Antibiotic effects on SO4(2-) uptake in human erythrocytes

The erythrocyte is a cell highly exposed to oxygen pressure that, in turn, provokes oxidative stress involving loss of SH-groups, cell shrinkage by activation of K(+)-Cl(-) cotransport (KCC) and membrane destabilization which plays an important role in the premature haemolysis of red blood cells (RBCs). Oxidative stress provoked by chemicals frequently occurs in human erythrocytes. The aim of this study was to test whether the antibiotics alter the redox state and investigate their influences on band 3 protein that is involved in the facilitated electro neutral exchange of Cl(-) for HCO(3)(-) across the membrane of mammalian erythrocytes. Normal erythrocytes were treated with some antibiotics and thiol oxidizing agent N-ethylmaleimide (NEM) and tested for sulphate uptake, K(+) efflux and for glutathione (GSH) concentration as an index of oxidative stress. The rate constant of SO(4)(=) uptake measured in erythrocytes treated with antibiotics as well as NEM was decreased with respect to control cells as a result of band 3 SH-groups oxidation or the stress-induced K(+)-Cl(-) symport-mediated cell shrinkage. In fact, this hypothesis was verified by increased K(+) efflux and decreased GSH values measured in treated erythrocytes compared to controls.     

Incorporation of 3H-N-ethylmaleimide into sheep red cell membrane thiol groups following protection by diamide-induced oxidation

The thiol oxidant diazene dicarboxylic acid bis [N,N-dimethylamide] (diamide) is known to reversibly activate K-Cl cotransport in sheep red blood cells [1]. Although the detailed mechanism of activation is unknown, functional thiols at the membrane or at the cytoplasmic level are recognized as important. To search for membrane bound thiols involved in the regulation of K-Cl cotransport, sheep red cells were first exposed to diamide at concentrations activating K-Cl cotransport, and then to the alkylating agent N-ethylmaleimide (NEM) in order to block non-oxidized thiols. White ghosts, prepared by osmotic lysis from these cells, were again treated with NEM followed by reduction of the diamide-induced dithiols with dithio-threitol (DTT) concentrations known to reverse the diamide-induced K-Cl flux [1]. Maximum ³H-NEM incorporation into the DTT-reduced thiols occurred at 50 M DTT. Saturation labelling by ³H-NEM of about 2 × 10⁴ diamide-protected thiols/cell occurred at 25 M NEM. Diamide protected about 0.1% of all membrane thiols chemically determined earlier [2]. Membranes from high K (HK) and low K (LK) sheep red cells did not differ significantly in the number of diamide-protected thiols, and polyacrylamide gels revealed a similar protein distribution of 3H-NEM-labelled thiols. Since diamide is known to stimulate K-Cl flux in LK cells ten times more than in HK cells this finding is consistent with the hypothesis of a cytoplasmic control effecting different K-Cl flux activities in the membranes of the two cation genotypic red blood cells.     

Oxidative Activation of K-Cl Cotransport by Diamide in Erythrocytes from Humans with Red Cell Disorders,and from Several Other Mammalian Species

Red blood cells (RBCs) from different mammalian species were investigated for the presence of diamide-induced oxidative activation of K-Cl cotransport reported to be present in sheep but absent in human RBCs. K efflux was measured in RBCs from human with hemoglobin (Hb) A or S, glucose-phosphate dehydrogenase (G6PDH) and a cytoskeletal deficiency, and from rat, mouse and rabbit. RBCs were incubated with diamide (0–1.0 mm) in K-free Cl or NO₃ media of variable osmolalities (200–450 mOsM). Cl-dependent K efflux or K-Cl cotransport (estimated as the difference between K efflux rate constants in Cl and NO₃) was activated by diamide in a sigmoidal fashion. Relative maximum K-Cl cotransport followed the sequence: human HbA (1) < rabbit (1.8) < sheep (6.9) < human HbS (9.5) ∼ rat (9.7). Relative diamide concentrations for half maximal activation of K-Cl cotransport followed the sequence: sheep (1.9) > human Hb A (1) > rabbit (0.75) > human HbS and rat (0.67). Cell swelling in 200 mOsM doubled K-Cl cotransport in diamide, both in human HbA and S cells but reduced that in rat RBCs. In contrast, cell shrinkage at 450 mOsM obliterated K-Cl cotransport in human HbA and S but not in rat RBCs. Human RBCs with G6PDH and a cytoskeleton deficiency behaved like HbA RBCs. In mouse RBCs, diamide-activated K-Cl cotransport was 30% higher in isotonic than in hypotonic medium. In human HbA and S, and in low or high K sheep RBCs fractionated by Percoll density gradient, diamide increased the activity of K-Cl cotransport, an effect inversely correlated with cell density. Analysis of pooled data reveals that K-Cl cotransport accounted for about 80% of all K flux in Cl. There was a statistically significant correlation between K-Cl cotransport and K efflux in Cl (P < 0.00001) and in NO₃ (P < 0.00001). In conclusion, a diamide-activated K-Cl cotransport was present in human RBCs and in all other mammalian RBCs tested, with a large inter-, and for human and sheep, intraspecies variability for its maximum activity. Received: 5 June 1996/Revised: 4 October 1996     

