Elevated intracellular Ca2+ affects Lii-Nao countertransport in human red blood cells

Changes in cytoplasmic Ca2+ concentration and in Lii-Nao countertransport activity have been shown to be associated with essential hypertension. Elevated intracellular free [Ca2+], as well as abnormalities of Ca2+ binding and transport have been reported in cells from different tissues of hypertensive laboratory animals and essential hypertensive patients. Similarly, enhanced rates of Lii-Nao countertransport and the modified pattern of the temperature dependence of this activity in red blood cells from essential hypertensive patients have been previously demonstrated. The aim of the present study was to investigate possible interaction between changes in intracellular free [Ca2+] and the Lii-Nao exchange in human red blood cells. The ionophore ionomycin was used to allow Ca2+ incorporation into the cells in a dose-dependent manner. The elevation of intracellular [Ca2+], in turn, resulted in enhanced Li+ efflux from the cells. At 3 microM, ionomycin selectively and significantly enhanced the Lii-Nao countertransport but not Li+ leakage from the cells. EGTA totally abolished the effect of ionomycin, indicating that the effect is directly related to Ca2+. As low as 0.4 microM Ca2+ caused a statistically significant effect. The maximal effect of Ca2+ on the Lii-Nao countertransport was achieved around the external pH range of 6.8-7.5. In contrast, the leakage of Li+ was significantly enhanced by Ca2+ at a pH of 7.4 and above. Ca2+ did not affect the Km of the Lii-Nao countertransport for Li+. Amiloride, which inhibits Na+/H+ exchange, inhibited only 10% of the Ca2+-enhanced countertransport. It is concluded that Ca2+ may play a role in the regulation of Lii-Nao countertransport in erythrocytes.     

Reincorporated plasma membrane Ca2+-ATPase can mediate B-Type Ca2+ channels observed in native membrane of human red blood cells

Recently, we reported indirect evidence that plasma membrane Ca2+-ATPase (PMCA) can mediate B-type Ca2+ channels of cardiac myocytes. In the present study, in order to bring more direct evidence, purified PMCA from human red blood cells (RBC) was reconstituted into giant azolectin liposomes amenable to the patch-clamp technique. Purified RBC PMCA was used because it is available pure in larger quantity than cardiac PMCA. The presence of B-type Ca2+ channels was first investigated in native membranes of human RBC. They were detected and share the characteristics of cardiac myocytes. They spontaneously appeared in scarce short bursts of activity, they were activated by chlorpromazine (CPZ) with an EC50 of 149 mmole/l or 1 mmole/l vanadate, and then switched off by 10 mmole/l eosin or dose-dependently blocked by 1-5 mmole/l ATP. Independent of membrane potential, the channel gating exhibited complex patterns of many conductance levels, with three most often observed conductance levels of 22, 47 and 80 pS. The activation by vanadate suggests that these channels could play a role in the influx of extracellular Ca2+ involved in the vanadate-induced Gardos effect. In PMCA-reconstituted proteoliposomes, nearly half of the ATPase activity was retained and clear "channel-like" openings of Ba2+- or Ca2+-conducting channels were detected. Channel activity could be spontaneously present, lasting the patch lifetime or, when previously quiescent, activity could be induced by application of 50 mmole/l CPZ only in presence of 25 U/ml calmodulin (CaM), or by application of 1 mmole/l vanadate alone. Eosin (10 mmole/l) and ATP (5 mmole/l) significantly reduced spontaneous activity. Channel gating characteristics were similar to those of RBC, with main conductance levels of 21, 40 and 72 pS. The lack of direct activation by CPZ alone might be attributed to a purification-induced modification or absence of unidentified regulatory component(s) of PMCA. Despite a few differences in results between RBC and reincorporated PMCA, most probably attributable to the decrease in ATPase activity following the procedure of reincorporation, the present experimental conditions appear to reveal a channel-mode of the PMCA that shares many similarities with the B-type Ca2+ channel.     

The thapsigargin-sensitive intracellular Ca2+ pool is more important in plasma membrane Ca2+ entry than the IP3-sensitive intracellular Ca2+ pool in neuronal cell lines

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.     

