INTRODUCTION — The volume and hemoglobin concentration of human red blood cells are determined by the red cell content of hemoglobin, ions, and water.
The transport systems involved in regulating the volume and composition of red cells, and which affect the cation, anion, and water content of the red cell, will be discussed here. A brief discussion of the role of ion transport in the pathogenesis of sickle cell disease will also be presented.
CONTROL OF HYDRATION — The water, cation, and anion contents are continuously regulated by the activity and interactions of several ion transport systems, as summarized in the figure (figure 1) [1,2]. The importance of these systems is indicated by the various diseases occurring when red cell ion transport is impaired (table 1).
Cation content — In human red cells (and other cells), the activity of the sodium-K-ATPase pump maintains a low sodium, high potassium milieu. The outward potassium gradient and the inward sodium gradient can be used by several passive (gradient-driven) transport systems, which are sensitive to changes in pH, volume, or membrane integrity.
Human red cells have a high content of total magnesium but a lower content of free magnesium. Magnesium is an important regulator of several cellular functions; its export from the red cells is regulated by a gradient-driven Na-Mg exchanger, while little is known about the mechanisms involved in controlling the entry of magnesium into the human erythrocyte.
A powerful membrane Ca-ATPase is responsible for the extremely low Ca content of human erythrocytes.
Anion content — The chloride content of the human red cell varies according to the cation content (permeant cations), the net charge of the non-permeant ions (eg, hemoglobin and 2,3-bisphosphoglycerate [2,3-BPG]), and intracellular pH. The chloride-bicarbonate anion exchanger (AE1 or band 3 protein) is the major pathway for the movement of these anions in human red cells.
Water content — Water can move across the red cell membrane by passive diffusion and also via a specific water channel aquaporin-1 (AQP-1), which is the prototype of the aquaporin family of transporters [3]. Passive diffusion and water channels allow the red cell to be in osmotic equilibrium with the plasma. As a result, the water content of the human red cell is determined by the intracellular sodium, potassium, and Cl content and the osmolality of the plasma. (See 'Water channels' below.)
ENERGY-DRIVEN SYSTEMS — There are two active transport systems in the red cell: the Na-K-ATPase pump and the calcium-ATPase pump.
N-K-ATPase pump — Sodium-potassium ATPase is an enzyme formed by three subunits: the alpha-1 catalytic subunit (1020 amino acids) involved in ion transport, the beta-1 subunit (302 amino acids) involved in the interaction with membrane and cytoskeletal components [4,5], and the gamma-1 proteolipid subunit. Each red cell contains a mean of 228 to 470 Na-K-ATPase units [6].
The Na-K-ATPase pump extrudes sodium in exchange for potassium with a 3:2 stoichiometry. Tosteson and Hoffman provided theoretical and experimental evidence for the pump-leak model, which explains how active transport works in parallel with passive transport to determine the sodium and potassium content of cells [7]. The cell sodium content is determined by the balance between the entry of sodium and its active extrusion by the Na-K pump.
A demonstration of this model was provided by studies of the red cells of a patient with a 10 to 20-fold increase in the number of pump sites per cell [8]. The red cell membranes had normal passive permeability to sodium and, due to increased Na-K-ATPase activity, a reduction in the cell sodium concentration and an elevation in the cell potassium concentration were present.
Loss-of-function mutations in the nonerythroid alpha-2 subunit gene of the Na-K pump have been associated with familial hemiplegic migraine type 2 [9,10], while missense mutations in the nonerythroid alpha-3 subunit gene have been associated with rapid-onset dystonia parkinsonism [11,12].
Ca-ATPase pump — The red cell membrane also contains a Ca-ATPase pump that maintains free cytosolic levels of calcium below 0.1 µmol/L [13]. Calcium efflux via the Ca-ATPase pump takes place in conjunction with hydrogen influx, with a 1:1 stoichiometry between ATP consumption and calcium extrusion.
The intracellular calcium concentration is increased two- to threefold during deoxygenation in sickle cell anemia red cells [14]. This change is associated with an increase in passive calcium influx and a reduction in Ca-ATPase pump activity. It may contribute to the sickling process by activating a major dehydration pathway via the calcium-sensitive potassium channel (see 'Calcium-activated potassium channel (Gardos channel)' below).
There is great variability in the transport capacity of the Ca-ATPase pump within the same person's red blood cells, with the presence of sub-populations of red cells in which the activity can vary six to ninefold [15]. It has been shown that aging of normal human red cells is associated with a monotonic decrease in Ca-ATPase activity [16].
PASSIVE GRADIENT-DRIVEN SYSTEMS — Several passive, gradient-driven systems, including electroneutral cotransporters and ion- or water-specific channels, utilize the inward sodium and outward potassium gradients generated by the Na-K pump to transport ions across the red cell membrane. Because of the dependence upon the Na-K pump, this process is also called secondary active transport.
