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 mecha nism on the brush-border membrane (BBM). The present study demonstrate s 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, spe cifically labeled three groups of proteins in flounder intestinal micr osomal membranes (MM): a similar to 180-kDa peptide, prominently label ed, and diffuse bands at similar to 110-70 and 50 kDa, less intensely labeled. Subcellular fractionation revealed a single prominently label ed protein of similar to 170 kDa in BBM but not in basolateral membran es (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 floun der 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'-cy clic monophosphate-dependent protein kinase. In addition, phosphorylat ion of the protein is not increased by protein kinase C or Ca2+ /calmo dulin-dependent protein kinase but is increased by the phosphatase inh ibitor calyculin A. Finally, calyculin A preserves the inhibitory effe ct of cGMP on ion transport, even in the absence of the nucleotide, su ggesting that phosphoryla tion-dephosphorylation mechanisms are crucia l in cotransporter regulation. Thus the flounder intestinal cotranspor ter is a similar to 175-kDa BBM protein that can be regulated by phosp horylation.