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Increased cytosolic Ca(2+) concentrations activate Gardos K(+) channels in human erythrocytes with membrane hyperpolarization, efflux of K(+), Cl⁻, and osmotically obliged H₂O resulting in cell shrinkage, a phenomenon referred to as Gardos effect. We tested whether the Gardos effect delays colloid osmotic hemolysis of injured erythrocytes from mice lacking the Ca(2+)-activated K(+) channel K(Ca)3.1. To this end, we applied patch clamp and flow cytometry and determined in vitro as well as in vivo hemolysis. As a result, erythrocytes from K(Ca)3.1-deficient (K(Ca)3.1(-/-)) mice lacked Gardos channel activity and the Gardos effect. Blood parameters, reticulocyte count, or osmotic erythrocyte resistance, however, did not differ between K(Ca)3.1(-/-) mice and their wild-type littermates, suggesting low or absent Gardos channel activity in unstressed erythrocytes. Oxidative stress-induced Ca(2+) entry and phospholipid scrambling were significantly less pronounced in K(Ca)3.1(-/-) than in wild-type erythrocytes. Moreover, in vitro treatment with α-toxin from Staphylococcus aureus, which forms pores in the cellular membrane, resulted in significantly stronger hemolysis of K(Ca)3.1(-/-) than of wild-type erythrocytes. Intravenous injection of α-toxin induced more profound hemolysis in K(Ca)3.1(-/-) than in wild-type mice. Similarly, intra-peritoneal application of the redox-active substance phenylhydrazine, an agent for the induction of hemolytic anemia, was followed by a significantly stronger decrease of hematocrit in K(Ca)3.1(-/-) than in wild-type mice. Finally, malaria infection triggered the activation of K(Ca)3.1 and transient shrinkage of the infected erythrocytes. In conclusion, K(Ca)3.1 channel activity and Gardos effect counteract hemolysis of injured erythrocytes, thus decreasing hemoglobin release into circulating blood.

Annexin A7 is a ubiquitously expressed Ca(2+)- and phospholipid-binding protein. Erythrocytes from mice lacking annexin A7 (anxA7(-/-)) are deformed and relatively resistant to osmotic swelling. In normal erythrocytes, hyperosmotic shock, Cl(-) removal, and energy depletion (glucose removal) trigger PGE(2) formation, which stimulates Ca(2+)-permeable cation channels, increases cytosolic Ca(2+) activity ([Ca(2+)](i)), and thus triggers suicidal death of erythrocytes or eryptosis, characterized by scrambling of the cell membrane with phosphatidylserine exposure at the cell surface. The present experiments explored the influence of annexin A7 deficiency on eryptosis. In erythrocytes from annexin A7-deficient mice (anxA7(-/-)) and wild-type mice (anxA7(+/+)), PGE(2) formation was determined utilizing an immunoassay, ion channel activity by whole-cell patch clamp recording, [Ca(2+)](i) by fluo3 fluorescence, and phosphatidylserine exposure by binding of annexin A5 in fluorescence activated cell sorter (FACS) analysis. Erythrocyte number and hematocrit were significantly smaller in blood from anx7(-/-) than in anx7(+/+) mice. Cl(-)-removal (replacement with gluconate) stimulated PGE(2)-formation, activated cation currents, increased [Ca(2+)](i), and triggered phosphatidylserine exposure, effects significantly more pronounced in anx7(-/-) than in anx7(+/+) erythrocytes. Hyperosmotic shock (addition of 400 mM sucrose) and glucose depletion (removal of glucose) similarly increased cytosolic Ca(2+) activity and triggered phosphatidylserine exposure, effects again significantly more pronounced in anx7(-/-) than in anx7(+/+) erythrocytes. The effects of Cl(-) removal on PGE(2) formation and the cation current, as well as the effect of hypertonic cell shrinkage on [Ca(2+)](i) and cell membrane scrambling, were blunted following inhibition of cyclooxygenase by aspirin or diclofenac. In conclusion, lack of annexin A7 sensitizes the erythrocytes for "proapoptotic" Ca(2+) overload, an effect shortening the life span of the affected erythrocytes and, thus, leading to anemia.

