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• W1995294810 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 α, β, γ, and δ. ENaCδ can associate with β and γ subunits and generate a constitutive current that is 2 orders of magnitude larger than that of homomeric ENaCδ. However, the distribution pattern of ENaCδ is not consistent with that of the β and γ subunits. ENaCδ is expressed mainly in the brain in contrast to β and γ subunits, which are expressed in non-neuronal tissues. To explain this discrepancy, we searched for novel functional properties of homomeric ENaCδ and investigated the detailed tissue distribution in humans. When human ENaCδ 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 ∼10% 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 ENaCδ mRNA was widely distributed throughout the brain and was also expressed in the heart, kidney, and pancreas in humans. Northern blotting confirmed that ENaCδ was expressed in the cerebellum and the hippocampus. In conclusion, human ENaCδ activity is regulated by protons, indicating that it may contribute to the pH sensation and/or pH regulation in the human brain. 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 α, β, γ, and δ. ENaCδ can associate with β and γ subunits and generate a constitutive current that is 2 orders of magnitude larger than that of homomeric ENaCδ. However, the distribution pattern of ENaCδ is not consistent with that of the β and γ subunits. ENaCδ is expressed mainly in the brain in contrast to β and γ subunits, which are expressed in non-neuronal tissues. To explain this discrepancy, we searched for novel functional properties of homomeric ENaCδ and investigated the detailed tissue distribution in humans. When human ENaCδ 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 ∼10% 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 ENaCδ mRNA was widely distributed throughout the brain and was also expressed in the heart, kidney, and pancreas in humans. Northern blotting confirmed that ENaCδ was expressed in the cerebellum and the hippocampus. In conclusion, human ENaCδ 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 Na+ channel (ENaC) 1The abbreviations used are: ENaC, epithelial Na+ channel; hENaCδ, human ENaCδ; MES, 2-(N-morpholino)ethanesulfonic acid; CHO, Chinese hamster ovary; ASIC, rat acid-sensing ion channel; rASIC2a, rat acid-sensing ion channel 2a. 1The abbreviations used are: ENaC, epithelial Na+ channel; hENaCδ, human ENaCδ; MES, 2-(N-morpholino)ethanesulfonic acid; CHO, Chinese hamster ovary; ASIC, rat acid-sensing ion channel; rASIC2a, rat acid-sensing ion channel 2a. subunits (α, β, γ, and δ), members of the degenerin/epithelial Na+ channel superfamily, have been cloned in mammals (1Canessa C.M. Horisberger J.D. Rossier B.C. Nature. 1993; 361: 467-470Crossref PubMed Scopus (816) Google Scholar, 2McDonald F.J. Snyder P.M. McCray Jr., P.B. Welsh M.J. Am. J. Physiol. 1994; 266: L728-L734Crossref PubMed Google Scholar, 3Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1739) Google Scholar, 4McDonald F.J. Price M.P. Snyder P.M. Welsh M.J. Am. J. Physiol. 1995; 268: C1157-C1163Crossref PubMed Google Scholar, 5Waldmann R. Champigny G. Bassilana F. Voilley N. Lazdunski M. J. Biol. Chem. 1995; 270: 27411-27414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). There is an overall ∼37% amino acid identity between the α, β, γ, and δ subunits. The δ subunit of ENaC was originally described as mainly being expressed in the human brain (5Waldmann R. Champigny G. Bassilana F. Voilley N. Lazdunski M. J. Biol. Chem. 1995; 270: 27411-27414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). ENaCδ can associate with β and γ subunits to form a heteromeric channel because the coexpression of these three subunits increases the Na+ current (5Waldmann R. Champigny G. Bassilana F. Voilley N. Lazdunski M. J. Biol. Chem. 1995; 270: 27411-27414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). The tissue distribution pattern of ENaCδ, however, is quite different from that of β and γ subunits. ENaCδ is expressed mainly in the brain, pancreas, testis, and ovary (5Waldmann R. Champigny G. Bassilana F. Voilley N. Lazdunski M. J. Biol. Chem. 1995; 270: 27411-27414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar), whereas β and γ subunits are expressed mainly in the kidney, lung, and colon (3Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1739) Google Scholar, 4McDonald F.J. Price M.P. Snyder P.M. Welsh M.J. Am. J. Physiol. 