The human red cell voltage-dependent cation channel. Part III: Distribution homogeneity and pH dependence.

The homogeneity of the distribution of the non-selective voltage-dependent cation channel (the NSVDC channel) in the human erythrocyte, and the pH dependence was investigated. Activation of this channel caused a uniform cellular dehydration, which was characterized by the changes in the erythrocyte osmotic resistance profiles: after 1/2 h of activation, the osmolarity at 50% hemolysis changed from 73 mM (control) to 34 mM NaCl, corresponding to 0.48% and 0.21% NaCl respectively. Unchanging standard deviations show participation of the entire erythrocyte population, which implies an even distribution of the NSVDC channel among the cells. Inactivation of the NSVDC channel with N-ethyl-maleimide (NEM) or blocking of the Cl(-) conductance with NS1652 retarded the migration of the resistance profiles towards lower osmolarities. The NSVDC channel activation was blocked by a decrease of the intracellular -- but not the extracellular -- pH. The apparent pK(A) value for the effect was estimated to be 6.5, and the specific histidine reagent 2.4'-dibromoacetophenone (DBAB) inactivated the NSVDC channel.

The human red cell voltage-regulated cation channel. The interplay with the chloride conductance, the Ca(2+)-activated K(+) channel and the Ca(2+) pump.

The activation/deactivation kinetics of the human erythrocyte voltage-dependent cation channel was characterized at the single-channel level using inside-out patches. It was found that the time dependence for voltage activation after steps to positive membrane potentials was slow ( t(1/2) about 30 s), whereas the deactivation was fast ( t(1/2) about 15 ms). Both activation and deactivation of this channel were also demonstrated in intact red cells in suspension. At very positive membrane potentials generated by suspension in extracellular low Cl(-) concentrations, the cation conductance switched on with a time constant of about 2 min. Deactivation of the cation channel was clearly demonstrated during transient activation of the Gárdos channel elicited by Ca(2+) influx via the cation channel and ensuing efflux via the Ca(2+) pump. Thus, the voltage-dependent cation channel, the Gárdos channel and the Ca(2+) pump constitute a coupled feedback-regulated system that may become operative under physiological conditions.

Calcium-dependent association of annexins with lipid bilayers modifies gramicidin A channel parameters.

In order to examine whether calcium-dependent binding of annexin to acidic phospholipids could change the lipid bilayer environment sufficiently to perturb channel-mediated transmembrane ion-transport, gramicidin A channel activity in planar lipid bilayers was investigated in the presence of calcium and annexins II, III or V. The experiments were performed with membranes consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in 300 mM KCl solution buffered to pH 7.4 and with either 0.1 or 1 mM calcium added to the solution. Annexin (1 microM) was subsequently applied to the cis side of the membrane. All three annexins (II, III and V) when tested at 1 mM calcium decreased the gramicidin single-channel conductance. Annexins II and III increased the mean lifetime of the channels whereas annexin V seemed to have no influence on the mean lifetime. Since the lifetime of gramicidin A channels is a function of the rate constant for dissociation of the gramicidin dimer, which is dependent on the physical properties of the lipid phase, binding of annexins II and III seems to stabilize the gramicidin channel owing to a change of the bilayer structure.

Treatment with NS3623, a novel Cl-conductance blocker, ameliorates erythrocyte dehydration in transgenic SAD mice: a possible new therapeutic approach for sickle cell disease.