In neurons alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are heteromeric cation channels composed of different sub-units, including GluA1-GluA4. When expressed without GluA2, AMPA receptors function as Ca(2+)-permeable cation channels. In erythrocytes, activation of Ca(2+)-permeable cation channels triggers suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with subsequent exposure of phosphatidylserine at the cell surface. Activators of the channels and thus eryptosis include removal of extracellular Cl(-) (replaced by gluconate) and energy depletion (removal of glucose). The present study explored whether GluA1 is expressed in human erythrocytes and whether pharmacological AMPA receptor inhibition modifies Ca(2+) entry and suicidal death of human erythrocytes. GluA1 protein abundance was determined by confocal microscopy, phosphatidylserine exposure was estimated from annexin V binding, cell volume from forward scatter in FACS analysis, cytosolic Ca(2+) concentration from Fluo3 fluorescence and channel activity by whole-cell patch-clamp recordings. As a result, GluA1 is indeed expressed in the erythrocyte cell membrane. The AMPA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide) inhibited the cation channels following Cl(-) removal and the eryptosis following Cl(-) removal or energy depletion. The present study reveals a novel action of AMPA receptor antagonists and raises the possibility that GluA1 or a pharmacologically related protein participates in the regulation of Ca(2+) entry into and suicidal death of human erythrocytes.

Infection with the malaria parasite Plasmodium falciparum induces osmolyte and anion channels in the host erythrocyte membrane involving ATP release and autocrine purinergic signaling. P. falciparum-parasitized but not unstimulated uninfected erythrocytes released ATP in a 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; 7 microM)-sensitive and serum album (SA; 0.5% w/v)-stimulated manner. Since Plasmodium infection of human erythrocytes induces SA-dependent outwardly (OR) and SA-independent inwardly rectifying (IR) anion conductances, we tested whether the infection-induced OR channels directly generate an ATP release pathway. P. falciparum-parasitized erythrocytes were recorded in whole-cell mode with either Cl(-) or ATP as the only anion in the bath or pipette. In parasitized cells with predominant OR activity, replacement of bath NaCl by Na-ATP (NMDG-Cl pipette solution) shifted the current reversal potential (V (rev)) from -2 +/- 1 to +51 +/- 3 mV (n = 15). In cells with predominant IR activity, in contrast, the same maneuver induced a shift of V (rev) to significantly larger (p < or = 0.05, two-tailed t test) values (from -3 +/- 1 to +66 +/- 8 mV; n = 5) and an almost complete inhibition of outward current. The anion channel blocker NPPB reversibly decreased the ATP-generated OR currents from 1.1 +/- 0.1 nS to 0.2 +/- 0.05 nS and further shifted V (rev) to +87 +/- 7 mV (n = 12). The NPPB-sensitive fraction of the OR reversed at +48 +/- 4 mV suggesting a relative permeability of P (ATP)/P (Cl) approximately 0.01. Together, these data raise the possibility that the OR might be the electrophysiological correlate of an erythrocyte ATP release pathway.

Peptidoglycans (PGNs) from bacterial cell walls belong to 'pathogen-associated molecular patterns' (PAMP), which modify the course of an infection with bacterial pathogens. Bacterial infections may lead to anaemia, which at least partially could result from accelerated erythrocyte death. The present study explored the effect of PGNs on eryptosis, a stress-induced suicidal death of erythrocytes, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Eryptotic cells are phagocytosed and thus rapidly cleared from circulating blood. Eryptosis is triggered by an increase in the cytosolic Ca(2+) concentration and by formation of ceramide. Erythrocyte Ca(2+) activity was estimated from Fluo3 fluorescence, ceramide formation by fluorescent antibodies, phosphatidylserine exposure from annexin V-binding, and erythrocyte volume from forward scatter in fluorescence activated cell sorting (FACS) analysis. Exposure of erythrocytes to PGNs increased cytosolic Ca(2+) concentration, increased ceramide formation, enhanced the percentage of annexin V-binding erythrocytes, decreased erythrocyte forward scatter, and lowered the intracellular ATP concentration. The effect of peptidoglycans was significantly blunted in the absence of extracellular Ca(2+). The clearance of erythrocytes exposed to PGNs was significantly enhanced in vivo. In conclusion, peptidoglycans induce eryptosis at least partially through an increase in the cytosolic Ca(2+) concentration, an effect presumably contributing to the development of anaemia during bacterial infections.