1995; 268: C1157-C1163Crossref PubMed Google Scholar). In addition, the expressed sequence tag data base shows that an ENaCδ gene has been found in humans and chimpanzees (GenBank™ accession numbers U38254 and O46547, respectively), but for now, there is no evidence for the orthologues in rats and mice. This suggests that ENaCδ associates with unknown subunits or that homomeric ENaCδ has its own unknown physiological function in humans. Our goal was to identify the novel functional properties of human ENaCδ (hENaCδ) using electrophysiological techniques and to investigate the more detailed tissue distribution of ENaCδ in humans by Northern blot and RNA dot-blot analyses. Here we describe how ENaCδ is expressed widely in the human brain, such as in the cerebellum and hippocampus, and how the channel activity is enhanced by external protons in both Xenopus oocyte and Chinese hamster ovary (CHO-K1) cell expression systems. These results provide the novel profile that ENaCδ responds to acidification in humans. Molecular Biology—All experiments were approved by the Ethics Committee of the Nagoya City University Graduate School of Medical Sciences and were conducted in accordance with the Declaration of Helsinki. Two expressed sequence tag clones (GenBank™ accession numbers BI520370 and AI199647) were obtained from the Medical Research Council Geneservice (Babraham, UK) to prepare a full-length hENaCδ containing a 1,914-nucleotide open reading frame. Rat acid-sensing ion channel 2a (rASIC2a; U53211) was isolated as described previously (6Ugawa S. Minami Y. Guo W. Saishin Y. Takatsuji K. Yamamoto T. Tohyama M. Shimada S. Nature. 1998; 395: 555-556Crossref PubMed Scopus (148) Google Scholar). Xenopus Oocyte Electrophysiology—The coding sequence of hENaCδ or rASIC2a was subcloned into a pBluescript vector (Stratagene, La Jolla, CA) with 5′- and 3′-untranslated regions of Xenopus β-globin added at the multicloning site to promote stable mRNA expression in Xenopus oocytes. Using an mMESSAGE mMACHINE T3 kit (Ambion, Austin, TX), cRNAs were synthesized and 5 ng was injected into Xenopus oocytes. The control oocytes were injected with an equal volume of diethyl dicarbonate-treated water and are described as native oocytes throughout. Electrophysiological recordings were taken 48–72 h after injection using a two-electrode voltage clamp technique with a CEZ-1200 amplifier (Nihon Kohden, Tokyo, Japan) and a MacLab A/D converter (ADInstruments, Colorado Springs, CO). The recording solution had an ionic composition of 96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, and 5 mm HEPES. The pH of the solution was adjusted to 7.5 with NaOH. The recording solution for pH experiments was prepared with the equivalent MES (pKa = 6.15) instead of HEPES (pKa = 7.55) and adjusted to a suitable pH with either NaOH or HCl. The recording chamber was continuously perfused with solution at a flow rate of 5 ml/min. All electrophysiological experiments were carried out at room temperature (24 ± 1 °C). CHO-K1 Cell Electrophysiology—The coding sequence of hENaCδ was subcloned in a pTracer-CMV2 vector (Invitrogen, Carlsbad, CA) and transiently transfected into CHO-K1 cells using LipofectAMINE 2000 (Invitrogen). The control cells were transfected with the vector alone, described as native cells throughout. Experiments were carried out 48–72 h later using the whole-cell patch clamp technique with an Axopatch 200B amplifier and pCLAMP 8 software (Axon Instruments, Foster City, CA). The transiently transfected cells were identified from green fluorescence protein expression. The extracellular solution had an ionic composition of 137 mm NaCl, 5.9 mm KCl, 2.2 mm CaCl2, 1.2 mm MgCl2, 14 mm glucose, and 10 mm MES. The pH of the solution was adjusted to 7.4 with NaOH. The pipette solution contained 140 mm KCl, 1 mm MgCl2, 10 mm MES, 2 mm Na2ATP, and 5 mm EGTA. The pH was adjusted to 7.2 with KOH. RNA Dot-Blot—A human multiple