A comparative functional analysis of plasma membrane Ca2+ pump isoforms in intact cells

The four basic isoforms of the plasma membrane Ca2+ pump and the two C-terminally truncated spliced variants PMCA4CII(4a) and 3CII(3a) were transiently overexpressed in Chinese hamster ovary cells together with aequorin targeted to the cytosol, the endoplasmic reticulum, and the mitochondria. As PMCA3CII(3a) had not yet been cloned and studied, it was cloned for this study, partially purified, and characterized. At variance with the corresponding truncated variant of PMCA4, which had been studied previously, PMCA3CII(3a) had very high calmodulin affinity. All four basic pump variants influenced the homeostasis of Ca2+ in the native intracellular environment. The level of [Ca2+] in the endoplasmic reticulum and the height of the [Ca2+] transients generated in the cytosol and in the mitochondria by the emptying of the endoplasmic reticulum store by inositol 1,4,5-trisphosphate were all reduced by the overexpression of the pumps. The effects were much greater with the neuron-specific PMCA2 and PMCA3 than with the ubiquitously expressed isoforms 1 and 4. Unexpectedly, the truncated PMCA3 and PMCA4 were as effective as the full-length variants in influencing the homeostasis of Ca2+ in the cytosol and the organelles. In particular, PMCA4CII(4a) was as effective as PMCA4CI(4b), even if its affinity for calmodulin is much lower. The results indicate that the availability of calmodulin may not be critical for the modulation of PMCA pumps in vivo.     

Normal Ca2+ extrusion by the Ca2+ pump of intact red blood cells exposed to high glucose concentrations

The ATPase activity of the plasma membrane Ca2+ pump (PMCA) has been reported to be inhibited by exposure of red blood cell (RBC) PMCA preparations to high glucose concentrations. It has been claimed that this effect could have potential pathophysiological relevance in diabetes. To ascertain whether high glucose levels also affect PMCA transport function in intact RBCs, Ca2+ extrusion by the Ca2+-saturated pump [PMCA maximal velocity (V(max))] was measured in human and rat RBCs exposed to high glucose in vivo or in vitro. Preincubation of normal human RBCs in 30-100 mM glucose for up to 6 h had no effect on PMCA V(max). The mean V(max) of RBCs from 15 diabetic subjects of 12.9 +/- 0.7 mmol. 340 g Hb(-1). h(-1) was not significantly different from that of controls (14.3 +/- 0.5 mmol. 340 g Hb(-1). h(-1)). Similarly, the PMCA V(max) of RBCs from 11 streptozotocin-diabetic rats was not affected by plasma glucose levels more than three times normal for 6-8 wk. Thus exposure to high glucose concentrations does not affect the ability of intact RBCs to extrude Ca2+.     

Asymmetric effects of divalent cations and protons on active Ca2+ efflux and Ca2+-ATPase in intact red blood cells

Summary The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca²⁺ transport have been examined in intact human red blood cells. Active Ca²⁺ efflux was determined from the initial rate of⁴⁵Ca²⁺ loss after CoCl₂ was added to block Ca²⁺ loading via the ionophore A23187. Ca²⁺-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca²⁺ in the presence of A23187. The apparent Ca affinity of active Ca²⁺ efflux (K _0.5=30–40 mol/liter cells) was significantly lower than that measured by the Ca²⁺-ATPase assay (K _0.5=0.4 m). Possible reasons for this apparent difference are considered. Both active Ca²⁺ efflux and Ca²⁺-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells · hr) in Mg²⁺-depleted cells, and completely restored by reintroduction of intracellular Mg²⁺. Active Ca²⁺ efflux was inhibited almost completely by raising external CaCl₂ (but not MgCl₂) to 20mm, probably by interaction of Ca²⁺ at the externally oriented E₂P conformation of the pump. Cd²⁺ was more potent than Ca²⁺ in this inhibition, while Mn²⁺ was less potent and 10mm Ba²⁺ was without effect. A Ca²⁺: proton exchange mechanism for active Ca²⁺ efflux was supported by the results, as external protons (pH 6–6.5) stimulated active Ca²⁺ efflux at least twofold above the efflux rate at pH 7.8 Ca²⁺ transport was not affected by decreasing the membrane potential across the red cell.     

Role of magnesium in the (Ca2+ + Mg2+)-stimulated membrane ATPase of human red blood cells.

