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 CaCO3precipitate 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 HCO3−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+/HCO3−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+/ nHCO3−cotransport; HCO3−independent, DIDS-insensitive transport; and increased basal intracellular Na+accumulation. fNBCe1 is a voltage-dependent Na+/ nHCO3−cotransporter that rectifies, independently from the extracellular Na+or HCO3−concentration, around −60 mV. Na+removal (0Na+prepulse) is necessary to produce the true HCO3−-elicited current. HCO3−addition results in huge outward currents with quick current decay. Kinetic analysis of HCO3−currents reveals that fNBCe1 has a much higher transport capacity (higher maximum current) and lower affinity (higher Km) than human kidney NBCe1 (hkNBCe1) does in the physiological range (membrane potential = −80 mV; [HCO3−] = 10 mM). In this state, fNBCe1 is in favor of operating as transepithelial HCO3−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.