tissue expression array (version 2) was purchased from Clontech (Palo Alto, CA). A 32P-labeled hENaCδ probe was prepared from the full-length hENaCδ cDNA fragment using a Ready-To-Go DNA labeling beads (-dCTP) kit (Amersham Biosciences). The membrane was prehybridized in a modified Church's buffer (0.5 m Na2HPO4, pH 7.2, 10 mm EDTA, and 7% SDS) for 6 h at 65 °C and then hybridized in a buffer containing 1.5 × 106 cpm/ml of a 32P-labeled probe for 12 h at 65 °C. After hybridization, the membrane was repeatedly washed in 2× SSC and 1% SDS for 20 min at 65 °C followed by two additional washes with a solution containing 0.1× SSC and 0.5% SDS for 20 min at 55 °C, and exposed to Kodak X-AR film for 1 week at -80 °C. Northern Blot—Human samples free from neurological disorders were taken within 24 h after death with permission of the deceased persons' families. (Ethical principles and considerations were observed regarding forensic and related research using human organs and fluids obtained from autopsies.) Human cerebellums and hippocampi were rapidly frozen on dry ice immediately after their removal at autopsy and were kept at -80 °C until use. From freshly frozen tissues, 2 μg of poly(A)+ RNAs were obtained using a FastTrack 2.0 kit (Invitrogen). The membrane was prehybridized in a hybridization buffer (5× saline/sodium phosphate/EDTA, 5× Denhardt's solution, 50% formamide, 20 μg/ml salmon sperm DNA, and 1% SDS) for 6 h at 42 °C and hybridized in a buffer containing 2.0 × 106 cpm/ml of the 32P-labeled hENaCδ probe for 12 h at 42 °C. After hybridization, the membrane was washed twice in 2× SSC at room temperature followed by two additional washes with a solution containing 1× SSC and 0.4% SDS for 30 min at 50 °C, rinsed in 2× SSC at room temperature, and exposed to film for 1 week at -80 °C. Statistics—Pooled data are shown as the mean ± S.E. Statistical significance between the two groups and among groups was determined by Student's t test and Scheffé's test after one way analysis of variance, respectively. Significant difference is expressed in the figures (*, p < 0.05; ** or ##, p < 0.01). The data on the relationships between proton concentrations and current responses were fitted using the following equation (see Fig. 2B): IpH = Imax/{1+(Kd/[H+]o)n} + C, where Imax is the maximum amplitude of the acid-evoked current, Kd is the apparent dissociation constant of protons, [H+]o is the concentration of extracellular protons, n is the Hill coefficient, and C is the constant. Characterization of hENaCδ Current in Xenopus Oocytes— When hENaCδ was expressed in Xenopus oocytes, an inward current was induced at a holding potential of -60 mV, and the current was mostly inhibited by 100 μm amiloride (Fig. 1A). The mean amplitude of the amiloride-sensitive current in hENaCδ-expressed oocytes was 160 ± 9 nA at -60 mV (n = 27, p < 0.01 versus native of 4 ± 1 nA, n = 22). The macroscopic properties of the hENaCδ current in our experimental system were similar to those reported previously (5Waldmann R. Champigny G. Bassilana F. Voilley N. Lazdunski M. J. Biol. Chem. 1995; 270: 27411-27414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). It has been reported that hENaCδ itself can induce some currents when expressed in Xenopus oocytes, but the heteromultimeric channel with β and γ subunits produces a larger current (5Waldmann R. Champigny G. Bassilana F. Voilley N. Lazdunski M. J. Biol. Chem. 1995; 270: 27411-27414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar), as is the case with ENaCα (3Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1739) Google Scholar, 4McDonald F.J. Price M.P. Snyder P.M. Welsh M.J. Am. J. Physiol. 1995; 268: C1157-C1163Crossref PubMed Google Scholar). However, unlike ENaCα, which plays a pathophysiological role as an ENaCαβγ complex in epithelia such as the kidney, lung, and colon (3Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1739) Google Scholar, 4McDonald F.J. Price M.P. Snyder P.M. Welsh M.J. Am. J. Physiol. 1995; 268: C1157-C1163Crossref PubMed Google Scholar, 6Ugawa S. Minami Y. Guo W. Saishin Y. Takatsuji K. Yamamoto T. Tohyama M. Shimada S. Nature. 1998; 395: 555-556Crossref PubMed Scopus (148) Google Scholar, 7Alvarez de la Rosa D. Canessa C.M. Fyfe G.K. Zhang P. Annu. Rev. Physiol. 