(Ca2+ + Mg2+)-stimulated ATPase of human red cell membranes as a function of ATP concentration was measured at fixed Ca2+ concentration and at two different but constant Mg2+ concentrations. Under the assumption that free ATP rather than Mg-ATP is the substrate, a value for Km (for ATP) of 1-2 micron is found which is in good agreement with the value obtained in the phosphorylation reaction by A.F. Rega and P.J. Garrahan (1975. J. Membrane Biol. 22:313). Mg2+ increases both the maximal rate and the affinity for ATP, whereas Ca2+ increases the maximal rate without affecting Km for ATP. As a by-product of these experiments, it was shown that after thorough removal of intracellular proteins the adenylate kinase reaction at approximately 1 mM substrate concentration is several times faster than maximal rate of (Ca2+ + Mg2+)ATPase in red cell membranes.     

On the functional use of the membrane compartmentalized pool of ATP by the Na+ and Ca++ pumps in human red blood cell ghosts

Previous evidence established that a sequestered form of adenosine triphosphate (ATP pools) resides in the membrane/cytoskeletal complex of red cell porous ghosts. Here, we further characterize the roles these ATP pools can perform in the operation of the membrane''s Na⁺ and Ca²⁺ pumps. The formation of the Na⁺- and Ca²⁺-dependent phosphointermediates of both types of pumps (E_Na-P and E_Ca-P) that conventionally can be labeled with trace amounts of [γ-³P]ATP cannot occur when the pools contain unlabeled ATP, presumably because of dilution of the [γ-³P]ATP in the pool. Running the pumps forward with either Na⁺ or Ca²⁺ removes pool ATP and allows the normal formation of labeled E_Na-P or E_Ca-P, indicating that both types of pumps can share the same pools of ATP. We also show that the halftime for loading the pools with bulk ATP is 10–15 minutes. We observed that when unlabeled “caged ATP” is entrapped in the membrane pools, it is inactive until nascent ATP is photoreleased, thereby blocking the labeled formation of E_Na-P. We also demonstrate that ATP generated by the membrane-bound pyruvate kinase fills the membrane pools. Other results show that pool ATP alone, like bulk ATP, can promote the binding of ouabain to the membrane. In addition, we found that pool ATP alone functions together with bulk Na⁺ (without Mg²⁺) to release prebound ouabain. Curiously, ouabain was found to block bulk ATP from entering the pools. Finally, we show, with red cell inside-outside vesicles, that pool ATP alone supports the uptake of ⁴⁵Ca by the Ca²⁺ pump, analogous to the Na⁺ pump uptake of ²²Na in this circumstance. Although the membrane locus of the ATP pools within the membrane/cytoskeletal complex is unknown, it appears that pool ATP functions as the proximate energy source for the Na⁺ and Ca²⁺ pumps.     

Endothelin mobilizes Ca2+ from a caffeine- and ryanodine-insensitive intracellular pool in rat atrial cells

Endothelin-1 is a powerful inotropic peptide for the rat atrium. Its action can develop in the absence of L-type Ca2+ channel activity provided that the external Ca2(+)-concentration has been raised to supraphysiological concentrations. Endothelin stimulates phosphatidylinositol hydrolysis in new born rat atrial cells via a mechanism that is insensitive to pertussis toxin. The diacylglycerol/protein kinase C signaling pathway cannot account for the contractile action of endothelin but its activation by phorbol esters induces a partial desensitization of phospholipase C activity. Endothelin-1 and the related peptides, endothelin-2, endothelin-3, and sarafotoxin S6b, raise intracellular Ca2+ levels in rat atrial cells. The actions of endothelin-1, endothelin-2, and sarafotoxin on [Ca2+]i are mutually exclusive, suggesting that they act at the same receptor site. The rise in [Ca2+]i induced by endothelins results both from the mobilization of intracellular stores and from Ca2+ entry through the sarcolemma via a pathway that is not voltage-dependent L-type Ca2+ channels. The Ca2+ store that is mobilized in response to endothelin retains its Ca2+ content when cells were incubated for long periods of time in a 50 nM Ca2+ solution. It is insensitive to caffeine and ryanodine. These two properties distinguish it from the sarcoplasmic reticulum. Contraction experiments in which the pacing rate has been altered to favor Ca2+ accumulation into terminal cisternae of the sarcoplasmic reticulum also suggest that the Ca2+ load of the sarcoplasmic reticulum is increased in endothelin treated rat atria.     

