Protons Activate the δ-Subunit of the Epithelial Na+ Channel in Humans

Abstract

The amiloride-sensitive epithelial Na ؉ channel (ENaC) controls Na ؉ transport into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as ␣, ␤, ␥, and ␦. ENaC␦ can associate with ␤ and ␥ subunits and generate a constitutive current that is 2 orders of magnitude larger than that of homomeric ENaC␦. However, the distribution pattern of ENaC␦ is not consistent with that of the ␤ and ␥ subunits. ENaC␦ is expressed mainly in the brain in contrast to ␤ and ␥ subunits, which are expressed in non-neuronal tissues. To explain this discrepancy, we searched for novel functional properties of homomeric ENaC␦ and investigated the detailed tissue distribution in humans. When human ENaC␦ was expressed in Xenopus oocytes and Chinese hamster ovary cells, a reduction of extracellular pH activated this channel (half-maximal pH for an activation of 5.0), and the acid-induced current was abolished by amiloride. The most striking finding was that the desensitization of the acid-evoked current was much slower (by ϳ10% 120 s later), dissociating from the kinetics of acid-sensing ion channels in the degenerin/epithelial Na ؉ channel family, which were rapidly desensitized during acidification. RNA dot-blot analyses showed that ENaC␦ mRNA was widely distributed throughout the brain and was also expressed in the heart, kidney, and pancreas in humans. Northern blotting confirmed that ENaC␦ was expressed in the cerebellum and the hippocampus. In conclusion, human ENaC␦ activity is regulated by protons, indicating that it may contribute to the pH sensation and/or pH regulation in the human brain. Four homologous epithelial Naϩ channel (ENaC) 1 subunits (␣, ␤, ␥, and ␦), members of the degenerin/epithelial Na ϩ channel superfamily, have been cloned in mammals (1-5). There is an overall ϳ37% amino acid identity between the ␣, ␤, ␥, and ␦ subunits. The ␦ subunit of ENaC was originally described as mainly being expressed in the human brain (5). ENaC␦ can associate with ␤ and ␥ subunits to form a heteromeric channel because the coexpression of these three subunits increases the Na ϩ current (5). The tissue distribution pattern of ENaC␦, however, is quite different from that of ␤ and ␥ subunits. ENaC␦ is expressed mainly in the brain, pancreas, testis, and ovary (5), whereas ␤ and ␥ subunits are expressed mainly in the kidney, lung, and colon (3, 4). In addition, the expressed sequence tag data base shows that an ENaC␦ gene has been found in humans and chimpanzees (GenBank TM accession numbers U38254 and O46547, respectively), but for now, there is no evidence for the orthologues in rats and mice. This suggests that ENaC␦ associates with unknown subunits or that homomeric ENaC␦ has its own unknown physiological function in humans. Our goal was to identify the novel functional properties of human ENaC␦ (hENaC␦) using electrophysiological techniques and to investigate the more detailed tissue distribution of ENaC␦ in humans by Northern blot and RNA dot-blot analyses.Here we describe how ...

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

“…Xenopus Oocyte Electrophysiology-Electrophysiological studies in Xenopus oocytes, using a two-electrode voltage clamp technique, were performed as described previously (11). In brief, cRNA(s) (2 ng for homomeric channel or each 0.02 ng for co-expression) was injected into Xenopus oocytes, whereas the control oocytes were injected with an equal volume of diethyl dicarbonate-treated water, described as native throughout.…”

“…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).…”

“…When the hENaC␦ homomer was expressed in Xenopus oocytes, a 100 M amiloride-sensitive inward current was induced at Ϫ60 mV (69 Ϯ 6 nA, n ϭ 5; Fig. 5A), as described previously (11). The application of 30 M capsazepine significantly evoked an inward current in hENaC␦-expressing oocytes (by 67 Ϯ 3 nA, n ϭ 5, p Ͻ 0.01), as well as in hENaC␦␤␥-injected oocytes (by 1133 Ϯ 80 nA, n ϭ 21, p Ͻ 0.01; Fig.…”

“…There is an overall ϳ37% amino acid identity between the ␣, ␤, ␥, and ␦ subunits. ENaC␣ is expressed mainly in epithelia, such as the kidney, lung, and colon, and binds with these ␤ and ␥ subunits to play a pathophysiological role (2,4), whereas the ␦ subunit is widely distributed throughout the brain and is also expressed in the heart, kidney, and pancreas (10,11). More recently, we have demonstrated that protons activate ENaC␦, indicating that it may contribute to pH sensation and/or pH regulation in the human brain (11).…”

“…ENaC␣ is expressed mainly in epithelia, such as the kidney, lung, and colon, and binds with these ␤ and ␥ subunits to play a pathophysiological role (2,4), whereas the ␦ subunit is widely distributed throughout the brain and is also expressed in the heart, kidney, and pancreas (10,11). More recently, we have demonstrated that protons activate ENaC␦, indicating that it may contribute to pH sensation and/or pH regulation in the human brain (11). Most interestingly, the expressed sequence tag and genome project data bases show that an ENaC␦ gene has been found only in humans and chimpanzees (GenBank TM accession numbers U38254 and O46547, respectively), and there is no evidence of the orthologues in rats and mice.…”