The dehydration of sickle red blood cells (RBCs) through the Ca-activated K channel depends on the parallel movement of Cl ions. To study whether Cl-conductance block might prevent dehydration of sickle RBCs, a novel Cl-conductance inhibitor (NS3623) was characterized in vitro using RBCs from healthy donors and sickle cell patients and in vivo using normal mice and a transgenic mouse model of sickle cell disease (SAD mice). In vitro, NS3623 reversibly blocked human RBC Cl-conductance (g(Cl)) with an IC(50) value of 210 nmol/L and a maximal block of 95%. In vivo, NS3623 inhibited RBC g(Cl) after oral administration to normal mice (ED(50) = 25 mg/kg). Although g(Cl), at a single dose of 100 mg/kg, was still 70% inhibited 5 hours after dosing, the inhibition disappeared after 24 hours. Repeated administration of 100 mg/kg twice a day for 10 days caused no adverse effects; therefore, this regimen was chosen as the highest dosing for the SAD mice. SAD mice were treated for 3 weeks with 2 daily administrations of 10, 35, and 100 mg/kg NS3623, respectively. The hematocrit increased, and the mean corpuscular hemoglobin concentration decreased in all groups with a concomitant increase in the intracellular cation content. A loss of the densest red cell population was observed in conjunction with a shift from a high proportion of sickled to well-hydrated discoid erythrocytes, with some echinocytes present at the highest dosage. These data indicate feasibility for the potential use of Cl-conductance blockers to treat human sickle cell disease.

Novel therapies for prevention of erythrocyte dehydration in sickle cell anemia.

Sickle cell anemia is a genetic disorder characterized by mutant hemoglobin (Hb) polymerization and resultant cell deformation (sickling) under conditions of reduced oxygen tension. The disease is caused by mutation of wild-type Glu to Val in position 6 of the beta-chain of hemoglobin, yielding hemoglobin S (HbS). The sickling process is markedly accelerated when the intracellular concentration of HbS is increased. A variable fraction of dehydrated erythrocytes is seen in the majority of patients, and these cells are believed to play an important role in the pathophysiology of the vasoocclusive events of sickle cell disease. Therapy of sickle cell disease is extremely limited in range and efficacy. Many patients still receive treatment only for symptomatic relief of sickle crises, painful episodes due to vasoocclusion by sickled cells. The last 15 years, however, have seen the identification of the principal transport pathways that mediate sickle erythrocyte dehydration, and the last 6 years have witnessed promising clinical tests of specific inhibitors of these pathways, with the intent of reducing cell sickling via inhibition of red cell dehydration. This review discusses the pathophysiology of sickle cell dehydration and explores current and future treatment options for in vivo prevention of sickle cell dehydration.

The non-selective voltage-activated cation channel in the human red blood cell membrane: reconciliation between two conflicting reports and further characterisation.

Using the patch-clamp technique, the non-selective, voltage-activated cation channel in the human red blood cell (RBC) membrane was further characterised. Activity of the cation channel could be demonstrated at a range of salt concentrations with the current-voltage characteristics for monovalent cations going from linear to superlinear functions, depending on the cation concentration in the range of 100-500 mM. The non-selective voltage-activated cation channel was demonstrated to be permeable to the divalent cations Ca2+ and Ba2+, and even Mg2+. The current-voltage relations for the divalent cations were superlinear even at 75 mM salt concentration, but indicated outward rectification in contrast to the I-V curve for monovalent cations. The degree of activation at a given membrane potential depended strongly on the prehistory of the channel. The gating exhibited hysteretic-like behaviour, since the quasi steady-state deactivation and activation curves were displaced by approximately 25 mV. This result fully explains apparent discrepancies between V0.5-values previously obtained by slightly different experimental protocols. The possible physiological/pathophysiological role of the channel is discussed in the context of the demonstrated permeability for divalent cations.

Volume control in sickle cells is facilitated by the novel anion conductance inhibitor NS1652.