Gramicidin S inhibition of the Ca2+-ATPase of human red blood cells

The cationic amphiphilic polypeptide gramicidin S inhibits the Ca2+-ATPase of human red-cell membranes by lowering the maximum velocity of the high-affinity component and the apparent affinity of the low-affinity component of the velocity-versus-ATP concentration curve of the enzyme. Gramicidin S does not alter the apparent affinity of the Ca2+-ATPase for Ca2+. Calmodulin is not essential for the inhibition, but increases the sensitivity of the enzyme to the inhibitor. The effects of gramicidin S on the Ca2+-ATPase can be reversed with phosphatidylcholine vesicles but not with buffer solutions, suggesting that gramicidin S acts from the lipid phase of the membrane.     

Stimulation of human red blood cells leads to Ca2+-mediated intercellular adhesion

Red blood cells (RBCs) are a major component of blood clots, which form physiologically as a response to injury or pathologically in thrombosis. The active participation of RBCs in thrombus solidification has been previously proposed but not yet experimentally proven. Holographic optical tweezers and single-cell force spectroscopy were used to study potential cell-cell adhesion between RBCs. Irreversible intercellular adhesion of RBCs could be induced by stimulation with lysophosphatidic acid (LPA), a compound known to be released by activated platelets. We identified Ca²⁺ as an essential player in the signaling cascade by directly inducing Ca²⁺ influx using A23187. Elevation of the internal Ca²⁺ concentration leads to an intercellular adhesion of RBCs similar to that induced by LPA stimulation. Using single-cell force spectroscopy, the adhesion of the RBCs was identified to be approximately 100 pN, a value large enough to be of significance inside a blood clot or in pathological situations like the vasco-occlusive crisis in sickle cell disease patients.     

Redistribution of intracellular Ca2+ in mitogen-stimulated human peripheral blood lymphocytes

The fluorescent probe chlortetracycline (CTC) was used to investigate redistribution of intracellular Ca2+ in concanavalin A (Con A)-stimulated human peripheral blood lymphocytes. The addition of the mitogen to CTC-equilibrated lymphocytes induced (within 10 to 15 minutes) a Con A-concentration dependent decrease in CTC fluorescence indicating the release of membrane-bound Ca2+. The effect was independent of the level of extracellular Ca2+ and could be observed in the presence of EGTA; it was suppressed by the metabolic inhibitors FCCP, antimycin and sodium cyanide. Analysis of the excitation spectra of CTC fluorescence indicated that the observed effect is caused by redistribution of intracellular Ca2+ rather than Mg2+. Thus the lectin interaction with the lymphocyte plasma membrane results in Ca2+ release into the cytosol from the intracellular stores.     

Mitochondrial Ca2+ homeostasis in intact cells

Ca2+ is a key regulator not only of multiple cytosolic enzymes, but also of a variety of metabolic pathways occurring within the lumen of intracellular organelles. Until recently, no technique to selectively monitor the Ca2+ concentration within defined cellular compartments was available. We have recently proposed the use of molecularly engineered Ca(2+)-sensitive photoproteins to obtain such a result and demonstrated the application of this methodology to the study of mitochondrial and nuclear Ca2+ dynamics. We here describe in more detail the use of chimeric recombinant aequorin targeted to the mitochondria. The technique can be applied with equivalent results to different cell models, transiently or permanently transfected. In all the cell types we analyzed, mitochondrial Ca2+ concentration ([Ca2+]m) increases rapidly and transiently upon stimulation with agonists coupled to InsP3 generation. We confirm that the high speed of mitochondrial Ca2+ accumulation with this type of stimuli depends on the generation of local gradients of Ca2+ in the cytosol, close to the channels sensitive to InsP3. In fact, only activation of these channels, but not the simple release from internal stores, as that elicited by blocking the intracellular Ca2+ ATPases, results in a fast mitochondrial Ca2+ accumulation. We also provide evidence in favor of a microheterogeneity among mitochondria of the same cells, about 30% of them apparently sensing the microdomains of high cytosolic Ca2+ concentration ([Ca2+]c). The changes in [Ca2+]m appear sufficiently large to induce a rapid activation of mitochondrial dehydrogenases, which can be followed by monitoring the level of NAD(P)H fluorescence. A general scheme can thus be envisaged by which the triggering of a plasma membrane receptor coupled to InsP3 generation raises the Ca2+ concentration both in the cytoplasm (thereby triggering energy-consuming processes, such as cell proliferation, motility, secretion, etc.) and in the mitochondria, where it activates the metabolic activity according to the increased cell needs.     

