The novel correlation of carbonic anhydrase II and anion exchanger 1 in gills of the spotted green pufferfish, Tetraodon nigrovirids

A novel relationship between branchial carbonic anhydrase II (CAII) and anion exchanger 1 (AE1) was investigated in the euryhaline spotted green pufferfish (Tetraodon nigrouiradis). The immunoblots revealed that AE1 was only detected in the membrane fraction of gills while CAII can be probed both in the membrane and cytosol fractions of gills. CAII protein abundance in the membrane fraction is salinity dependent. Immunological detection of the membrane fraction CAII protein in gills showed 3.9-fold higher in the hyposmotic (freshwater) group than the hyperosmotic (seawater;35 parts per thousand) group. In contrast, there was no change in the protein level of cytosolic CAII between seawater and freshwater groups. The whole-mount immunocytochemical staining demonstrated that both AE1 and CAII were colocalized to the Na+/K+-ATPas-eimmunoreactive cells in gill epithelium of the pufferfish. The interaction between CAII and AE1 was further identified by co-immunoprecipitation because AE1 was detected in the immunoprecipitates of CAII and vice versa. Our results showed that in pufferfish gills CAII was not only expressed in the cytosol to produce the substrate for AE1 transport during Cl- influx but also associated with the plasma membrane via AE1. Obviously, it is essential for the physiological function of AE1 to interact with CAII in the membrane of gill Na+/K+-ATPase-immunoreactive cells. To our knowledge, this is the first study to demonstrate the interaction of branchial CAII and AE1 in fish. The novel correlation proposed a new model of Cl-/HCO3- transport in gills of the teleosts.