The course of malaria does not only depend on the virulence of the parasite Plasmodium but also on properties of host erythrocytes. Here, we show that infection of erythrocytes from human sickle cell trait (HbA/S) carriers with ring stages of P. falciparum led to significantly enhanced PGE(2) formation, Ca(2+) permeability, annexin-A7 degradation, phosphatidylserine (PS) exposure at the cell surface, and clearance by macrophages. P. berghei-infected erythrocytes from annexin-A7-deficient (annexin-A7(-/-)) mice were more rapidly cleared than infected wildtype cells. Accordingly, P. berghei-infected annexin-A7(-/-) mice developed less parasitemia than wildtype mice. The cyclooxygenase inhibitor aspirin decreased erythrocyte PS exposure in infected annexin-A7(-/-) mice and abolished the differences of parasitemia and survival between the genotypes. Conversely, the PGE(2)-agonist sulprostone decreased parasitemia and increased survival of wild type mice. In conclusion, PS exposure on erythrocytes results in accelerated clearance of Plasmodium ring stage-infected HbA/S or annexin-A7(-/-) erythrocytes and thus confers partial protection against malaria in vivo., (2009 S. Karger AG, Basel.)

Zidovudine, a drug widely used in the treatment of AIDS, has been shown to influence cytosolic calcium activity in HIV-infected lymphocytes. Thus, zidovudine may modify the activity of Ca(2+)-permeable ion channels. In erythrocytes, activation of Ca(2+)-permeable cation channels stimulates eryptosis, the suicidal erythrocyte death. Eryptosis is characterized by cell shrinkage (apparent from a decrease of forward scatter) and phosphatidylserine (PS) exposure (apparent from annexin V-binding) at the erythrocyte surface. Triggers of eryptosis include isotonic cell shrinkage (Cl(-) replacement by gluconate), energy depletion (removal of glucose) or exposure to a variety of drugs including azathioprine. The present study explored, whether zidovudine influences the activity of erythrocytic Ca(2+)-permeable cation channels and eryptosis. Whole-cell patch-clamp recordings indeed revealed that zidovudine blocked the Ca(2+)-permeable cation channels activated by Cl(-) removal. In the presence of Cl(-) and glucose, the percentage of annexin V-binding cells was low and not significantly modified by the presence of zidovudine. Both, Cl(-) removal and glucose depletion increased annexin V-binding and decreased forward scatter, effects significantly blunted by zidovudine (2 microg/ml). According to Fluo3 fluorescence, zidovudine (2 microg/ml) did not significantly modify cytosolic Ca(2+) concentration under control conditions, but significantly blunted the increase in cytosolic Ca(2+) activity following glucose depletion. Furthermore, zidovudine significantly inhibited azathioprine-induced eryptosis. The present observations disclose a completely novel effect of zidovudine, i.e. its inhibitory influence on Ca(2+) entry and subsequent suicidal erythrocyte death during isotonic cell shrinkage or energy depletion.

Human erythrocytes express cation channels which contribute to the background leak of Ca(2+), Na(+) and K(+). Excessive activation of these channels upon energy depletion, osmotic shock, Cl(-) depletion, or oxidative stress triggers suicidal death of erythrocytes (eryptosis), characterized by cell-shrinkage and exposure of phosphatidylserine at the cell surface. Eryptotic cells are supposed to be cleared from circulating blood. The present study aimed to identify the cation channels. RT-PCR revealed mRNA encoding the non-selective cation channel TRPC6 in erythroid progenitor cells. Western blotting indicated expression of TRPC6 protein in erythrocytes from man and wildtype mice but not from TRPC6(-/-) mice. According to flow-cytometry, Ca(2+) entry into human ghosts prepared by hemolysis in EGTA-buffered solution containing the Ca(2+) indicator Fluo3/AM was inhibited by the reducing agent dithiothreitol and the erythrocyte cation channel blockers ethylisopropylamiloride and amiloride. Loading of the ghosts with antibodies against TRPC6 or TRPC3/6/7 but neither with antibodies against TRPM2 or TRPC3 nor antibodies pre-adsorbed with the immunizing peptides inhibited ghost Ca(2+) entry. Moreover, free Ca(2+) concentration, cell-shrinkage, and phospholipid scrambling were significantly lower in Cl(-)-depleted TRPC6(-/-) erythrocytes than in wildtype mouse erythrocytes. In conclusion, human and mouse erythrocytes express TRPC6 cation channels which participate in cation leak and Ca(2+)-induced suicidal death.