K-Cl cotransport in red blood cells from patients with KCC3 isoform mutants.

Red blood cells (RBCs) possess the K-Cl cotransport (KCC) isoforms 1, 3, and 4. Mutations within a given isoform may affect overall KCC activity. In a double-blind study, we analyzed, with Rb as a K congener, K fluxes (total flux, ouabain-sensitive Na+/K+ pump, and bumetanide-sensitive Na-K-2Cl cotransport, Cl-dependent, and ouabain- and bumetanide-insensitive KCC with or without stimulation by N-ethylmaleimide (NEM) and staurosporine or Mg removal, and basal channel-mediated fluxes, osmotic fragility, and ions and water in the RBCs of 8 controls, and of 8 patients with hereditary motor and sensory neuropathy with agenesis of corpus callosum (HMSN-ACC) with defined KCC3 mutations (813FsX813 and Phe529FsX532) involving the truncations of 338 and 619 C-terminal amino acids, respectively. Water and ion content and, with one exception, mean osmotic fragility, as well as K fluxes without stimulating agents, were similar in controls and HMSN-ACC RBCs. However, the NEM-stimulated KCC was reduced 5-fold (p < 0.0005) in HMSN-ACC vs control RBCs, as a result of a lower Vmax (p < 0.05) rather than a lower Km (p = 0.109), accompanied by corresponding differences in Cl activation. Low intracellular Mg activated KCC in 6 out of 7 controls vs 1 out of 6 HMSN-ACC RBCs, suggesting that regulation is compromised. The lack of differences in staurosporine-activated KCC indicates different action mechanisms. Thus, in HMSN-ACC patients with KCC3 mutants, RBC KCC activity, although indistinguishable from that of the control group, responded differently to biochemical stressors, such as thiol alkylation or Mg removal, thereby indirectly indicating an important contribution of KCC3 to overall KCC function and regulation.     

Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells

We have studiedthe regulation of the K-Cl cotransporter KCC1 and its functionalinteraction with the Na-K-Cl cotransporter. K-Cl cotransporter activitywas substantially activated in HEK-293 cells overexpressing KCC1(KCC1-HEK) by hypotonic cell swelling, 50 mM external K, andpretreatment with N-ethylmaleimide(NEM). Bumetanide inhibited ⁸⁶Rbefflux in KCC1-HEK cells after cell swelling [inhibition constant (K_i) ~190µM] and pretreatment with NEM(K_i ~60 µM).Thus regulation of KCC1 is consistent with properties of the red cellK-Cl cotransporter. To investigate functional interactions between K-Cland Na-K-Cl cotransporters, we studied the relationship between Na-K-Clcotransporter activation and intracellular Cl concentration([Cl]_i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activitythan controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells wasactivated <2-fold by low-Cl hypotonic prestimulation, compared with10-fold activation in HEK-293 cells and >20-fold activation in cellsoverexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cellshad lower resting[Cl]_i than HEK-293cells; cell volume was not different among cell lines. We found a steeprelationship between[Cl]_i and Na-K-Clcotransport activity within the physiological range, supporting aprimary role for [Cl]_iin activation of Na-K-Cl cotransport and in apical-basolateral crosstalk in ion-transporting epithelia.     

