypothesis that ENaC and CFTR regulate each other, and to identify the region(s) of ENaC involved in the interaction between CFTR and ENaC, rENaC and its mutants were co-expressed with CFTR in Xenopus oocytes. Whole cell macroscopic sodium currents revealed that wild type (wt) ␣␤␥-rENaC-induced Na ؉ current was inhibited by coexpression of CFTR, and further inhibited when CFTR was activated with a cAMP-raising mixture (CKT). Conversely, ␣␤␥-rENaC stimulated CFTR-mediated Cl ؊ currents up to ϳ6-fold. Deletion mutations in the intracellular tails of the three rENaC subunits suggested that the carboxyl terminus of the ␤ subunit was required both for the down-regulation of ENaC by activated CFTR and the up-regulation of CFTR by ENaC. However, both the carboxyl terminus of the ␤ subunit and the amino terminus of the ␥ subunit were essential for the down-regulation of rENaC by unstimulated CFTR. Interestingly, down-regulation of rENaC by activated CFTR was Cl ؊-dependent, while stimulation of CFTR by rENaC was not dependent on either cytoplasmic Na ؉ or a depolarized membrane potential. In summary, there appear to be at least two different sites in ENaC involved in the intermolecular interaction between CFTR and ENaC.