2000; 62: 573-594Crossref PubMed Scopus (286) Google Scholar), it is unclear whether ENaCδ associates with other subunits in vivo to play a role in physiological functions. It has been reported that ENaCδ is expressed mainly in the brain, pancreas, testis, and ovary (5Waldmann R. Champigny G. Bassilana F. Voilley N. Lazdunski M. J. Biol. Chem. 1995; 270: 27411-27414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar), whereas β and γ subunits are expressed mainly in the kidney, lung, and colon (3Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1739) Google Scholar, 4McDonald F.J. Price M.P. Snyder P.M. Welsh M.J. Am. J. Physiol. 1995; 268: C1157-C1163Crossref PubMed Google Scholar). Therefore we focused on homomeric hENaCδ in this study. Proton Activation of hENaCδ Current in Xenopus Oocytes— Interestingly, the activity of hENaCδ showed pH dependence in the Xenopus oocyte expression system. At a holding potential of -60 mV, the application of pH 5.0 induced an inward current in hENaCδ-injected oocytes (514 ± 41 nA, n = 17, p < 0.01 versus native of 62 ± 4 nA, n = 10; Fig. 1, B and C). The current-voltage relationship showed that the acidic pH stimuli enhanced the channel activity at all voltages examined in hENaCδ oocytes. The addition of 100 μm amiloride dramatically blocked the acid-evoked current (87 ± 2% decrease, n = 17, p < 0.01) and, moreover, significantly inhibited the current (to 62 ± 8 nA, n = 17, p < 0.01 versus the initial resting current of 175 ± 6 nA). During a sustained acidic pulse at pH 5.0, there was a slight decline in the amplitude of the acid-evoked current in hENaCδ oocytes (6 ± 1% decrease at 120 s, n = 17). In sharp contrast, rASIC2a was activated by extracellular acidification and rapidly desensitized even as the pH remained steady (90 ± 1% reduction, n = 3; Fig. 1, D and E). The activity of the acid-induced hENaCδ current was constitutive in the presence of an acidic medium, with clear distinction from the major properties of acid-sensitive degenerin/epithelial Na+ channels such as rASIC2a expressed in the brain (9Price M.P. Snyder P.M. Welsh M.J. J. Biol. Chem. 1996; 271: 7879-7882Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 10Waldmann R. Lazdunski M. Curr. Opin. Neurobiol. 1998; 8: 418-424Crossref PubMed Scopus (441) Google Scholar), which was rapidly activated by protons and desensitized even during acidic pH (Fig. 1, D and E, and Refs. 6Ugawa S. Minami Y. Guo W. Saishin Y. Takatsuji K. Yamamoto T. Tohyama M. Shimada S. Nature. 1998; 395: 555-556Crossref PubMed Scopus (148) Google Scholar, 10Waldmann R. Lazdunski M. Curr. Opin. Neurobiol. 1998; 8: 418-424Crossref PubMed Scopus (441) Google Scholar, and 11Ugawa S. Ueda T. Minami Y. Horimoto M. Shimada S. Neuroreport. 2001; 12: 2141-2145Crossref PubMed Scopus (8) Google Scholar). The kinetics of desensitization during acidification on ASIC1a and ASIC2a are faster; the pH 6.0-evoked ASIC1a current is desensitized to the resting level within 30 s (12Waldmann R. Champigny G. Bassilana F. Heurteaux C. Lazdunski M. Nature. 1997; 386: 173-177Crossref PubMed Scopus (1106) Google Scholar, 13Sutherland S.P. Benson C.J. Adelman J.P. McCleskey E.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 711-716Crossref PubMed Scopus (326) Google Scholar), and the pH 5.0-induced ASIC2a current is reduced to ∼30% of peak amplitude within 30 s (9Price M.P. Snyder P.M. Welsh M.J. J. Biol. Chem. 1996; 271: 7879-7882Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 11Ugawa S. Ueda T. Minami Y. Horimoto M. Shimada S. Neuroreport. 2001; 12: 2141-2145Crossref PubMed Scopus (8) Google Scholar, 14Champigny G. Voilley N. Waldmann R. Lazdunski M. J. Biol. Chem. 1998; 273: 15418-15422Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). On the other hand, pH-sensitive ASIC3 current possesses unique biphasic kinetics or a transient larger current followed by a sustained current of smaller amplitude (13Sutherland S.P. Benson C.J. Adelman J.P. McCleskey E.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 711-716Crossref PubMed Scopus (326) Google Scholar, 15Waldmann R. Bassilana F. de Weille J. Champigny G. