CHARACTERIZATION OF THE PROTEINS OF THE INTESTINAL NA+-K+-2CL- COTRANSPORTER

Absorptive intestinal epithelia, such as that of the winter flounder, absorb salt via a bumetanide-sensitive Na+-K+-2Cl(-) cotransport mechanism on the brush-border membrane (BBM). The present study demonstrates the first molecular characterization of the intestinal Na+-K+-2Cl(-) cotransporter and its unique regulation. The photoaffinity bumetanide analogue, 4-[H-3]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid, specifically labeled three groups of proteins in flounder intestinal microsomal membranes (MM): a similar to 180-kDa peptide, prominently labeled, and diffuse bands at similar to 110-70 and 50 kDa, less intensely labeled. Subcellular fractionation revealed a single prominently labeled protein of similar to 170 kDa in BBM but not in basolateral membranes (BLM) and little or no labeling of proteins of similar to 110-70 or 50 kDa. Polyclonal antiserum raised against the Ehrlich ascites cell cotransporter identified a 180-kDa peptide in MM and a 175-kDa peptide (pI similar to 5.4) in BBM but none in BLM or in the cytosol of flounder intestine. As predicted from the regulation of cotransport in this tissue, phosphorylation of this protein is increased by guanosine 3',5'-cyclic monophosphate (cGMP)-dependent but not by adenosine 3',5'-cyclic monophosphate-dependent protein kinase. In addition, phosphorylation of the protein is not increased by protein kinase C or Ca2+ /calmodulin-dependent protein kinase but is increased by the phosphatase inhibitor calyculin A. Finally, calyculin A preserves the inhibitory effect of cGMP on ion transport, even in the absence of the nucleotide, suggesting that phosphoryla tion-dephosphorylation mechanisms are crucial in cotransporter regulation. Thus the flounder intestinal cotransporter is a similar to 175-kDa BBM protein that can be regulated by phosphorylation.