Functional homogeneity of the non-mitochondrial Ca2+ pool in intact mouse lacrimal acinar cells.

In the absence of extracellular Ca2+, treatment of mouse lacrimal acinar cells with maximal concentrations of methacholine released Ca2+ from intracellular stores. No additional Ca2+ was mobilized by subsequent application of the intracellular Ca(2+)-ATPase inhibitor, thapsigargin, the stable inositol 1,4,5-trisphosphate ((1,4,5)IP3) analog, inositol 2,4,5-trisphosphate ((2,4,5)IP3) (by microinjection), or the Ca2+ ionophore, ionomycin. However, following prolonged activation of cells by methacholine in the presence of extracellular Ca2+, Ca2+ accumulated into a pool which was released by ionomycin but not by thapsigargin. This latter accumulation was blocked by prior microinjection of ruthenium red, indicating that it represents mitochondrial uptake. In saponin-permeabilized lacrimal cells, two Ca(2+)-sequestering pools were detected: (i) a ruthenium red-sensitive, thapsigargin-insensitive pool, presumed to be the mitochondria; and (ii) a ruthenium red-insensitive, thapsigargin-sensitive pool. Only the thapsigargin-sensitive pool accumulated Ca2+ at concentrations similar to those in unstimulated cells. The thapsigargin-sensitive Ca2+ pool was sensitive to (1,4,5)IP3; however, in contrast to findings in intact cells, only 44% of this pool was releasable by (1,4,5)IP3 or (2,4,5)IP3. These data indicate that, in intact lacrimal acinar cells, all exchangeable (ionomycin-sensitive) Ca2+ residues in a pool which responds homogeneously to agonists, (1,4,5)IP3, and thapsigargin. Prolonged elevation of [Ca2+]i results in Ca2+ accumulation into a second, ruthenium red-sensitive pool, presumably mitochondria. Finally, permeabilization of the cells fragments the non-mitochondrial pool, resulting in two pools, one sensitive and one insensitive to (1,4,5)IP3.     

The plasma membrane Ca2+-ATPase protein from red blood cells is not modified in preeclampsia

Plasma membrane Ca2+-ATPase activity diminishes by about 50% in red blood cells during preeclampsia. We investigated whether the number of Ca2+-ATPase molecules is modified in red cell membranes from preeclamptic pregnant women by measuring the specific phosphorylated intermediate of this enzyme. Also, we isolated the Ca2+-ATPase protein from both normotensive and preeclamptic pregnant women and estimated its molecular weight, and its cross-reactions with specific polyclonal and monoclonal (5F10) antibodies against it. We measured the Ca2+-ATPase activity in a purified state and the effect of known modulators of this ATPase. It was found that the phosphorylated intermediate associated with PMCA is similar for red cell ghosts from normotensive and preeclamptic women, suggesting a similar number of ATPase molecules in these membranes. The molecular weight of the Ca2+-ATPase is around 140 kDa for both normotensive and preeclamptic membranes, and its cross-reactions with specific antibodies is similar, suggesting that the protein structure remains intact in preeclampsia. Calmodulin, ethanol, or both calmodulin plus ethanol, stimulated the Ca2+-ATPase activity to the same extent for both normotensive and preeclamptic preparations. Our results showed that the reduced Ca2+-ATPase activity of the red cell membranes from preeclamptic women is not associated with a defective enzyme, but rather with a high level of lipid peroxidation.     

