Opposite effects of Ni2+ on Xenopus and rat ENaCs expressed in Xenopus oocytes

• Published in: American Journal of Physiology: Cell Physiology 2005-10, Vol.289 (4), p.C946-C958
• Main Authors: Cucu, Dana, Simaels, Jeannine, Eggermont, Jan, Van Driessche, Willy, Zeiske, Wolfgang
• Format: Article
• Language: English
• Subjects: Amiloride - pharmacology; Animals; Diuretics - pharmacology; Electrophysiology; Epithelial Sodium Channels; Membrane Potentials - drug effects; Membrane Potentials - physiology; Mutation; Nickel - pharmacology; Oocytes - metabolism; Rats; Recombinant Proteins - biosynthesis; RNA, Messenger - metabolism; Sodium Channels - biosynthesis; Sodium Channels - drug effects; Sodium Channels - genetics; Sodium Channels - physiology; Species Specificity; Xenopus
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00419.2004

1 Laboratory of Physiology, Department of Molecular Cell Biology, K. U. Leuven, Campus Gasthuisberg O & N, Leuven, Belgium; 2 Division of Animal Physiology, Department of Biology/Chemistry, University of Osnabrück, Osnabrück, Germany Submitted 25 August 2004 ; accepted in final form 30 May 2005 Opposite effects of Ni 2+ on Xenopus and rat ENaCs expressed in Xenopus oocytes. Am J Physiol Cell Physiol 289: C946–C958, 2005. First published June 8, 2005; .—The epithelial Na + channel (ENaC) is modulated by various extracellular factors, including Na + , organic or inorganic cations, and serine proteases. To identify the effect of the divalent Ni 2+ cation on ENaCs, we compared the Na + 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 2+ , 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 + conductance was assessed using the dual electrode voltage-clamp (TEVC) technique. The inhibitory effect of Ni 2+ on amiloride binding is not species dependent, because Ni 2+ decreased the affinity (mainly reducing the association rate constant) of the blocker in both species in competition with Na + . 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 2+ , whereas the secondary voltage-activated current component remained unaffected. In WT rENaCs, only a voltage-dependent block by Ni 2+ was obtained. To further study the origin of the xENaC stimulation by Ni 2+ , and based on the rationale of the well-known high affinity of Ni 2+ for histidine residues, we designed -subunit mutants of xENaCs by substituting histidines that were expressed in oocytes, together with WT - and -subunits. Changing His 215 to Asp in one putative amiloride-binding domain (WYRFHY) in the extracellular loop between Na + channel membrane segments M1 and M2 had no influence on the stimulatory effect of Ni 2+ , and neither did complete deletion of this segment. Next, we mutated His 416 flanked by His 411 and Cys 417 , a unique site for possible