A low cation conductance and a high anion conductance are characteristic of normal erythrocytes. In sickle cell anemia, the polymerization of hemoglobin S (HbS) under conditions of low oxygen tension is preceded by an increase in cation conductance. This increase in conductance is mediated in part through Ca(++)-activated K(+) channels. A net efflux of potassium chloride (KCl) leads to a decrease in intracellular volume, which in turn increases the rate of HbS polymerization. Treatments minimizing the passive transport of ions and solvent to prevent such volume depletion might include inhibitors targeting either the Ca(++)-activated K(+) channel or the anion conductance. NS1652 is an anion conductance inhibitor that has recently been developed. In vitro application of this compound lowers the net KCl loss from deoxygenated sickle cells from about 12 mmol/L cells/h to about 4 mmol/L cells/h, a value similar to that observed in oxygenated cells. Experiments performed in mice demonstrate that NS1652 is well tolerated and decreases red cell anion conductance in vivo. (Blood. 2000;95:1842-1848)

The feasibility of pharmacological volume control of sickle cells is dependent on the quantization of the transport pathways. A model study.

Normal erythrocytes are under physiological conditions characterized by low cation and high anion conductance. However, in the case of sickle cell anemia the erythrocytes contain a modified haemoglobin, HbS, which under low oxygen tension gives rise to sickling. This condition is preceded by an increase in cation conductance, especially due to the Ca2+-activated K+-channels, leading to net-efflux of KCI and thereby decreased cellular volume, which is part of the pathological condition.A possible symptomatic treatment could be application of conductance blockers, targeting the Ca2+-activated K+-channel or the anion conductance in order to minimize the passive transport of ions and solvent. It has been argued, that due to the high anion conductance, solute loss depended at moderately increased cation conductances on the cation only. Consequently the Ca2+-activated K+-conductance should be the target for attempts to modify solute loss.It is shown that: knowledge of mean conductances (time averages) for pathways showing fluctuations are insufficient to predict the quantitative effect of conductance inhibitors, since inhibition is strongly dependent on the kinetics of the mechanisms mediating the translocation and a block of the high conductance anion pathway can be as effective as inhibition of the Ca+-activated K+-conductance with regard to net salt loss.

Annexins from Ehrlich ascites cells inhibit the calcium-activated chloride current in Xenopus laevis oocytes.

The effect of annexins II, III and V, purified from different species, on the calcium-activated chloride current across the stage-V to stage-VI Xenopus laevis oocyte membrane was tested either directly, using calcium entry mediated by depolarization, by A23187 permeabilization of oocytes or indirectly by quisqualate stimulation of a metabotropic glutamate receptor in the membrane expressed by the oocyte after injection of mRNA. The annexins isolated from the Ehrlich ascites cell, which is a mouse tumor cell, were found to be potent inhibitors of the chloride current, showing half-maximal inhibition at 50 nM, whereas no block was found using bovine or porcine annexins isolated from lung tissue. Of the annexins tested, we found annexin III to be naturally occurring in the oocyte, while only trace amounts of annexins II and V could be demonstrated. The inhibition pattern varied somewhat according to the stimulus method, the inhibition being more complete when an indirect stimulus via the metabotropic receptor was applied compared to a direct calcium stimulus.

The voltage-gated non-selective cation channel from human red cells is sensitive to acetylcholine.

Using patch-clamp technique it is demonstrated, that the voltage-gated non-selective cation channel present in the human red cell is coupled to an acetylcholine receptor of nicotinic type. The concentration dependence of carbachol, the potency of selected agonists and an estimate of the numbers of channels/red cell are reported.

Evidence for a voltage-gated, non-selective cation channel in the human red cell membrane.

Using the patch clamp technique we have identified a voltage-dependent, non-selective cation channel in the human red cell membrane. Basic properties of this channel are reported, and it is proposed that it may be involved in the increased transport of cations which is seen when intact human red cells are suspended in a depolarising media.

The gating of human red cell Ca2(+)-activated K(+)-channels is strongly affected by the permeant cation species.

Using inside-out patches, the effect of various permeant cations on the gating behaviour of the human red cell Ca2(+)-activated K(+)-channel was examined. For symmetric solutions the dwell time histograms indicated two shut and two open states. Mean open times as well as the open-state probability were affected by the permeant cation species: Rb+ stabilised the channel in the open configuration, whereas NH4+ had a destabilising effect. Intermediate stability was obtained in K+ solutions. Bi-ionic experiments indicated that the gating was influenced by the ion species occupying the channel, rather than by ions bound to external modifier sites.

