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 approximately 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 WO(3.3), 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(5) and tm(6), and a large intracellular carboxyl terminus containing protein kinase C phosphorylation sites. Northern blot analysis uncovers an approximately 3.7-kilobase pair transcript present in muscle, placenta, brain, and kidney. With regard to function, WO(3. 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, WO(3.3) specifically inhibits human NKCC1-mediated (86)Rb(+) flux. In addition, coimmunoprecipitation studies using lysates from WO(3. 3)-transfected HEK-293 cells suggest a direct interaction of WO(3.3) 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.