Ammonia exposure increases the expression of Na+:K+:2Cl− cotransporter 1a in the gills of the giant mudskipper, Periophthalmodon schlosseri
The giant mudskipper, Periophthalmodon schlosseri, is an obligate air-breathing teleost that can actively excrete ammonia against high concentrations of environmental ammonia. This study aimed to clone and sequence the Na+:K+:2Cl−cotransporter1 (nkcc1) from the gills of P. schlosseri, and to determine the effects of ammonia exposure on its mRNA expression and protein abundance after pre-acclimation to slightly brackish water (salinity 3; SBW) for 2 weeks. The complete coding cDNA sequences of nkcc1a consisted of 3453 bp, coding for 1151 amino acid with an estimated molecular mass of 125.4 kDa. Exposure to 75 mmol l−1 NH4Cl in SBW had no effect on the mRNA expression of nkcc1a. However, western blotting revealed a significant increase in the protein abundance of multiple T4-immunoreactive bands of molecular mass 170–250 kDa in the gills of P. schlosseri exposed to ammonia. Furthermore, immunofluorescence microscopy demonstrated the colocalization of the increased T4-immunoreactive protein with Na+/K+-ATPase (Nka) α-subunit to the basolateral membrane of certain ionocytes in the gills of the ammonia-exposed fish. As Nkcc1 is known to have a basolateral localization, it can be concluded that ammonia exposure led to an increase in the expression of glycosylated Nkcc1, the molecular masses of which were reduced upon enzymatic deglycosylation, in the gills of P. schlosseri. The dependency on post-transcriptional and post-translational regulation of branchial Nkcc1 in P. schlosseri would facilitate prompt responses to changes in environmental condition. As NH4+ can replace K+, NH4+ could probably enter ionocytes through the basolateral Nkcc1a during active ammonia excretion, but increased influx of Na+, NH4+ and 2Cl− would alter the transmembrane Na+ gradient. Consequently, exposure of P. schlosseri to ammonia would also result in an increase in branchial activity of Nka with decreased NH4+ affinity so as to maintain intracellular Na+ and K+ homeostasis as reported elsewhere.