Na-K-Cl cotransport — Na-K-Cl cotransport was first described in human red cells in 1974 [17] and is widely distributed in other cell types, including the apical and basolateral membranes of the thick ascending limb of the loop of Henle. The major erythroid form of this electroneutral transporter, called NKCC, has a 1Na:1K:2Cl stoichiometry. As with its counterpart in the basolateral membrane of the thick ascending limb, it is inhibited with high affinity by loop diuretics, which bind to the Cl site, and belongs to the family of cation-chloride cotransporters (CCC) [18-20]; other members of this family are the thiazide-sensitive Na-Cl cotransporter (NCC) [20], the volume-sensitive K-Cl cotransporter (KCC), and the apical Na-K-Cl cotransporter of the renal thick limb NKCC2, which typically has a lower affinity for loop diuretics such as bumetanide [21].
In human red cells, Na-K-Cl cotransport results in a small net extrusion of sodium, potassium, and chloride under physiologic conditions [22]. It plays only a minor role in the regulation of human red cell volume.
K-Cl cotransport — The electroneutral K-Cl cotransporter belongs to the family of chloride-cation transporters (CCC). At least four major isoforms of this transporter have been identified: KCC1 [23], KCC2 [24], KCC3 [25], and KCC4 [26]. The importance of these transporters has been shown in the following situations:
●The KCC2 (-/-) mouse dies perinatally due to respiratory failure induced by the loss of synaptic inhibitory input [27].
●The KCC3 (-/-) mouse shows impaired cell volume regulation in neurons and kidney tubular cells, with deafness, reduced seizure threshold, peripheral neurologic disease, and arterial hypertension [28].
●Human KCC3 mutations are associated with agenesis of the corpus callosum and peripheral neuropathy (ACCPN or Andermann syndrome) [29].
●The KCC4 (-/-) mouse exhibits deafness and renal tubular acidosis [30].
KCC isoforms 1, 3, and 4 are present in human erythrocytes. K-Cl cotransport mediates extrusion of potassium and chloride from the red cell (figure 1). The functions of this transporter have been studied in several species (sheep, dog, and duck) [31], in red cells of patients homozygous for hemoglobin C disease [32], in sickle cell disease [33], thalassemia [34,35], and in normal human reticulocytes [36].
The most relevant functional properties of K-Cl cotransport are:
●Volume-dependence (with activation by cell swelling)
●pH-dependence (with optimum pH of 6.7 to 7.2)
●Inhibition by intracellular magnesium and other divalent cations, including zinc [37]
●Activation by hydrostatic pressure [38]
●Activation by urea and oxidation [39-41]
●Activation by positively charged hemoglobin variants [32,42-44]
Role in sickle erythrocytes — Erythrocyte dehydration is thought to play an important role in the pathophysiology of the chronic organ damage and acute vaso-occlusive events of sickle cell disease. Dehydrated cells have increased cellular hemoglobin (HbS) concentration, which in turn leads to a marked increase in the polymerization of HbS and sickling.
The K-Cl cotransport system plays an important role in the pathologic dehydration of sickle red cells, either alone or in conjunction with the calcium-activated potassium (Gardos) channel [45-48]. (See 'Calcium-activated potassium channel (Gardos channel)' below and "Pathophysiology of sickle cell disease", section on 'Effects on the RBC'.)
It is believed that every time sickle erythrocytes are exposed to the renal medullary environment with high urea concentrations and/or to low pH, they lose potassium, chloride, and water via K-Cl cotransport. Sickle erythrocytes exhibit a marked increase in K-Cl cotransport, an effect that is most likely due to interaction between the relatively positively charged HbS and the cell membrane or the regulatory machinery of the transporter [42], since this has also been shown for cells containing HbC [44].
K-Cl cotransport in normal erythrocytes is inhibited by low oxygen tension and is refractory to stimulation by low pH or urea; in contrast, the activity of the transporter in sickle erythrocytes is independent of oxygen tension and retains the ability to be activated by low pH and urea under hypoxic conditions [49-51]. As a result, the K-Cl cotransporter in sickle erythrocytes can induce significant potassium, chloride, and water loss even at a low pO2 (as occurs, for example, in the renal medulla). In a transgenic mouse model of sickle cell disease, expression of an activated form of the K-Cl cotransporter KCC1 was demonstrated to increase morbidity and mortality substantially [52]. (See "Sickle cell disease effects on the kidney".)
Prevention of K-Cl cotransport-induced red cell dehydration is a potential therapeutic strategy for sickle cell disease [53].
