MOLECULAR-CLONING AND EXPRESSION OF A CHLORIDE CHANNEL-ASSOCIATED PROTEIN PI(CLN) IN HUMAN YOUNG RED-BLOOD-CELLS - ASSOCIATION WITH ACTIN

We report the cloning and sequencing from human reticulocytes of cDNA coding for the Cl- channel-associated protein, pI(Cln). Human reticulo cyte pI(Cln) (HRpI(Cln)) cDNA encodes a protein (predicted molecular m ass 26 293 Dal identical with human nonpigmented ciliary epithelial ce ll pI(Cln). By using full-length HRpI(Cln) cDNA (approx. 1.2 kb) to pr obe human lymphocyte metaphase-chromosome spreads, the location of the human I-Cln gene was mapped to 11q13 by fluorescence in situ hybridiz ation analysis. Polyclonal antibodies to recombinant HRpI(Cln) detecte d bands at approx. 43 kDa and approx. 37 kDa in both normal (AA) and s ickle (SS) red blood cell (RBC) ghost membranes. In SS ghosts, and in ghosts from a patient with autoimmune haemolytic anaemia with 9.8% ret iculocytes, the amount of HRpI(Cln) was increased compared with AA gho sts, suggesting that the expression or membrane assembly of HRpI(Cln) is cell age-dependent. Laser scanning confocal fluorescent microscopy immunolocalized HRpI(Cln) largely to the RBC membrane. The increased s taining intensity of HRpI(Cln) in a reticulocyte-enriche AA RBC densit y-separated fraction is consistent with a dependence of HRpI(Cln) memb rane content on cell age. HRpI(Cln) and beta-actin form stable complex es in vivo, demonstrated with the yeast two-hybrid system. Low-ionic-s trength extraction of ghost membranes, which results in the extraction of the spectrin-actin cytoskeleton, also results in the extraction of HRpI(Cln), consistent with the possibility for the association of the se proteins in RBCs in vivo. The results presented here establish the presence of the Cl- channel-associated protein, pI(Cln), in human RBCs , and raises the possibility that this protein has a role in RBC Cl- t ransport and volume regulation in young RBCs. Moreover the association of RBC pI(Cln) with actin offers a model in which to test interaction s between RBC ion channels and the cytoskeleton.