{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,19]],"date-time":"2026-02-19T19:49:34Z","timestamp":1771530574843,"version":"3.50.1"},"reference-count":37,"publisher":"American Physiological Society","issue":"4","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["American Journal of Physiology-Cell Physiology"],"published-print":{"date-parts":[[2005,10]]},"abstract":"<jats:p> Opposite effects of Ni<jats:sup>2+<\/jats:sup> on Xenopus and rat ENaCs expressed in Xenopus oocytes. Am J Physiol Cell Physiol 289: C946\u2013C958, 2005. First published June 8, 2005; .\u2014The epithelial Na<jats:sup>+<\/jats:sup> channel (ENaC) is modulated by various extracellular factors, including Na<jats:sup>+<\/jats:sup>, organic or inorganic cations, and serine proteases. To identify the effect of the divalent Ni<jats:sup>2+<\/jats:sup> cation on ENaCs, we compared the Na<jats:sup>+<\/jats:sup> permeability and amiloride kinetics of Xenopus ENaCs (xENaCs) and rat ENaCs (rENaCs) heterologously expressed in Xenopus oocytes. We found that the channel cloned from the kidney of the clawed toad Xenopus laevis [wild-type (WT) xENaC] was stimulated by external Ni<jats:sup>2+<\/jats:sup>, whereas the divalent cation inhibited the channel cloned from the rat colon (WT rENaC). The kinetics of amiloride binding were determined using noise analysis of blocker-induced fluctuation in current adapted for the transoocyte voltage-clamp method, and Na<jats:sup>+<\/jats:sup> conductance was assessed using the dual electrode voltage-clamp (TEVC) technique. The inhibitory effect of Ni<jats:sup>2+<\/jats:sup> on amiloride binding is not species dependent, because Ni<jats:sup>2+<\/jats:sup> decreased the affinity (mainly reducing the association rate constant) of the blocker in both species in competition with Na<jats:sup>+<\/jats:sup>. Importantly, using the TEVC method, we found a prominent difference in channel conductance at hyperpolarizing voltage pulses. In WT xENaCs, the initial ohmic current response was stimulated by Ni<jats:sup>2+<\/jats:sup>, whereas the secondary voltage-activated current component remained unaffected. In WT rENaCs, only a voltage-dependent block by Ni<jats:sup>2+<\/jats:sup> was obtained. To further study the origin of the xENaC stimulation by Ni<jats:sup>2+<\/jats:sup>, and based on the rationale of the well-known high affinity of Ni<jats:sup>2+<\/jats:sup> for histidine residues, we designed \u03b1-subunit mutants of xENaCs by substituting histidines that were expressed in oocytes, together with WT \u03b2- and \u03b3-subunits. Changing His<jats:sup>215<\/jats:sup> to Asp in one putative amiloride-binding domain (WYRFHY) in the extracellular loop between Na<jats:sup>+<\/jats:sup> channel membrane segments M1 and M2 had no influence on the stimulatory effect of Ni<jats:sup>2+<\/jats:sup>, and neither did complete deletion of this segment. Next, we mutated His<jats:sup>416<\/jats:sup> flanked by His<jats:sup>411<\/jats:sup> and Cys<jats:sup>417<\/jats:sup>, a unique site for possible heavy metal ion chelation, and, with this quality, most proximal (\u223c100 amino acids upstream of the second putative amiloride binding site at the pore entrance), was found localized at M2. Replacing His<jats:sup>416<\/jats:sup> with arginine, aspartate, tyrosine, and alanine clearly affected amiloride binding in all cases, as well as Na<jats:sup>+<\/jats:sup> conductance, as expressed in the xENaC current-voltage relationship, especially with regard to aspartate and tyrosine. However, similarly to those obtained with the WYRFHY stretch, none of these mutations could either abolish the stimulating effect of Ni<jats:sup>2+<\/jats:sup> or reverse it to an inhibitory type. <\/jats:p>","DOI":"10.1152\/ajpcell.00419.2004","type":"journal-article","created":{"date-parts":[[2005,6,9]],"date-time":"2005-06-09T00:14:21Z","timestamp":1118276061000},"page":"C946-C958","source":"Crossref","is-referenced-by-count":11,"title":["Opposite