Molecular cloning and expression of a chloride channel-associated protein pICln 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, pICln. Human reticulocyte pICln (HRpICln) cDNA encodes a protein (predicted molecular mass 26293Da) identical with human non-pigmented ciliary epithelial cell pICln. By using full-length HRpICln cDNA (approx. 1.2 kb) to probe human lymphocyte metaphase-chromosome spreads, the location of the human ICln gene was mapped to 11q13 by fluorescence in situ hybridization analysis. Polyclonal antibodies to recombinant HRpICln detected bands at approx. 43 kDa and approx. 37 kDa in both normal (AA) and sickle (SS) red blood cell (RBC) ghost membranes. In SS ghosts, and in ghosts from a patient with autoimmune haemolytic anaemia with 9.8% reticulocytes, the amount of HRpICln was increased compared with AA ghosts, suggesting that the expression or membrane assembly of HRpICln is cell age-dependent. Laser scanning confocal fluorescent microscopy immunolocalized HRpICln largely to the RBC membrane. The increased staining intensity of HRpICln in a reticulocyte-enriched AA RBC density-separated fraction is consistent with a dependence of HRpICln membrane content on cell age. HRpICln and beta-actin form stable complexes in vivo, demonstrated with the yeast two-hybrid system. Low-ionic-strength extraction of ghost membranes, which results in the extraction of the spectrin-actin cytoskeleton, also results in the extraction of HRpICln, consistent with the possibility for the association of these proteins in RBCs in vivo. The results presented here establish the presence of the Cl- channel-associated protein, pICln, in human RBCs, and raises the possibility that this protein has a role in RBC Cl- transport and volume regulation in young RBCs. Moreover the association of RBC pICln with actin offers a model in which to test interactions between RBC ion channels and the cytoskeleton.
Schwartz
R S
RS
Division of Hematology, Albert Einstein College of Medicine-Montefiore Medical Center, 111 East 210th Street, Bronx, NY 10467, USA.
Rybicki
A C
AC
Nagel
R L
RL
D000199:Actins / Q000097:blood*; D000328:Adult; D000595:Amino Acid Sequence; D000755:Anemia, Sickle Cell / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D018118:Chloride Channels / Q000096:biosynthesis / Q000097:blood* / Q000235:genetics; D002874:Chromosome Mapping; D002880:Chromosomes, Human, Pair 11; D003001:Cloning, Molecular; D017931:DNA Primers; D004285:Dogs; D004905:Erythrocyte Aging; D004910:Erythrocyte Membrane / Q000378:metabolism; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007473:Ion Channels; D007621:Karyotyping; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012156:Reticulocytes / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014981:Xenopus; D029867:Xenopus Proteins
D000199:Actins; C498731:CLNS1A protein, Xenopus; C410035:CLNS1A protein, human; D018118:Chloride Channels; C498729:Clns1a protein, rat; D017931:DNA Primers; D007473:Ion Channels; D011994:Recombinant Proteins; D029867:Xenopus Proteins