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 20975-20978Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). The desensitization of the acid-evoked hENaCδ current was much slower than that of ASICs, and no significant desensitization was observed during acidification on an acid-elicited hENaCδ current. In hENaCδ-expressed oocytes, the gradual decrease in pH from 7.5 to 3.0 showed that the hENaCδ current was increased by acidic pH below 6.5 in a proton concentration-dependent manner (704 ± 75 nA at pH 3.0, n = 13, p < 0.01) and was mostly saturated at pH 4.0 (Fig. 2). The half-maximal pH for activation of the hENaCδ current was 5.0, and the Hill coefficient was 0.6. The proton-induced currents were from the hENaCδ channel because in control oocytes minimal acid-evoked currents were observed at lower than pH 4.0 (122 ± 56 nA at pH 3.0, n = 7) and the currents were insensitive to 100 μm amiloride (n = 5). Acid-evoked hENaCδ Currents in CHO-K1 Cells—Further evidence for the acid-evoked activation of hENaCδ was provided by whole-cell voltage clamp studies of CHO-K1 cells transiently expressing hENaCδ (Fig. 3). At a holding potential of -60 mV, 100 μm amiloride-sensitive inward currents were observed in hENaCδ-transfected cells (193 ± 15 pA, n = 9, p < 0.01 versus native) but not in native cells (6 ± 2 pA, n = 9). Extracellular acidification induced larger amiloride-sensitive currents in an acidic pH-dependent manner in hENaCδ cells (504 ± 58 pA at pH 5.0, n = 9, p < 0.01) than any observed in native cells (47 ± 10 pA, n = 9). The pH response profile in CHO-K1 cells was very similar to that obtained in the oocytes. Although both ENaC and ASIC families belong to the degenerin/epithelial Na+ channel superfamily, the functional properties of ENaCs have been thought to differ from those of ASICs. ENaCs are constitutively active channels, whereas ASICs require acidic stimulation for activation (7Alvarez de la Rosa D. Canessa C.M. Fyfe G.K. Zhang P. Annu. Rev. Physiol. 2000; 62: 573-594Crossref PubMed Scopus (286) Google Scholar, 8Kellenberger S. Schild L. Physiol. Rev. 2002; 82: 735-767Crossref PubMed Scopus (839) Google Scholar, 10Waldmann R. Lazdunski M. Curr. Opin. Neurobiol. 1998; 8: 418-424Crossref PubMed Scopus (441) Google Scholar). The members of the ASIC family have been implicated as transducers of pH stimuli because these channels are activated by acidification in an extracellular compartment (8Kellenberger S. Schild L. Physiol. Rev. 2002; 82: 735-767Crossref PubMed Scopus (839) Google Scholar, 10Waldmann R. Lazdunski M. Curr. Opin. Neurobiol. 1998; 8: 418-424Crossref PubMed Scopus (441) Google Scholar). The profile of pH responsiveness on ASICs varies with these channels (8Kellenberger S. Schild L. Physiol. Rev. 2002; 82: 735-767Crossref PubMed Scopus (839) Google Scholar, 10Waldmann R. Lazdunski M. Curr. Opin. Neurobiol. 1998; 8: 418-424Crossref PubMed Scopus (441) Google Scholar); the half-maximal pH values for the activation of heterologously expressed ASIC1a, ASIC2a, and ASIC3 are 6.2–6.4 (12Waldmann R. Champigny G. Bassilana F. Heurteaux C. Lazdunski M. Nature. 1997; 386: 173-177Crossref PubMed Scopus (1106) Google Scholar, 13Sutherland S.P. Benson C.J. Adelman J.P. McCleskey E.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 711-716Crossref PubMed Scopus (326) Google Scholar), 4.35 (11Ugawa S. Ueda T. Minami Y. Horimoto M. Shimada S. Neuroreport. 2001; 12: 2141-2145Crossref PubMed Scopus (8) Google Scholar, 14Champigny G. Voilley N. Waldmann R. Lazdunski M. J. Biol. Chem. 1998; 273: 15418-15422Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), and 6.5–6.7/3.5 (for peak/sustained current) (13Sutherland S.P. Benson C.J. Adelman J.P. McCleskey E.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 711-716Crossref PubMed Scopus (326) Google Scholar, 15Waldmann R. Bassilana F. de Weille J. Champigny G. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 20975-20978Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar), respectively. On the other hand, ENaCα is blocked by acidic pH (16Chalfant M.L. Denton J.S. Berdiev B.K. Ismailov I.I. Benos D.J. Stanton B.A. Am. J. Physiol. 