Listeriolysin, the secreted cytolysin of the facultative intracellular bacterium Listeria monocytogenes, is its major virulence factor. Previously, non-lytic concentrations of listeriolysin were shown to induce Ca2+-permeable nonselective cation channels in human embryonic kidney cells. In erythrocytes, Ca2+ entry is followed by activation of K+ channels resulting in K+-exit as well as by membrane scrambling resulting in phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing erythrocytes are recognized by macrophages, engulfed, degraded and thus cleared from circulating blood. Phosphatidylserine exposure is a key event of eryptosis, the suicidal death of erythrocytes. The present study utilized patch-clamp technique, Fluo3-fluorescence, and annexin V-binding in FACS analysis to determine the effect of listeriolysin on cell membrane conductance, cytosolic free Ca2+ concentration, and phosphatidylserine exposure, respectively. Within 30 minutes, exposure of human peripheral blood erythrocytes to low concentrations of listeriolysin (which were non-hemolytic for the majority of cells) induced a Ca2+-permeable cation conductance in the erythrocyte cell membrane, increased cytosolic Ca2+ concentration, and triggered annexin V-binding. Increase of extracellular K+ concentration blunted, but did not prevent, listeriolysin-induced annexin V-binding. In conclusion, listeriolysin triggers suicidal death of erythrocytes, an effect at least partially due to depletion of intracellular K+. Listeriolysin induced suicidal erythrocyte death could well contribute to the pathophysiology of L. monocytogenes infection.

Anti-A IgG antibodies have previously been shown to stimulate Ca(2+) entry into red blood cells. Increased cytosolic free Ca(2+) concentration is known to trigger eryptosis, i.e. suicidal erythrocyte death, characterized by exposure of phosphatidylserine at the erythrocyte surface. As macrophages are equipped with phosphatidylserine receptors, they bind, engulf and degrade phosphatidylserine exposing cells. The present experiments have been performed to explore whether anti-A IgGs trigger phosphatidylserine exposure of erythrocytes. Phosphatidylserine exposure was estimated from annexin-V binding as determined in FACS analysis. Exposure to anti-A IgGs (0.5 microg/ml) indeed significantly increased annexin-V binding in erythrocytes with blood group A, but not in erythrocytes with blood group 0. According to Fluo3 fluorescence, anti-A IgGs increased cytosolic Ca(2+) concentration. Whole cell patch clamp recordings revealed the activation of a Ca(2+)-permeable cation channel following treatment with anti-A-IgGs. Annexin-V binding following anti-A IgG exposure was blunted by Ca(2+) removal while anti-A IgG-stimulated cation channel activity was not dependent on extracellular Ca(2+). Osmotic shock (exposure of erythrocytes to 850 mOsm) increased annexin binding, an effect further enhanced by exposure to anti-A IgGs. In conclusion, anti-A IgGs activate erythrocyte cation channels leading to Ca(2+) entry and subsequent erythrocyte cell membrane scrambling. The effect most likely contributes to the elimination of erythrocytes following an immune reaction against the A antigen., (Copyright (c) 2007 S. Karger AG, Basel.)

