title: Na+/H+ and Na+/NH+4 activities of zebrafish NHE3b expressed in Xenopus oocytes.
abstract: Zebrafish Na(+)/H(+) exchanger 3b (zNHE3b) is highly expressed in the apical membrane of ionocytes where Na(+) is absorbed from ion-poor fresh water against a concentration gradient. Much in vivo data indicated that zNHE3b is involved in Na(+) absorption but not leakage. However, zNHE3b-mediated Na(+) absorption has not been thermodynamically explained, and zNHE3b activity has not been measured. To address this issue, we overexpressed zNHE3b in Xenopus oocytes and characterized its activity by electrophysiology. Exposure of zNHE3b oocytes to Na(+)-free media resulted in significant decrease in intracellular pH (pH(i)) and intracellular Na(+) activity (aNa(i)). aNa(i) increased significantly when the cytoplasm was acidified by media containing CO₂-HCO₃(-) or butyrate. Activity of zNHE3b was inhibited by amiloride or 5-ethylisopropyl amiloride (EIPA). Although the activity was accompanied by a large hyperpolarization of ∼50 mV, voltage-clamp experiments showed that Na(+)/H(+) exchange activity of zNHE3b is electroneutral. Exposure of zNHE3b oocytes to medium containing NH₃/NH₄(+) resulted in significant decreases in pH(i) and aNa(i) and significant increase in intracellular NH₄(+) activity, indicating that zNHE3b mediates the Na(+)/NH₄(+) exchange. In low-Na(+) (0.5 mM) media, zNHE3b oocytes maintained aNa(i) of 1.3 mM, and Na(+)-influx was observed when pHi was decreased by media containing CO₂-HCO₃(-) or butyrate. These results provide thermodynamic evidence that zNHE3b mediates Na(+) absorption from ion-poor fresh water by its Na(+)/H(+) and Na(+)/NH₄(+) exchange activities.
authors.affiliation: Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan; and.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Ito
authors.forename: Akira
authors.initials: A
authors.lastname: Kato
authors.forename: Taku
authors.initials: T
authors.lastname: Hirata
authors.forename: Shigehisa
authors.initials: S
authors.lastname: Hirose
authors.forename: Michael F
authors.initials: MF
authors.lastname: Romero
mesh_terms: D062646:Acid Sensing Ion Channel Blockers / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D064751:Ammonium Compounds / Q000378:metabolism*; D000818:Animals; D055724:Electrophysiological Phenomena; D005618:Fresh Water / Q000737:chemistry; D005786:Gene Expression Regulation / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D008839:Microelectrodes; D009865:Oocytes / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D014981:Xenopus; D015027:Zebrafish
chemical_list: D062646:Acid Sensing Ion Channel Blockers; D064751:Ammonium Compounds; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D012964:Sodium; C039614:ethylisopropylamiloride
keywords: NHE3; Na+/H+ exchange; Na+/NH4+ exchange; Xenopus oocyte electrophysiology; ammonium; intracellular pH; zebrafish