KCl Cotransport Regulation and Protein Kinase G in Cultured Vascular Smooth Muscle Cells

K-Cl cotransport is activated by vasodilators in erythrocytes and vascular smooth muscle cells and its regulation involves putative kinase/phosphatase cascades. N-ethylmaleimide (NEM) activates the system presumably by inhibiting a protein kinase. Nitrovasodilators relax smooth muscle via cGMP-dependent activation of protein kinase G (PKG), a regulator of membrane channels and transporters. We investigated whether PKG regulates K-Cl cotransport activity or mRNA expression in normal, PKG-deficient-vector-only-transfected (PKG-) and PKG-catalytic-domain-transfected (PKG+) rat aortic smooth muscle cells. K-Cl cotransport was calculated as the Cl-dependent Rb influx, and mRNA was determined by semiquantitative RT-PCR. Baseline K-Cl cotransport was higher in PKG+ than in PKG- cells (p <0.01). At 0.5 mM, NEM stimulated K-Cl cotransport by 5-fold in PKG- but not in PKG+ cells. However, NEM was more potent although less effective to activate K-Cl cotransport in normal (passage 1-3) and PKG+ than in PKG- cells. In PKG- cells, [(dihydroindenyl) oxy] alkanoic acid (300 mM) but not furosemide (1 mM) inhibited K-Cl cotransport. Furthermore, no difference in K-Cl cotransport mRNA expression was observed between these cells. In conclusion, this study shows that manipulation of PKG expression in vascular smooth muscle cells affects K-Cl cotransport activity and its activation by NEM.     

Erythrocytes anion transport and oxidative change in β‐thalassaemias

β‐Thalassaemia is characterized by a decrease in globin β‐chain synthesis and an excess in free α‐globin chains. This induces alterations in membrane lipids and proteins resulting from a reduction in spectrin/band 3 ratio, partial oxidation of band 4.1 and clustering of band 3. The membrane injury provokes hyperhaemolysis and bone marrow hyperplasia. The pathophysiology of thalassaemia is associated with iron overload that generates oxygen free radicals and oxidative tissue injury with ocular vessel alterations. The aim of this research is to investigate the influence of oxidative stress on band 3 efficiency, which is an integral membrane protein of RBCs (red blood cells). Band 3 protein, of which there are more than 1 million copies per cell, is the most abundant membrane protein in human RBCs. It mediates the anion exchange and acid–base equilibrium through the RBC membrane. Some experiments were performed on thalassaemic cells and β‐thalassaemia‐like cells and tested for sulfate uptake. To test the antioxidant effect of Mg²⁺, other experiments were performed using normal and pathological cells in the presence of Mg²⁺. The oxidant status in thalassaemic cells was verified by increased K⁺ efflux, by lower GSH levels and by increased G6PDH (glucose‐6‐phosphate dehydrogenase) activity. The rate constant of SO₄ ^2? uptake decreases in thalassaemic cells as well as in β‐thalassaemia‐like cells when compared with normal cells. It increases when both cells are incubated with Mg²⁺. Our data show that oxidative stress plays a relevant role in band 3 function of thalassaemic cells and that antioxidant treatment with Mg²⁺ could reduce oxidative damage to the RBC membrane and improve the anion transport efficiency regulated by band 3 protein.     

Volume-sensitive K(+)/Cl(-) cotransport in rabbit erythrocytes. Analysis of the rate-limiting activation and inactivation events

The kinetics of activation and inactivation of K(+)/Cl(-) cotransport (KCC) have been measured in rabbit red blood cells for the purpose of determining the individual rate constants for the rate-limiting activation and inactivation events. Four different interventions (cell swelling, N-ethylmaleimide [NEM], low intracellular pH, and low intracellular Mg(2+)) all activate KCC with a single exponential time course; the kinetics are consistent with the idea that there is a single rate-limiting event in the activation of transport by all four interventions. In contrast to LK sheep red cells, the KCC flux in Mg(2+)-depleted rabbit red cells is not affected by cell volume. KCC activation kinetics were examined in cells pretreated with NEM at 0 degrees C, washed, and then incubated at higher temperatures. The forward rate constant for activation has a very high temperature dependence (E(a) approximately 32 kCal/mol), but is not affected measurably by cell volume. Inactivation kinetics were examined by swelling cells at 37 degrees C to activate KCC, and then resuspending at various osmolalities and temperatures to inactivate most of the transporters. The rate of transport inactivation increases steeply as cell volume decreases, even in a range of volumes where nearly all the transporters are inactive in the steady state. This finding indicates that the rate-limiting inactivation event is strongly affected by cell volume over the entire range of cell volumes studied, including normal cell volume. The rate-limiting inactivation event may be mediated by a protein kinase that is inhibited, either directly or indirectly, by cell swelling, low Mg(2+), acid pH, and NEM.     