Eryptosis or apoptosis-like death of erythrocytes is characterized by phosphatidylserine exposure and erythrocyte shrinkage, both typical features of nucleated apoptotic cells. Eryptosis is triggered by activation of nonselective Ca(2+)-permeable cation channels with subsequent entry of Ca(2+) and stimulation of Ca(2+)-sensitive scrambling of the cell membrane. The channels are activated and thus eryptosis is triggered by Cl(-) removal, osmotic shock, oxidative stress, or glucose deprivation. The present study has been performed to compare cation channel activity and susceptibility to eryptosis in neonatal and adult erythrocytes. Channel activity was determined by patch-clamp analysis, cytosolic Ca(2+) activity by fluo-3 fluorescence, phosphatidylserine exposure by FITC-labeled annexin V binding, and cell shrinkage by decrease in forward scatter in fluorescence-activated cell sorting analysis. Prostaglandin E(2) (PGE(2)) formation, cation channel activity, Ca(2+) entry, annexin V binding, and decreased forward scatter were triggered by removal of Cl(-) in both adult and neonatal erythrocytes. The effects were, however, significantly blunted in neonatal erythrocytes. Osmotic shock, PGE(2,) and platelet-activating factor similarly increased annexin V binding and decreased forward scatter, effects again significantly reduced in neonatal erythrocytes. On the other hand, spontaneous and oxidative (addition of tert-butylperoxide) stress-induced eryptosis was significantly larger in neonatal erythrocytes. In conclusion, cation channel activity, Ca(2+) leakage, and thus channel-dependent triggering of eryptosis are blunted, whereas spontaneous and oxidative stress-induced eryptosis is more pronounced in neonatal erythrocytes.

The prostaglandin PGE(2), a metabolite of the cyclooxygenase pathway, activates Ca(2+)-permeable cation channels in erythrocyte cell membranes leading to entry of Ca(2+) with subsequent eryptosis, i.e. cell shrinkage, breakdown of phosphatidylserine (PS) asymmetry and membrane blebbing, all features typical for apoptosis in nucleated cells. PS exposing cells are recognized by macrophages, engulfed, degraded and thus cleared from circulating blood. The present study explored whether the specific lipoxygenase inhibitor Bay-Y5884 influences eryptosis. As determined by competitive ELISA, Bay-Y5884 (20 microM) enhanced the release of PGE(2) from human erythrocytes. According to whole-cell patch-clamp, Bay-Y5884 (20 microM) activated nonselective cation channels. The effect of Bay-Y5884 on cation channels was abolished by the cyclooxygenase inhibitor diclophenac (10 microM). Bay-Y5884 (30-40 microM) significantly increased erythrocyte free Ca(2+) concentration and PS exposure as analyzed in flow cytometry by Fluo3 fluorescence and annexin-V binding, respectively. PS exposure triggered by 20 microM (but not by 40 microM) Bay-Y5884 was blunted by cyclooxygenase inhibitors acetylsalicylic acid (50 microM) and diclophenac (10 microM). In conclusion, the lipoxygenase inhibitor Bay-Y5884 enhances erythrocyte PGE(2) formation with subsequent activation of non-selective cation channels, Ca(2+) entry and phospholipid scrambling., (Copyright (c) 2006 S. Karger AG, Basel.)

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.

Infection of human erythrocytes with the malaria parasite Plasmodium falciparum induces new permeability pathways (NPPs) in the host cell membrane. Isotopic flux measurements demonstrated that the NPP are permeable to a wide variety of molecules, thus allowing uptake of nutrients and release of waste products. Recent patch-clamp recordings demonstrated the infection-induced up-regulation of an inwardly and an outwardly rectifying Cl(-) conductance. The present experiments have been performed to explore the sensitivity to cell volume and the organic osmolyte permeability of the two conductances. It is shown that the outward rectifier has a high relative lactate permeability (P(lactate)/P(Cl) = 0.4). Sucrose inhibited the outward-rectifier and abolished the infection-induced hemolysis in isosmotic sorbitol solution but had no or little effect on the inward-rectifier. Furosemide and NPPB blocked the outward-rectifying lactate current and the sorbitol hemolysis with IC(50)s in the range of 0.1 and 1 microM, respectively. In contrast, the IC(50)s of NPPB and furosemide for the inward-rectifying current were >10 microM. Osmotic cell-shrinkage inhibited the inwardly but not the outwardly rectifying conductance. In conclusion, the parasite-induced outwardly-rectifying anion conductance allows permeation of lactate and neutral carbohydrates, whereas the inward rectifier seems largely impermeable to organic solutes. All together, these data should help to resolve ongoing controversy regarding the number of unique channels that exist in P. falciparum-infected erythrocytes.