effects of Ni<sup>2+<\/sup> on <i>Xenopus<\/i> and rat ENaCs expressed in <i>Xenopus<\/i> oocytes"],"prefix":"10.1152","volume":"289","author":[{"given":"Dana","family":"Cucu","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jeannine","family":"Simaels","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jan","family":"Eggermont","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Willy","family":"Van Driessche","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wolfgang","family":"Zeiske","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"24","reference":[{"key":"R1","doi-asserted-by":"crossref","unstructured":"Amuzescu B, Segal A, Flonta ML, Simaels J, and Van Driessche W. Zinc is a voltage-dependent blocker of native and heterologously expressed epithelial Na+ channels. Pfl\u00fcgers Arch 466: 69\u201377, 2003.","DOI":"10.1007\/s00424-002-0998-3"},{"key":"R2","doi-asserted-by":"crossref","unstructured":"Awayda MS. Specific and nonspecific effects of protein kinase C on the epithelial Na+ channel. J Gen Physiol 115: 559\u2013570, 2000.","DOI":"10.1085\/jgp.115.5.559"},{"key":"R3","doi-asserted-by":"crossref","unstructured":"Awayda MS, Boudreaux MJ, Reger RL, and Hamm LL. Regulation of the epithelial Na+ channel by extracellular acidification. Am J Physiol Cell Physiol 279: C1896\u2013C1905, 2000.","DOI":"10.1152\/ajpcell.2000.279.6.C1896"},{"key":"R4","doi-asserted-by":"crossref","unstructured":"Baud C, Kado RT, and Marcher K. Sodium channels induced by depolarization of the Xenopus laevis oocyte. Proc Natl Acad Sci USA 79: 3188\u20133192, 1982.","DOI":"10.1073\/pnas.79.10.3188"},{"key":"R5","doi-asserted-by":"crossref","unstructured":"Carrington PE, Chivers PT, Al-Mjeni F, Sauer RT, and Maroney MJ. Nickel coordination is regulated by the DNA-bound state of NikR. Nat Struct Biol 10: 126\u2013130, 2003.","DOI":"10.1038\/nsb890"},{"key":"R6","doi-asserted-by":"crossref","unstructured":"Chra\u00efbi A and Horisberger JD. Na+ self inhibition of human epithelial Na+ channel: temperature dependence and effect of extracellular proteases. J Gen Physiol 120: 133\u2013145, 2002.","DOI":"10.1085\/jgp.20028612"},{"key":"R7","doi-asserted-by":"crossref","unstructured":"Cucu D, Simaels J, Jans D, and Van Driessche W. The transoocyte voltage clamp: a non-invasive technique for electrophysiological experiments with Xenopus laevis oocytes. Pfl\u00fcgers Arch 447: 934\u2013942, 2004.","DOI":"10.1007\/s00424-003-1171-3"},{"key":"R8","doi-asserted-by":"crossref","unstructured":"Cucu D, Simaels J, Van Driessche W, and Zeiske W. External Ni2+ and ENaC in A6 cells: Na+ current stimulation by competition at a binding site for amiloride and Na+. J Membr Biol 194: 33\u201345, 2003.","DOI":"10.1007\/s00232-003-2023-y"},{"key":"R9","doi-asserted-by":"crossref","unstructured":"Flonta ML, De Beir-Simaels J, Mesotten D, and Van Driessche W. Cu2+ reveals different binding sites of amiloride and CDPC on the apical Na channel of frog skin. Biochim Biophys Acta 1370: 169\u2013174, 1998.","DOI":"10.1016\/S0005-2736(97)00261-7"},{"key":"R10","doi-asserted-by":"crossref","unstructured":"Grinstein S, Candia O, and Erlij D. Nonhormonal mechanisms for the regulation of transepithelial sodium transport: the roles of surface potential and cell calcium. J Membr Biol 40: 261\u2013280, 1978.","DOI":"10.1007\/BF02026010"},{"key":"R11","unstructured":"Harasztosi C, Rusznak Z, Kovacs L, and Szucs G. Effects of divalent cations on voltage-gated Ca2+ channels and depolarization-induced [Ca2+], transients of freshly isolated pyramidal cells of the rat dorsal cochlear nucleus. Gen Physiol Biophys 20: 349\u2013360, 2001."