1999; 276: C477-C486Crossref PubMed Google Scholar, 17Zhang P. Fyfe G.K. Grichtchenko I.I. Canessa C.M. Biophys. J. 1999; 77: 3043-3051Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 18Konstas A.A. Mavrelos D. Korbmacher C. Pflügers Arch. Eur. J. Physiol. 2000; 441: 341-350Crossref PubMed Scopus (20) Google Scholar). In this investigation, using two different expression systems, we found that the activity of hENaCδ was clearly enhanced by extracellular acidification and the response was influenced by amiloride. Expression of ENaCδ in Human Tissues—The ENaCδ distribution was analyzed using a human RNA dot-blot array. The RNA dot-blot analyses demonstrated that ENaCδ mRNAs were widely expressed in various human brain regions such as the cerebellum, cerebral cortex, hippocampus, caudate nucleus, and putamen (Fig. 4A). Moreover, strong blotting signals were also found in the heart, kidney, and pancreas as well as in the fetal brain, heart, and kidney. Next we performed Northern blot analyses (Fig. 4B) using the poly(A)+ RNAs obtained from freshly frozen human cerebellum and hippocampus, where an intense signal was shown among brain in RNA dot-blot analysis. The expression of ∼5.5-kb mRNA was observed in the cerebellum. Moreover, a faint signal of ∼5.5 kb was detected in the hippocampus. Both RNA dot-blot and Northern blot analyses showed that ENaCδ mRNA was expressed in human brain tissue such as the cerebellum and hippocampus. Neuronal activity is well known to be associated with pH fluctuations (19Chesler M. Kaila K. Trends Neurosci. 1992; 15: 396-402Abstract Full Text PDF PubMed Scopus (471) Google Scholar). In this investigation, hENaCδ started to open at pH 6.5 and induced larger amiloride-sensitive currents with more acidic pH. More recently, ASIC1a in the central nervous system has been implicated in long term potentiation, suggesting that minute fluxes in synaptic pH may activate ASICs to enhance synaptic plasticity, learning, and memory (20Bianchi L. Driscoll M. Neuron. 2002; 34: 337-340Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 21Wemmie J.A. Chen J. Askwith C.C. Hruska-Hageman A.M. Price M.P. Nolan B.C. Yoder P.G. Lamani E. Hoshi T. Freeman Jr., J.H. Welsh M.J. Neuron. 2002; 34: 463-477Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). These findings (21Wemmie J.A. Chen J. Askwith C.C. Hruska-Hageman A.M. Price M.P. Nolan B.C. Yoder P.G. Lamani E. Hoshi T. Freeman Jr., J.H. Welsh M.J. Neuron. 2002; 34: 463-477Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar) provide a starting point for a number of exciting follow up investigations into the role of the neuronal degenerin/epithelial Na+ channel family in the brain. Preliminary experiments into acquiring an ENaCδ clone from the neuronal tissues of other mammals, such as rats and mice, were unsuccessful. A further search of the expressed sequence tag data base showed that not even a partial fragment of ENaCδ has been found yet except for in humans and chimpanzees (GenBank™ accession numbers U38254 and O46547, respectively). Nevertheless, other ENaC subunits (α, β, and γ) have been isolated not only from mammals but also from amphibians and birds. The corresponding genomic assignments of ENaCδ were identified on human chromosome 1p36.3-p36.2 (22Waldmann R. Bassilana F. Voilley N. Lazdunski M. Mattei M. Genomics. 1996; 34: 262-263Crossref PubMed Scopus (14) Google Scholar). ENaCδ may be a unique gene constructed on the genome of only primates in the process of evolution. In this investigation, we found that hENaCδ was abundantly expressed throughout the human brain, such as in the cerebellum and hippocampus, and the channel activity was constitutively enhanced at a pH lower than 6.5 in a pH-dependent manner when expressed in Xenopus oocytes and CHO-K1 cells. Taken together, these data suggest a possible role for ENaCδ as a key component of proton-activated currents in the human brain. We thank Katsuyuki Tanaka and Kenji Kajita for technical assistance." @default.
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• W1995294810 date "2004-03-01" @default.
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• W1995294810 title "Protons Activate the δ-Subunit of the Epithelial Na+ Channel in Humans" @default.
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