Functional characterization and osmoregulatory role of the Na+/K+/2Cl--cotransporter (NKCC1) in the gill of sea lamprey (Petromyzon marinus), a basal vertebrate

The present study provides molecular and functional characterization of Na+/K+/2Cl- cotransporter (nkcc1/NKCC1) in the gills of sea lamprey, the most basal extant vertebrate with an osmoregulatory strategy. We report the full-length peptide sequence for the lamprey NKCC1, which we show to group strongly with and occupy a basal position among other vertebrate NKCC1 sequences. Lamprey nkcc1 mRNA were present in many tissues but was 5-fold higher in the gill than any other tissue. NKCC1 protein was only detected in the gill. Gill mRNA and protein abundances of NKCC1 and Na+/K+-ATPase (NKA) were significantly upregulated (20- to 200-fold) in late metamorphosis in freshwater, coinciding with the development of salinity tolerance, and were upregulated an additional 2-fold after acclimation to seawater. Immunohistochemistry revealed that NKCC1 in the gill is found in filamental ionocytes that develop during metamorphosis. Lamprey treated with bumetanide, a widely used pharmacological inhibitor of NKCC1, exhibited higher plasma Cl- and osmolality and reduced muscle water content after 24 h in seawater, but had no effect in FW. This work provides the first functional characterization of NKCC1 as having a functional role mechanism for branchial salt secretion in lampreys, providing evidence that this mode of Cl- secretion has been present among vertebrates for ~550 million years.