},{"key":"R12","doi-asserted-by":"crossref","unstructured":"Ho WK, Kim I, Lee CO, Youm JB, Lee SH, and Earm YE. Blockade of HERG channels expressed in Xenopus laevis oocytes by external divalent cations. Biophys J 76: 1959\u20131971, 1999.","DOI":"10.1016\/S0006-3495(99)77355-8"},{"key":"R13","doi-asserted-by":"crossref","unstructured":"Horisberger JD and Chra\u00efbi A. Epithelial sodium channel: a ligand-gated channel? Nephron Physiol 96: 37\u201341, 2004.","DOI":"10.1159\/000076406"},{"key":"R14","doi-asserted-by":"crossref","unstructured":"Ismailov II, Kieber-Emmons T, Lin C, Berdiev BK, Shlyonsky VG, Patton HK, Fuller CM, Worrell R, Zuckerman JB, Sun W, Eaton DC, Benos DJ, and Kleyman TR. Identification of an amiloride binding domain within the \u03b1-subunit of the epithelial Na+ channel. J Biol Chem 272: 21075\u201321083, 1997.","DOI":"10.1074\/jbc.272.34.21075"},{"key":"R15","doi-asserted-by":"crossref","unstructured":"Jans D, Simaels J, Cucu D, Zeiske W, and Van Driessche W. Effects of extracellular Mg2+ on transepithelial capacitance and Na+ transport in A6 cells under different osmotic conditions. Pfl\u00fcgers Arch 439: 504\u2013512, 2000.","DOI":"10.1007\/s004249900194"},{"key":"R16","doi-asserted-by":"crossref","unstructured":"Kellenberger S, Gautschi I, and Schild L. An external site controls closing of the epithelial Na+ channel ENaC. J Physiol 543: 413\u2013424, 2002.","DOI":"10.1113\/jphysiol.2002.022020"},{"key":"R17","doi-asserted-by":"crossref","unstructured":"Kellenberger S, Gautschi I, and Schild L. Mutations in the epithelial Na+ channel ENaC outer pore disrupt amiloride block by increasing its dissociation rate. Mol Pharmacol 64: 848\u2013856, 2003.","DOI":"10.1124\/mol.64.4.848"},{"key":"R18","doi-asserted-by":"crossref","unstructured":"Kellenberger S and Schild L. Epithelial sodium channel\/degenerin family of ion channels: a variety of functions for a shared structure. Physiol Rev 82: 735\u2013767, 2002.","DOI":"10.1152\/physrev.00007.2002"},{"key":"R19","doi-asserted-by":"crossref","unstructured":"Kelly O, Lin C, Ramkumar M, Saxena NC, Kleyman TR, and Eaton DC. Characterization of an amiloride binding region in the \u03b1-subunit of ENaC. Am J Physiol Renal Physiol 285: F1279\u2013F1290, 2003.","DOI":"10.1152\/ajprenal.00094.2003"},{"key":"R20","doi-asserted-by":"crossref","unstructured":"Khater KA and Rakowski RF. Effect of pH on charge movement by the Na\/K pump in Xenopus oocytes. Ann NY Acad Sci 834: 350\u2013353, 1997.","DOI":"10.1111\/j.1749-6632.1997.tb52268.x"},{"key":"R21","doi-asserted-by":"crossref","unstructured":"Magistretti J, Brevi S, and de Curtis M. Ni2+ slows the activation kinetics of high-voltage-activated Ca2+ currents in cortical neurons: evidence for a mechanism of action independent of channel-pore block. J Membr Biol 179: 243\u2013262, 2001.","DOI":"10.1007\/s002320010050"},{"key":"R22","doi-asserted-by":"crossref","unstructured":"Oh Y and Benos DJ. Single-channel characteristics of a purified bovine renal amiloride-sensitive Na+ channel in planar lipid bilayers. Am J Physiol Cell Physiol 264: C1489\u2013C1499, 1993.","DOI":"10.1152\/ajpcell.1993.264.6.C1489"},{"key":"R23","doi-asserted-by":"crossref","unstructured":"Palmer LG. Interactions of amiloride and other blocking cations with the apical Na channel in the toad urinary bladder. J Membr Biol 87: 191\u2013199, 1985.","DOI":"10.1007\/BF01871218"},{"key":"R24","doi-asserted-by":"crossref","unstructured":"Palmer LG, Corthesy-Theulaz I, Gaeggeler HP, Kraehenbuhl JP, and Rossier B. Expression of epithelial Na channels in Xenopus oocytes. J Gen Physiol 96: 23\u201346, 1990.","DOI":"10.1085\/jgp.96.1.23"},{"key":"R25","doi-asserted-by":"crossref","unstructured":"Schild L, Schneeberger E, Gautschi I, and Firsov D. Identification of amino acid residues in the \u03b1, \u03b2, and \u03b3 subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation. J Gen Physiol 109: 15\u201326, 1997.","DOI":"10.1085\/jgp.109.1.15"},{"key":"R26","doi-asserted-by":"crossref","unstructured":"Segal A, Awayda MS, Eggermont J, Van Driessche W, and Weber WM. Influence of voltage and extracellular Na+ on amiloride block and transport kinetics of rat epithelial Na+ channel expressed