The route of Ca2+ entry during reloading of the intracellular Ca2+ pool in pancreatic acini

To trace the route of Ca2+ entry and the role of the cytosolic Ca2+ pool in reloading of the internal stores of pancreatic acinar cells, Mn2+ influx into Fura 2-loaded cells and the effect of 1,2-bis(2-aminophenoxyethane-N,N,N',N'-tetraacetic acid (BAPTA) on Ca2+ storage in intracellular stores and reloading were examined. Treatment of acini suspended in Ca2(+)-free medium with carbachol (cell stimulation) or carbachol and atropine (reloading period) resulted in 2-fold increase in the rate of Mn2+ influx. Increasing Ca2+ permeability of the plasma membrane by elevation of extracellular pH from 7.4 to 8.2 further increased the rate of Mn2+ influx observed during cell stimulation and the reloading period. Loading the acini with BAPTA by incubation with 50 microM of the acetomethoxy form of BAPTA (BAPTA/AM) was followed by a transient reduction in free cytosolic Ca2+ concentration ((Ca2+]i). To compensate for the increased Ca2+ buffering capacity in the cytosol the acini incorporated Ca2+ from the external medium. Although BAPTA prevented changes in free cytosolic Ca2+ concentration during carbachol and atropine treatment, it had no apparent effect on Ca2+ content of the internal stores or the ability of agonists to release Ca2+ from these stores. Loading the cytosol with BAPTA considerably reduced the rate of Ca2+ reloading. These observations are not compatible with direct communication between the medium and the inositol 1,4,5-trisphosphate releasable pool and provide direct evidence for Ca2+ entry into the cytosol prior to its uptake into the intracellular pool, both during cell stimulation and the Ca2+ reloading.     

Rapid functional assays of intracellular Ca2+ channels

Functional assays of intracellular Ca2+ channels, such as the inositol 1,4,5-trisphosphate receptor (IP3R), have generally used 45Ca2+-flux assays, fluorescent indicators loaded within either the cytosol or the endoplasmic reticulum (ER) of single cells, or electrophysiological analyses. None of these methods is readily applicable to rapid, high-throughput quantitative analyses. Here we provide a detailed protocol for high-throughput functional analysis of native and recombinant IP3Rs. A low-affinity Ca2+ indicator (mag-fluo-4) trapped within the ER of permeabilized cells is shown to report changes in luminal free Ca2+ concentration reliably. An automated fluorescence plate reader allows rapid measurement of Ca2+ release from intracellular stores mediated by IP3R. The method can be readily adapted to other cell types or to the analysis of other intracellular Ca2+ channels. This protocol can be completed in 2-3 h.     

The intracellular Ca2+ channels of membrane traffic

Regulation of organellar fusion and fission by Ca²⁺ has emerged as a central paradigm in intracellular membrane traffic. Originally formulated for Ca²⁺-driven SNARE-mediated exocytosis in the presynaptic terminals, it was later expanded to explain membrane traffic in other exocytic events within the endo-lysosomal system. The list of processes and conditions that depend on the intracellular membrane traffic includes aging, antigen and lipid processing, growth factor signaling and enzyme secretion. Characterization of the ion channels that regulate intracellular membrane fusion and fission promises novel pharmacological approaches in these processes when their function becomes aberrant. The recent identification of Ca²⁺ permeability through the intracellular ion channels comprising the mucolipin (TRPMLs) and the two-pore channels (TPCs) families pinpoints the candidates for the Ca²⁺ channel that drive intracellular membrane traffic. The present review summarizes the recent developments and the current questions relevant to this topic.     

Age decline in the activity of the Ca2+-sensitive K+ channel of human red blood cells

The Ca(2+)-sensitive K(+) channel of human red blood cells (RBCs) (Gardos channel, hIK1, hSK4) was implicated in the progressive densification of RBCs during normal senescence and in the mechanism of sickle cell dehydration. Saturating RBC Ca(2+) loads were shown before to induce rapid and homogeneous dehydration, suggesting that Gardos channel capacity was uniform among the RBCs, regardless of age. Using glycated hemoglobin as a reliable RBC age marker, we investigated the age-activity relation of Gardos channels by measuring the mean age of RBC subpopulations exceeding a set high density boundary during dehydration. When K(+) permeabilization was induced with valinomycin, the oldest and densest cells, which started nearest to the set density boundary, crossed it first, reflecting conservation of the normal age-density distribution pattern during dehydration. However, when Ca(2+) loads were used to induce maximal K(+) fluxes via Gardos channels in all RBCs (F(max)), the youngest RBCs passed the boundary first, ahead of the older RBCs, indicating that Gardos channel F(max) was highest in those young RBCs, and that the previously observed appearance of uniform dehydration concealed a substantial degree of age scrambling during the dehydration process. Further analysis of the Gardos channel age-activity relation revealed a monotonic decline in F(max) with cell age, with a broad quasi-Gaussian F(max) distribution among the RBCs.