Annexin A7 is a ubiquitously expressed Ca2+- and phospholipid-binding protein. Erythrocytes from mice lacking annexin A7 ( anxA7 −/−) are deformed and relatively resistant to osmotic swelling. In normal erythrocytes, hyperosmotic shock, Cl− removal, and energy depletion (glucose removal) trigger PGE2 formation, which stimulates Ca2+-permeable cation channels, increases cytosolic Ca2+ activity ([Ca2+]i), and thus triggers suicidal death of erythrocytes or eryptosis, characterized by scrambling of the cell membrane with phosphatidylserine exposure at the cell surface. The present experiments explored the influence of annexin A7 deficiency on eryptosis. In erythrocytes from annexin A7-deficient mice ( anxA7 −/−) and wild-type mice ( anxA7 +/+), PGE2 formation was determined utilizing an immunoassay, ion channel activity by whole-cell patch clamp recording, [Ca2+]i by fluo3 fluorescence, and phosphatidylserine exposure by binding of annexin A5 in fluorescence activated cell sorter (FACS) analysis. Erythrocyte number and hematocrit were significantly smaller in blood from anx7 −/− than in anx7 +/+ mice. Cl−-removal (replacement with gluconate) stimulated PGE2-formation, activated cation currents, increased [Ca2+]i, and triggered phosphatidylserine exposure, effects significantly more pronounced in anx7 −/− than in anx7 +/+ erythrocytes. Hyperosmotic shock (addition of 400 mM sucrose) and glucose depletion (removal of glucose) similarly increased cytosolic Ca2+ activity and triggered phosphatidylserine exposure, effects again significantly more pronounced in anx7 −/− than in anx7 +/+ erythrocytes. The effects of Cl− removal on PGE2 formation and the cation current, as well as the effect of hypertonic cell shrinkage on [Ca2+]i and cell membrane scrambling, were blunted following inhibition of cyclooxygenase by aspirin or diclofenac. In conclusion, lack of annexin A7 sensitizes the erythrocytes for “proapoptotic” Ca2+ overload, an effect shortening the life span of the affected erythrocytes and, thus, leading to anemia. [ABSTRACT FROM AUTHOR]

Infection with the malaria parasite Plasmodium falciparum induces osmolyte and anion channels in the host erythrocyte membrane involving ATP release and autocrine purinergic signaling. P. falciparum-parasitized but not unstimulated uninfected erythrocytes released ATP in a 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; 7 µM)-sensitive and serum album (SA; 0.5% w/ v)-stimulated manner. Since Plasmodium infection of human erythrocytes induces SA-dependent outwardly (OR) and SA-independent inwardly rectifying (IR) anion conductances, we tested whether the infection-induced OR channels directly generate an ATP release pathway. P. falciparum-parasitized erythrocytes were recorded in whole-cell mode with either Cl− or ATP as the only anion in the bath or pipette. In parasitized cells with predominant OR activity, replacement of bath NaCl by Na–ATP (NMDG–Cl pipette solution) shifted the current reversal potential ( Vrev) from −2 ± 1 to +51 ± 3 mV ( n = 15). In cells with predominant IR activity, in contrast, the same maneuver induced a shift of Vrev to significantly larger ( p ≤ 0.05, two-tailed t test) values (from −3 ± 1 to +66 ± 8 mV; n = 5) and an almost complete inhibition of outward current. The anion channel blocker NPPB reversibly decreased the ATP-generated OR currents from 1.1 ± 0.1 nS to 0.2 ± 0.05 nS and further shifted Vrev to +87 ± 7 mV ( n = 12). The NPPB-sensitive fraction of the OR reversed at +48 ± 4 mV suggesting a relative permeability of PATP/ PCl ≈ 0.01. Together, these data raise the possibility that the OR might be the electrophysiological correlate of an erythrocyte ATP release pathway. [ABSTRACT FROM AUTHOR]