Steady-state and transient membrane potentials in human red cells determined by protonophore-mediated pH changes.

Protonophores have been used frequently to determine changes in membrane potential in suspensions of red cells, since such changes are reflected by changes in extracellular pH, due to proton and consequently protonophore reequilibration. In a previous paper (Bennekou, P. 1988, J. Membrane Biol. 102:225-234) a kinetic model for the translocation of a protonophore, CCCP, across the human red cell membrane was established. This model accounts for the protonophore reequilibration following abrupt changes in membrane potential. In this paper the limitations of the method with regard to the estimation of transient membrane potentials are examined, using the transport model to simulate changes in extracellular pH in response to noninstantaneous changes in membrane potential. The temperature and time resolution calculated from the model are reported. Furthermore, it is shown that the transport model established for CCCP is valid for another protonophore, TCS, thus indicating the general validity of the transport scheme for the entire class of protonophores.

The effect of ATP, intracellular calcium and the anion exchange inhibitor DIDS on conductive anion fluxes across the human red cell membrane.

The influence of ATP depletion, the intracellular ionized Ca-concentration, anion substitution and DIDS on the conductive anion fluxes across the human red cell membrane has been examined. Under physiological or near physiological conditions it is not possible to observe conductive anion fluxes across the erythrocyte membrane in that anions totally dominate the membrane conductance. Consequently anions are at electro-chemical equilibrium and the netflux is zero. However, conductive anion fluxes can be induced by raising the potassium conductance, either by addition of valinomycin, or by triggering the native calcium activated potassium channel by addition of the Ca2+ ionophore A23187 to cells suspended in a calcium containing medium. The interpretation of data from experiments with valinomycin induced netfluxes has normally been done according to a constant field model, and the results have consequently been given as permeabilities. Since it has been demonstrated recently, that these cation pathways do not conform to a constant field scheme (Bennekou, P. and Christophersen, P. (1986) J. Membr. Biol. 93, 221-227 and Vestergaard-Bogind, B., Stampe, P. and Christophersen, P. (1985) J. Membr. Biol. 88, 67-75), it has been chosen, instead of permeabilities, to calculate the ion conductances from net efflux data, using an independent estimate of the membrane potential. The main result reported, is that only one component is found for the conductive anion fluxes in the presence of DIDS using the latter theoretical framework, whereas a sizeable DIDS-insensitive component is found when the constant field analysis is used. Furthermore it is found that ATP and intracellular calcium do not influence the anion conductances.

Protonophore anion permeability of the human red cell membrane determined in the presence of valinomycin.

A transport model for translocation of the protonophore CCCP across the red cell membrane has been established and cellular CCCP binding parameters have been determined. The time course of the CCCP redistribution across the red cell membrane, following a jump in membrane potential induced by valinomycin addition, has been characterized by fitting values of preequilibrium extracellular pH vs. time to the transport model. It is demonstrated, that even in the presence of valinomycin, the CCCP-anion is "well behaved," in that the translocation can be described by simple electrodiffusion. The translocation kinetics conform to an Eyring transport model, with a single activation energy barrier, contrary to translocation across lipid bilayers, that is reported to follow a transport model with a plateau in the activation energy barrier. The CCCP anion permeability across the red cell membrane has been calculated to be close to 2.0 X 10(-4) cm/sec at 37 degrees C with small variations between donors. Thus the permeability of CCCP in the human red cell membrane deviates from that found in black lipid membranes, in which the permeability is found to be a factor of 10 higher.

Flux ratio of valinomycin-mediated K+ fluxes across the human red cell membrane in the presence of the protonophore CCCP.