in Xenopus oocytes. Pfl\u00fcgers Arch 443: 882\u2013891, 2002.","DOI":"10.1007\/s00424-001-0773-x"},{"key":"R27","doi-asserted-by":"crossref","unstructured":"Segal A, Cucu D, Van Driessche W, and Weber WM. Rat ENaC expressed in Xenopus laevis oocytes is activated by cAMP and blocked by Ni2+. FEBS Lett 515: 177\u2013183, 2002.","DOI":"10.1016\/S0014-5793(02)02464-X"},{"key":"R28","doi-asserted-by":"crossref","unstructured":"Sheng S, Perry CJ, and Kleyman TR. External nickel inhibits epithelial sodium channel by binding to histidine residues within the extracellular domains of \u03b1 and \u03b3 subunits and reducing channel open probability. J Biol Chem 277: 50098\u201350111, 2002.","DOI":"10.1074\/jbc.M209975200"},{"key":"R29","doi-asserted-by":"crossref","unstructured":"Sheng S, Perry CJ, and Kleyman TR. Extracellular Zn2+ activates epithelial Na+ channels by eliminating Na+ self-inhibition. J Biol Chem 279: 31687\u201331696, 2004.","DOI":"10.1074\/jbc.M405224200"},{"key":"R30","doi-asserted-by":"crossref","unstructured":"St\u00fchmer W. Electrophysiologic recordings from Xenopus oocytes. Methods Enzymol 293: 280\u2013300, 1998.","DOI":"10.1016\/S0076-6879(98)93019-1"},{"key":"R31","doi-asserted-by":"crossref","unstructured":"Van Driessche W and Erlij D. Noise analysis of inward and outward Na+ currents across the apical border of ouabain-treated frog skin. Pfl\u00fcgers Arch 398: 179\u2013188, 1983.","DOI":"10.1007\/BF00657149"},{"key":"R32","doi-asserted-by":"crossref","unstructured":"Van Driessche W and Van Deynse N. Analysis of transepithelial noise signals from ion channels: advantages and limitations of the method. In: Current Topics in Membrane Transport, edited by Bronner F, Helman SI, and Van Driessche W. San Diego, CA: Academic, 1990, p. 37\u201359.","DOI":"10.1016\/S0070-2161(08)60228-2"},{"key":"R33","doi-asserted-by":"crossref","unstructured":"Virginio C, Church D, North RA, and Surprenant A. Effects of divalent cations, protons and calmidazolium at the rat P2X7 receptor. Neuropharmacology 36: 1285\u20131294, 1997.","DOI":"10.1016\/S0028-3908(97)00141-X"},{"key":"R34","doi-asserted-by":"crossref","unstructured":"Watt RK and Ludden PW. Nickel-binding proteins. Cell Mol Life Sci 56: 604\u2013625, 1999.","DOI":"10.1007\/s000180050456"},{"key":"R35","unstructured":"Zeiske W. Die Na+-Aufnahme durch die apikale Membran des Froschhautepithels: ihr Mechanismus und ihre Steuerung durch Ionen und lipophile Substanzen. Saarbr\u00fccken, Germany: Universit\u00e4t des Saarlandes, 1979."},{"key":"R36","doi-asserted-by":"crossref","unstructured":"Zeiske W and Lindemann B. Chemical stimulation of Na+ current through the outer surface of frog skin epithelium. Biochim Biophys Acta 352: 323\u2013326, 1974.","DOI":"10.1016\/0005-2736(74)90223-5"},{"key":"R37","doi-asserted-by":"crossref","unstructured":"Zelenina M, Bondar AA, Zelenin S, and Aperia A. Nickel and extracellular acidification inhibit the water permeability of human aquaporin-3 in lung epithelial cells. J Biol Chem 278: 30037\u201330043, 2003.","DOI":"10.1074\/jbc.M302206200"}],"container-title":["American Journal of Physiology-Cell Physiology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.physiology.org\/doi\/pdf\/10.1152\/ajpcell.00419.2004","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,6,17]],"date-time":"2024-06-17T18:27:24Z","timestamp":1718648844000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.physiology.org\/doi\/10.1152\/ajpcell.00419.2004"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2005,10]]},"references-count":37,"journal-issue":{"issue":"4","published-print":{"date-parts":[[2005,10]]}},"alternative-id":["10.1152\/ajpcell.00419.2004"],"URL":"https:\/\/doi.org\/10.1152\/ajpcell.00419.2004","relation":{},"ISSN":["0363-6143","1522-1563"],"issn-type":[{"value":"0363-6143","type":"print"},{"value":"1522-1563","type":"electronic"}],"subject":[],"published":{"date-parts":[[2005,10]]}}}