Intracellular H⁺ regulates the α-subunit of ENaC, the epithelial Na⁺ channel

Abstract

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 NH2 Terminus of the Epithelial Sodium Channel Contains an Endocytic Motif

“…Isolation of Xenopus Oocytes and Injection of cRNA-Oocytes were isolated and injected with cRNA as described previously (20,21). Briefly, ovarian lobes were removed from Xenopus laevis and stored in calcium-free OR-2 solution.…”

Regulation of the Epithelial Na+ Channel by Cytosolic ATP

“…Previous patch clamp studies in native Na ϩ -absorptive epithelia have shown that the feedback inhibition involves changes in intracellular factors, including membrane potential, cytosolic pH, Ca 2ϩ , and Na ϩ concentrations, and cell metabolism (1). Furthermore, in recent studies using both molecular biological and electrophysiological techniques, cytosolic pH, Na ϩ , and/or Ca 2ϩ concentration have been also shown to affect the activity of the cloned rENaC when expressed in different heterologous expression systems (15)(16)(17)(18)(19)(20), although these cytosolic factors may not act on the ENaC protein directly (18).Focusing on the relationship between cell metabolism and Na ϩ channel activity, Palmer et al (21) and Garty et al (22) observed that inhibition of cell metabolism decreased the apical membrane Na ϩ permeability in the toad urinary bladder, although it is not known whether metabolites such as ATP were responsible for this effect. Frindt et al (23) also suggested a role for metabolic changes in mediating negative feedback regulation of amiloride-sensitive Na ϩ channels in rat cortical collecting tubules; they found that increasing Na ϩ entry into cells by the addition of cAMP led to a decrease in channel activity in cell-attached patches, where the channels were apparently pro-…”

“…Previous patch clamp studies in native Na ϩ -absorptive epithelia have shown that the feedback inhibition involves changes in intracellular factors, including membrane potential, cytosolic pH, Ca 2ϩ , and Na ϩ concentrations, and cell metabolism (1). Furthermore, in recent studies using both molecular biological and electrophysiological techniques, cytosolic pH, Na ϩ , and/or Ca 2ϩ concentration have been also shown to affect the activity of the cloned rENaC when expressed in different heterologous expression systems (15)(16)(17)(18)(19)(20), although these cytosolic factors may not act on the ENaC protein directly (18).…”

Capsazepine Is a Novel Activator of the δ Subunit of the Human Epithelial Na+ Channel

“…The reverse transcription (RT) was performed as follows. We heated the reaction mixture of total RNA (3 g) and 500 ng of an oligo(dT) [12][13][14][15][16][17][18] primer (Invitrogen) in 8 l of diethyl bicarbonate-treated water at 70°C for 10 min, chilled it for 1 min, added 11 l of the reaction buffer (as a final concentration, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl 2 , 10 mM dithiothreitol, and 0.5 mM individual dNTPs), and incubated it at 50°C for 2 min. The mixture was incubated at 50°C for 90 min after the addition of 200 units of SuperScript II reverse transcriptase (Invitrogen), and then heated at 70°C for 15 min.…”

“…In pharmacological profiles of ENaC, it is well known that the potassium-sparing diuretics, amiloride and benzamil, inhibit the activities of ENaC␣, ENaC␦, and the complexes with ␤ and ␥ subunits (6 -11). Extracellular protons reduce the activity of ENaC␣ (18), whereas ENaC␦ is activated by protons (11,19). In addition, ENaC␣␤␥ is activated by cAMP and blocked by Ni 2ϩ (20).…”