The ratio of valinomycin-mediated unidirectional K+ fluxes across the human red cell membrane, has been determined in the presence of the protonophore carbonylcyanide m-chlorophenylhydrazone, CCCP, using the K+ net efflux and 42K influx. The driving force for the net efflux (Vm - EK+) has been calculated from the membrane potential, estimated by the CCCP-mediated proton distribution and the Nernst potential for potassium ions across the membrane. An apparent driving potential for the K+ net efflux has been calculated from the K+ flux ratio, determined in experiments where the valinomycin and CCCP concentrations were varied systematically. This apparent driving force, in conjunction with the actual driving force calculated on basis of the CCCP estimated membrane potential, is used to calculate a flux ratio exponent, which represents an estimate of the deviation of valinomycin-mediated K+ transport from unrestricted electrodiffusion, when protonophore is present. In the present work, the flux ratio exponent is found to be 0.90 when the CCCP concentration is 5.0 microM and above, while the exponent decreases to about 0.50 when no CCCP is present. The influence of CCCP upon the rate constants in the valinomycin transport cycle is discussed. The significance of this result is that red cell membrane potentials are overestimated, when calculated from valinomycin-mediated potassium isotope fluxes, using a constant field equation.

K+-valinomycin and chloride conductance of the human red cell membrane. Influence of the membrane protonophore carbonylcyanide m-chlorophenylhydrazone.

Chloride ion conductance of the human red cell membrane has been calculated, as the ratio between ion net charge flux and driving potential. The proton carrier CCCP was used to monitor changes in membrane potential following addition of valinomycin in sufficient quantities to raise the K+ conductance to a level comparable to the Cl- conductance. A K+-specific electrode was used to monitor changes in extracellular K+ concentration, and an H+-sensitive glass electrode for changes in extracellular pH, reflecting changes in membrane potential. The effects of varied concentrations of valinomycin and CCCP upon K+ and Cl- conductances were studied. It was found that, within an experimental error of about 10% S.D., the chloride conductance was constant for valinomycin concentrations in the range 1.0 X 10(-8)-1.0 X 10(-6), and for CCCP-concentrations in the range 2.0 X 10(-7)-2.0 X 10(-5) mol per litre cell suspension, while at a constant concentration of valinomycin the induced K+ conductance was considerably augmented by addition of CCCP.

Calcium-induced oscillations in K+ conductance and membrane potential of human erythrocytes mediated by the ionophore A23187.

The time-dependence of ionophore A23187-induced changes in the conductance of the Ca2+-sensitive K+ channels of the human red cell has been monitored with ion-specific electrodes. The membrane potential was reflected in CCCP-mediated pH changes in a buffer-free extracellular medium, and changes in extracellular K+ activity and electrode potential of an extracellular Ca2+-electrode were recorded. Within a narrow range of ionophore-mediated Ca2+ influx, the above-mentioned parameters were found to oscillate when ionophore was added to a suspension of glucose-fed cells. The period of oscillation was about 2 min/cycle depending on ionophore concentration, and the amplitude of hyperpolarization was about 60 mV, corresponding to a maximal gK+ of the same magnitude as gCl-. Without CCCP present no oscillation in K+ conductance was observed. The Ca2+ affinity for the opening process was in the micromolar range. The closing of the K+ channels was a spontaneous process in that the depolarization was well under way before the Ca2+-ATPase-mediated Ca2+ net efflux started. Below the Ca2+ influx range for oscillations, no response was observed for up to 20 min after the addition of ionophore. Above the upper limit, a permanent hyperpolarization resulted with an extracellular K+ activity increasing monotonically as a function of time. In experiments with ATP-depleted cells, responses of the latter type ensued at all ionophore concentrations above the lower limit. Addition of surplus EGTA to suspensions of hyperpolarized cells restores the normal membrane potential in the case of glucose-fed cells, whereas the K+-channels in ATP-depleted cells remained open.