Cloning and functional characterization of a cation-Cl-cotransporter-interacting protein

To date, the cation-Cl- cotransporter (CCC) family comprises two branches of homologous membrane proteins. One branch includes the Na+-K+-Cl- cotransporters (NKCCs) and the Na+-Cl- cotransporter, and the other branch includes the K+-Cl- cotransporters. Here, we have isolated the first member of a third CCC family branch. This member shares similar to 25% identity in amino acid sequence with each of the other known mammalian CCCs. The corresponding cDNA, obtained from a human heart library and initially termed WO3.3 encodes a 914-residue polypeptide of 96.2 kDa (calculated mass). Sequence analyses predict a 12-transmembrane domain (tm) region, two N-linked glycosylation sites between tm, and tm,, and a large intracellular carboxyl terminus containing protein kinase C phosphorylation sites. Northern blot analysis uncovers an similar to 3.7-kilobase pair transcript present in muscle, placenta, brain, and kidney. With regard to function, WO3.3 expressed either in HEK-293 cells or Xenopus laevis oocytes does not increase Rb+-, Na+-, and Cl--coupled transport during 5-or 6-h fluxes, respectively. In the oocyte, however, WO3.3 specifically inhibits human NKCC1-mediated Rb-86(+) flux In addition, coimmunoprecipitation studies using lysates from WO3.3-transfected HEK-293 cells suggest a direct interaction of WO3.3 with endogenous NKCC. Thus, we have cloned and characterized the first putative heterologous CCC-interacting protein (CIP) known at present. CIP1 may be part of a novel family of proteins that modifies the activity or kinetics of CCCs through heterodimer formation.