Platelet-derived growth factor regulates K-Cl cotransport in vascular smooth muscle cells

Platelet-derived growth factor (PDGF), apotent serum mitogen for vascular smooth muscle cells (VSMCs), plays animportant role in membrane transport regulation and in atherosclerosis. K-Cl cotransport (K-Cl COT/KCC), the coupled-movement of K and Cl, isinvolved in ion homeostasis. VSMCs possess K-Cl COT activity and theKCC1 and KCC3 isoforms. Here, we report on the effect of PDGF on K-ClCOT activity and mRNA expression in primary cultures of rat VSMCs. K-ClCOT was determined as the Cl-dependent Rb influx and mRNA expression bysemiquantitative RT-PCR. Twenty four-hour serum deprivation inhibitedbasal K-Cl COT activity. Addition of PDGF increased total proteincontent and K-Cl COT activity in a time-dependent manner. PDGFactivated K-Cl COT in a dose-dependent manner, both acutely (10 min)and chronically (12 h). AG-1296, a selective inhibitor of the PDGFreceptor tyrosine kinase, abolished these effects. Actinomycin D andcycloheximide had no effect on the acute PDGF activation of K-Cl COT,suggesting posttranslational regulation by the drug. Furthermore, PDGFincreased KCC1 and decreased KCC3 mRNA expression in a time-dependentmanner. These results indicate that chronic activation of K-Cl COTactivity by PDGF may involve regulation of the two KCC mRNA isoforms,with KCC1 playing a dominant role in the mechanism of PDGF-mediated activation.

     

PDGF activates K-Cl cotransport through phosphoinositide 3-kinase and protein phosphatase-1 in primary cultures of vascular smooth muscle cells

K-Cl cotransport (K-Cl COT, KCC) is an electroneutrally coupled movement of K and Cl present in most cells. In this work, we studied the pathways of regulation of K-Cl COT by platelet-derived growth factor (PDGF) in primary cultures of vascular smooth muscle cells (VSMCs). Wortmannin and LY 294002 blocked the PDGF-induced K-Cl COT activation, indicating that the phosphoinositide 3-kinase (PI 3-K) pathway is involved. However, PD 98059 had no effect on K-Cl COT activation by PDGF, suggesting that the mitogen-activated protein kinase pathway is not involved under the experimental conditions tested. Involvement of phosphatases was also examined. Sodium orthovanadate, cyclosporin A and okadaic acid had no effect on PDGF-stimulated K-Cl COT. Calyculin A blocked the PDGF-stimulated K-Cl COT by 60%, suggesting that protein phosphatase-1 (PP-1) is a mediator in the PDGF signaling pathway/s. In conclusion, our results indicate that the PDGF-mediated pathways of K-Cl COT regulation involve the signaling molecules PI 3-K and PP-1.     

Effector-induced Syk-mediated phosphorylation in human erythrocytes

Band 3 (AE1), the most prominent polypeptide of the human erythrocyte membrane, becomes heavily tyrosine phosphorylated following treatment of intact cells with protein tyrosine phosphatase inhibitors such as diamide, pervanadate, vanadate, or N-ethylmaleimide (NEM). The mechanism underlying this tyrosine phosphorylation is thought to involve the sequential action of two protein tyrosine kinases, Syk (p72syk) and Lyn (p53/56lyn). While Lyn catalysed phosphorylation appears to be strictly dependent on prior phosphorylation of Tyr8 and 21 of band 3 by Syk, little is known about the mechanism of induction of Syk phosphorylation. Data presented here show that both the fraction of Syk that associates with the membrane and the extent of phosphorylation of band 3 differ in response to the above inhibitors. While diamide and NEM stimulate syk translocation to the membrane during their induction of band 3 tyrosine phosphorylation, pervanadate and vanadate induce no change in kinase distribution. Moreover, diamide and NEM-induced Syk recruitment to the membrane are phosphotyrosine independent and involve their preferential association with Triton X-100-insoluble membrane skeletons. Together these data reveal a complex process controlling the association and catalytic activity of protein tyrosine kinases syk and lyn with the human erythrocyte membrane.     

