title: Euryhaline pufferfish NBCe1 differs from nonmarine species NBCe1 physiology.
abstract: Marine fish drink seawater and eliminate excess salt by active salt transport across gill and gut epithelia. Euryhaline pufferfish (Takifugu obscurus, mefugu) forms a CaCO(3) precipitate on the luminal gut surface after transitioning to seawater. NBCe1 (Slc4a4) at the basolateral membrane of intestinal epithelial cell plays a major role in transepithelial intestinal HCO(3)(-) secretion and is critical for mefugu acclimation to seawater. We assayed fugu-NBCe1 (fNBCe1) activity in the Xenopus oocyte expression system. Similar to NBCe1 found in other species, fNBCe1 is an electrogenic Na(+)/HCO(3)(-) cotransporter and sensitive to the stilbene inhibitor DIDS. However, our experiments revealed several unique and distinguishable fNBCe1 transport characteristics not found in mammalian or other teleost NBCe1-orthologs: electrogenic Li(+)/nHCO(3)(-) cotransport; HCO(3)(-) independent, DIDS-insensitive transport; and increased basal intracellular Na(+) accumulation. fNBCe1 is a voltage-dependent Na(+)/nHCO(3)(-) cotransporter that rectifies, independently from the extracellular Na(+) or HCO(3)(-) concentration, around -60 mV. Na(+) removal (0Na(+) prepulse) is necessary to produce the true HCO(3)(-)-elicited current. HCO(3)(-) addition results in huge outward currents with quick current decay. Kinetic analysis of HCO(3)(-) currents reveals that fNBCe1 has a much higher transport capacity (higher maximum current) and lower affinity (higher K(m)) than human kidney NBCe1 (hkNBCe1) does in the physiological range (membrane potential = -80 mV; [HCO(3)(-)] = 10 mM). In this state, fNBCe1 is in favor of operating as transepithelial HCO(3)(-) secretion, opposite of hkNBCe1, from blood to the luminal side. Thus, fugu-NBCe1 represents the first ortholog-based tool to study amino acid substitutions in NBCe1 and how those change ion and voltage dependence.
authors.affiliation: Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA. chang.minhwang@mayo.edu
authors.forename: Min-Hwang
authors.initials: MH
authors.lastname: Chang
authors.forename: Consuelo
authors.initials: C
authors.lastname: Plata
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Kurita
authors.forename: Akira
authors.initials: A
authors.lastname: Kato
authors.forename: Shigehisa
authors.initials: S
authors.lastname: Hirose
authors.forename: Michael F
authors.initials: MF
authors.lastname: Romero
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D019943:Amino Acid Substitution / Q000187:drug effects; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D005260:Female; D006801:Humans; D007700:Kinetics; D008094:Lithium / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D012964:Sodium / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics / Q000378:metabolism*; D023741:Takifugu / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D001639:Bicarbonates; D027982:Sodium-Bicarbonate Symporters; D008094:Lithium; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid