The effect of environmental salinity on the protein expression of Na+/K+-ATPase, Na+/K+/2Cl− cotransporter, cystic fibrosis transmembrane conductance regulator, anion exchanger 1, and chloride channel 3 in gills of a euryhaline teleost, Tetraodon nigroviridis

Abstract

Chloride transport mechanisms in the gills of the estuarine spotted green pufferfish (Tetraodon nigroviridis) were investigated. Protein abundance of Na(+)/K(+)-ATPase (NKA) and the other four chloride transporters, i.e., Na(+)/K(+)/2Cl(-) cotransporter (NKCC), cystic fibrosis transmembrane conductance regulator (CFTR), Cl(-)/HCO(3)(-) anion exchanger 1 (AE1), and chloride channel 3 (CLC-3) in gills of the seawater- (SW; 35 per thousand) or freshwater (FW)-acclimatized fish were examined by immunoblot analysis. Appropriate negative controls were used to confirm the specificity of the antibodies to the target proteins. The relative protein abundance of NKA was higher (i.e., 2-fold) in gills of the SW group compared to the FW group. NKCC and CFTR were expressed in gills of the SW group but not in the FW group. In contrast, the levels of relative protein abundance of branchial AE1 and CLC-3 in the FW group were 23-fold and 2.7-fold higher, respectively, compared to those of the SW group. This study is first of its kind to provide direct in vivo evidence of the protein expression of CLC-3 in teleostean gills, as well as to examine the simultaneous protein expression of the Cl(-) transporters, especially AE1 and CLC-3 of FW- and SW-acclimatized teleosts. The differential protein expression of NKA, chloride transporters in gills of the FW- and SW-acclimatized T. nigroviridis observed in the present study shows their close relationship to the physiological homeostasis (stable blood osmolality), as well as explains the impressive ionoregulatory ability of this euryhaline species in response to salinity challenges.