The KCl cotransporter mediates volume reduction in normal (AA) reticulocytes, and its abnormal regulation in sickle (SS) reticulocytes contributes to cellular dehydration that facilitates Hb S sickling. mRNA for three KCC genes - KCC1, KCC3, and KCC4 - as well as a splicing isoform, KCC1ex1b, is present in reticulocytes (

Exp. Hem. 2005; 33:624
). Western blotting has demonstrated KCC1 in human RBC membranes (
Su et al, AJPhysiol. 1999;277:C899
) and KCC3 in sheep (
Lauf et al, CompBiochemPhysiol 2001;130:499
); KCC4 protein was found in hRBC membranes by proteomic analysis (
Pasini et al, Blood 2006;108:791
). We confirm here the presence of KCC3 protein in hRBC via western blotting using an antibody to an exon 3 epitope distal to known N-terminal splicing sites. We sought to characterize and compare human KCC isoforms expressed in human cells (HEK 293) and assess their similarity to KCC activity in RBC. cDNAs for human KCC1, KCC1ex1b, KCC3a, and KCC4, with N-terminal c-myc epitope tags were expressed in HEK 293 cells. Stable cell lines were selected by growth in neomycin, and expression monitored by quantitative PCR analysis of the expressed construct and other endogenous isoforms and by western blotting (anti-myc) of plasma membranes. KCC activity was measured as N-ethylmaleimide(NEM)-stimulated, Cl-dependent Rb uptake in cells grown to 75–90 % confluency. Cells were incubated at 37°C in isotonic saline media with various concentrations of Rb (Na replacement), plus 0.1 mM ouabain and 0.01 mM bumetanide. At 2 and 4 min cells were washed with iced Rb-free media, then lysed and assayed for Rb by flame emission, normalized to sample protein. Flux rates were calculated from Rb uptake at 2 and 4 min. The flux rate in Cl-free sulfamate media was subtracted from that in Cl-media to yield the Cl-dependent flux rate. Wild-type HEK cells showed no increase in Cl-dependent Rb influx when exposed to 1 mM NEM, but Cl-dependent, NEM-stimulated Rb uptake was apparent in cells expressing KCC isoforms. All isoforms were stimulated by hypotonic conditions (75 mOsm), but relative to NEM-stimulated activity, KCC3 was most responsive. Kinetic characteristics of Cl-dependent, NEM-stimulated Rb influx in HEK cell expressing human KCC1, KCC3a, and KCC4 isoforms isoforms are given below, compared to RBC KCC fluxes. A splice variant, KCC1ex1b, coding for a protein with a truncated N-terminus, exhibited 48 ± 7% of the activity of the full-length KCC1 isoform, but did not alter transport activity of coexpressed KCC3a. Thus, KCC isoforms expressed in HEK cells share some, but not all, characteristics with RBC KCC fluxes. The isoform kinetics also differ from those previously published in xenopus oocyte expression systems, suggesting that the membrane milieu in which KCC proteins are expressed may influence their functional characteristics. Likewise, KCC activity in RBC may represent an average of several transporters operating in parallel. The truncated KCC1ex1b isoform, expressed at higher levels in normal than in sickle reticulocytes, has lower activity than KCC1. KCC3 and KCC4 exhibit more robust transport activity than KCC1 and may mediate a substantial part of KCC activity in RBC.

KCC Kinetic Characteristics

VMax (μmol/mgprot./min)Km (ext. Rb, mM)Anion Selectivity
hKCC1 28.9 ± 5.6 16.7 ± 5.4 Cl > Br > I > SCN 
hKCC3a 107 ± 45 11.0 ± 7.1 Cl = Br > I > SCN 
hKCC4 206 ± 16 15.6 ± 8.2 Cl > Br >> I = SCN 
RBC NA 12 - 20 Cl = Br >> I = SCN 
VMax (μmol/mgprot./min)Km (ext. Rb, mM)Anion Selectivity
hKCC1 28.9 ± 5.6 16.7 ± 5.4 Cl > Br > I > SCN 
hKCC3a 107 ± 45 11.0 ± 7.1 Cl = Br > I > SCN 
hKCC4 206 ± 16 15.6 ± 8.2 Cl > Br >> I = SCN 
RBC NA 12 - 20 Cl = Br >> I = SCN 
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Topics:
erythrocytes, potassium chloride, protein isoforms, western blotting, epitopes, anions, antibodies, bumetanide, dehydration, ethylmaleimide

Disclosures: Clinton H. Joiner, consulting for Novartis, McNeil.

Author notes

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Corresponding author

2006, The American Society of Hematology
2006
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