Band 3 tyr-phosphorylation in normal and glucose-6-phospate dehydrogenase-deficient human erythrocytes

Haemolysis is usually episodic in glucose-6-phosphate dehydrogenase (G6PD) deficiency, often triggered by a period of oxidative stress. In the present work, we investigate a possible biochemical mechanism underlying the enhanced susceptibility of G6PD deficient red blood cells (RBC) to oxidative stress. We analysed eight male subjects with Mediterranean glucose-6P-dehydrogenase deficiency (G6PDd), class II, for their ability in phosphorylating erythrocyte membrane band 3 following oxidative and osmotic stress. Our findings show that this sensitivity is connected to an early membrane band 3 Tyr-phosphorylation in the presence of diamide. However, since both Syk, and Lyn kinases, and SHP-2 phosphatase, mostly implicated in the band 3 P-Tyr level regulation, are alike in content and activity in normal and patient erythrocytes, an alteration in the membrane organization is likely the cause of the anomalous response to the oxidant. We report, in fact, that hypertonic-induced morphological change in G6PDd erythrocyte induces a higher membrane band 3 Tyr-phosphorylation, suggesting a pre-existing membrane alteration, likely due to the chronic lowering of the redox systems in patients. We also report that 1-chloro-2,4-dinitrobenzene-pre-treatment of normal red cells can alter the normal protein-protein and protein-membrane interaction under hypertonic rather than oxidative stress, thus partially resembling the response in patients, and that RBC may utilize a wider range of redox defence, under oxidative conditions, including, but not exclusively, NADPH and glutathione. On the whole, these results would encourage a different approach to the evaluation of the effects of pharmacological administration to patients, giving more attention to the possible drug-induced membrane alteration evidenced by the abnormal band 3 Tyr-phosphorylation.     

Role of reactive oxygen species generated by NADPH oxidase in the mechanism of activation of K+-Cl--cotransport by N-ethylmaleimide in HepG2 human hepatoma cells

K⁺-Cl^--cotransport (KCC) is ubiquitously present in all cells, and plays an essential role in ion and volume regulation. In this study we investigated the role of reactive oxygen species (ROS) in regulation of KCC in HepG2 human hepatoblastoma cells. N-ethylmaleimide (NEM), a KCC activator, induced Cl^--dependent K⁺ efflux, which was markedly prevented by KCC inhibitors (calyculin-A, genistein and BaCl₂), indicating that KCC is activated by NEM in the HepG2 cells. Treatment with NEM also induced a sustained increase in the level of intracellular ROS assessed by 2',7'-dichlorofluorescein flourescence. Antioxidants, N-acetyl cysteine or N,N'-diphenyl-p-phenylenediamine significantly inhibited both ROS generation and KCC activation induced by NEM. The NEM-induced ROS production was significantly suppressed by inhibitors of NADPH oxidase (diphenylene iodonium, apocynin and neopterine). These inhibitors also significantly inhibited the NEM-induced KCC activation. Taken together, these results suggest that ROS generated by NADPH oxidase may mediate the NEM-induced activation of KCC in human hepatoma cells.     

Molecular identification of K-CL cotransporter in dog erythroid progenitor cells

KCC1 cDNA was cloned in dog erythroblasts that had differentiated from peripheral mononuclear cells. The size of the cDNA was 3,258 bp, the same as in pigs, but 3 bp longer than in humans and rodents. The dog KCC1 cDNA encodes for 1,086 amino acid residues with a calculated molecular mass of 120 kDa. The 560 bp cDNA fragment from position 679 to 1,238 in the full length cDNA from the dog erythroblasts was 100% identical to that in the kidney. Hydropathy analysis showed that the structure of dog KCC1 was similar to in other species; 12 trans membrane domains, four glycosylation sites in loop 5, and 17 consensus phosphorylation sites in the cytosol. However, there were variations in dog KCC1 compared to in other species; there was one CK2 phosphorylation site that was found only in dog KCC1. There were also substitutions of amino acids that affect pH sensitivity (His) and change acidic/basic residues or charged residues. In HEK 293 cells transfected with dog KCC1 cDNA (HEK-dKCC1), the Rb influx, which was ouabain-resistant, Cl-dependent, N-ethyl maleimide (NEM)- stimulative and Na-independent, was measured as for K-Cl cotransport, and the influx was found to be increased approximately 3 fold in HEK-dKCC1 compared to in the control. This ouabain-resistant Cl-dependent Rb influx was also volume-sensitive in hyposmotic medium, and the volume-sensitive component was inhibited by furosemide. Thus, the KCC1 cDNA cloned in dog erythroblasts encodes a volume-sensitive K-Cl cotransporter.     

Effect of intracellular magnesium and oxygen tension on K+-Cl- cotransport in normal and sickle human red cells.

In red cells from normal individuals (HbA cells), the K+-Cl- cotransporter (KCC) is inactivated by low O2 tension whilst in those from sickle cell patients (HbS cells), it remains fully active. Changes in free intracellular [Mg2+] have been proposed as a mechanism. In HbA cells, KCC activity was stimulated by Mg2+ depletion and inhibited by Mg2+ loading but the effect of O2 was independent of Mg2+. At all [Mg2+]is, the transporter was stimulated in oxygenated cells, minimally active in deoxygenated ones. By contrast, the stimulatory effects of O2 was abolished by inhibitors of protein (de)phosphorylation. HbS cells had elevated KCC activity, which was of similar magnitude in oxygenated and deoxygenated cells, regardless of Mg2+ clamping. In deoxygenated cells, the antisickling agent dimethyl adipimidate inhibited sickling, Psickle and KCC. Results indicate a role for protein phosphorylation in O2 dependence of KCC, with different activities of the relevant enzymes in HbA and HbS cells, probably dependent on Hb.     

Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation

Although K-Cl cotransporter (KCC1) mRNA is expressed in manytissues, K-Cl cotransport activity has been measured in few cell types,and detection of endogenous KCC1 polypeptide has not yet been reported.We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flankinggenomic regions and mapped the mKCC1 gene to chromosome 8. Threeanti-peptide antibodies raised against recombinant mKCC1 function asimmunoblot and immunoprecipitation reagents. The tissue distributionsof mKCC1 mRNA and protein are widespread, and mKCC1 RNA isconstitutively expressed during erythroid differentiation of ES cells.KCC1 polypeptide or related antigen is present in erythrocytes ofmultiple species in which K-Cl cotransport activity has beendocumented. Erythroid KCC1 polypeptide abundance is elevated inproportion to reticulocyte counts in density-fractionated cells, inbleeding-induced reticulocytosis, in mouse models of sickle celldisease and thalassemia, and in the corresponding human disorders.mKCC1-mediated uptake of ⁸⁶Rb intoXenopus oocytes requires extracellularCl, is blocked by thediureticR(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling,N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.

     

K-Cl co-transport: immunocytochemical and functional evidence for more than one KCC isoform in high K and low K sheep erythrocytes

K-Cl co-transport (COT) is significantly higher in low K (LK), L-antigen (L) positive, than in high K (HK), M-antigen (M) positive, sheep red blood cells (SRBCs) and is inhibited by sheep allo-anti-L1 antibody. To answer the question of whether this difference in K-Cl co-transport activity resides at the level of the transporter or its regulation, a combined immunocytochemical and functional approach was taken. At least four isoforms of K-Cl COT encoded by different KCC genes are known, with 12 transmembrane domains and cytoplasmic C- and N-terminal domains (Ctd and Ntd, respectively). Polyclonal anti-rat (rt)KCC1 antibodies against a fusion peptide with 77 amino acids from the Ctd of rtKCC1 and anti-human (h)KCC3 against an 18-aa peptide from the Ntd of hKCC3, were prepared in rabbits (rb). Two distinctly separate protein bands of 180 and 145 kDa molecular mass were detected in hemoglobin-free ghosts from RBCs of two LK (one homozygous LL and one heterozygous LM) and one HK (homozygous MM) sheep by Western blots with rb anti-rtKCC1 and rb anti-hKCC3. Confocal microscopy showed specific immunostaining of KCC1 with rb anti-rtKCC1, and of KCC3 with rb anti-hKCC3, in white ghosts from both LK and HK SRBCs. To test the functional heterogeneity of K-Cl COT, the effect of the anti-L1 antibody was assessed on K-Cl COT activated by the kinase inhibitor staurosporine. Incubation of LK SRBCs with anti-L1 serum inhibited by 30% staurosporine-stimulated K-Cl COT suggesting that approximately two-thirds of the transport activity is independent of the L1 antigen. That staurosporine altered the L1 antigen/antibody reaction is unlikely since the action of another antibody, anti-Lp, stimulating the Na/K pump flux, was not modified. The present results, in conjunction with earlier work, lead to the hypothesis that the partial anti-L1 inhibition of K-Cl COT may be related to the molecular KCC dimorphism, seen in these cells with anti-KCC1 and anti-KCC3 antibodies.