●Dietary magnesium supplements (eg, magnesium pidolate, available over-the-counter in France and other European countries, but not available in the United States) have been used to increase red cell magnesium content, which impairs K-Cl cotransport and reduces dehydration of sickle cells with minimal side effects [54-56]. However, a trial of intravenous magnesium administration to treat acute painful episodes in sickle cell disease did not show a benefit.
●In one study, addition of a sulfhydryl-reducing agent normalized the sensitivity of K-Cl cotransport activity to urea in sickle cell red cells, mitigating the urea-stimulated volume decrease in sickle cell reticulocytes [41]. This observation suggests that the dysfunctional activity was due, in part, to reversible sulfhydryl oxidation, providing rationale for use of sulfhydryl reducing agents in these patients. However, a clinical trial on the effects of orally administered antioxidant N-acetylcysteine in patients with sickle cell disease showed no significant effect on the frequency of daily pain [57].
Na-H exchanger — The NHE1 sodium-hydrogen exchanger (or antiporter) transports sodium into cells in exchange for hydrogen and accounts for a small fraction of the passive sodium influx [58]. In other cell types, but not in human red cells, Na-H exchange mediates the volume regulatory increase following hypertonic shrinkage and regulates cell pH.
Na-H exchange is functionally up-regulated in one form of genetically engineered murine spherocytosis, which lacks the cytoskeletal protein 4.1R, where it probably plays a role in determining the abnormally elevated cell Na content of the red cells [59]. 4.1R has been shown to bind and regulate NHE1 [60].
Na-Mg exchanger — The sodium-magnesium exchanger (or antiporter) plays a crucial role in controlling red cell Mg content by exporting Mg from the erythrocyte in exchange for Na [61]. This exchanger may be involved in generating the abnormally low Mg content observed in sickle erythrocytes [62]. The human gene SCL41A1 has been shown to encode for the Na-Mg exchanger [63].
Anion exchanger (band 3) — The protein mediating anion exchange (band 3) in human red cells is encoded by the AE1 gene on human chromosome 17 [64]. The 95 kDa human anion exchanger-1 protein (AE1) has two main structural and functional domains: the N-terminal cytoplasmic domain, involved in the interaction with the complex ankyrin, band 4.1, and band 4.2 and with hemoglobin and glycolytic enzymes; and the C-terminal membrane-spanning domain involved in ion transport. Each human red cell contains approximately 1.2 million copies of AE1 per cell.
The AE1 anion exchanger, which primarily functions as a chloride-bicarbonate exchanger, provides the basis for the high permeability of the red cells to chloride and bicarbonate ions. Its physiological function to facilitate CO2 transport from tissues to alveoli may be related to its ability to bind deoxyhemoglobin, but not oxyhemoglobin, with high affinity [65]. This interaction mediates the switch in glucose metabolism between the pentose phosphate and the glycolytic pathways, as well as ATP release from the red cell [66]. It also plays an important role in defining red cell shape and membrane stability, as shown by the association of genetic defects in band 3 with hereditary spherocytosis and hereditary stomatocytosis [67-69]. A comprehensive review of band 3 mutations in erythroid and other tissues is available [70]. (See "Red blood cell membrane: Structure, organization, and dynamics", section on 'Band 3'.)
Although absence of band 3 was thought to be incompatible with life, studies using targeted mutagenesis in mice suggest that complete band 3 deficiency is associated with chronic hemolytic anemia, and increased perinatal death rates [71]. The absence of band 3 was associated with normal cytoskeletal assembly but the mechanical stability of the red cell was greatly impaired. Band 3 also plays an important role in regulating mitosis in erythropoietic cells, and may be involved in dyserythropoietic disorders [72].
Homozygous mutations in band 3 have been generally believed to be lethal, due to extreme erythrocyte cytoskeletal instability and associated severe hemolytic anemia. However, three homozygous human band 3 mutations have been described, all in association with severe hemolysis [70]:
●Band 3 Coimbra (Val488Met), a variant associated with complete absence of band 3 in erythrocytes. This mutation was found in a newborn with severe hemolysis, hydrops, distal renal tubular acidosis (RTA), and nephrocalcinosis, who required chronic blood transfusion and daily bicarbonate [73,74]. Splenectomy resulted in transfusion independence for nine years, with regular transfusions plus iron chelation restarted at age 12. At age 19, the distal RTA was well controlled with normal renal function [75].
●Band 3 Neapolis, a T-to-C substitution at the +2 position in the donor splice site of intron 2, resulting in an 88 percent reduction of erythroid band 3, resulting in transfusion-dependent severe hemolytic anemia which markedly improved after splenectomy [76].
●Band 3 nullVIENNA (ser477X), a nonsense mutation that abolishes band 3 expression, associated with transfusion-dependent hemolysis, dyserythropoiesis, and complete distal RTA [75].
Calcium-activated potassium channel (Gardos channel) — The calcium-activated potassium channel of red cells (KCNN4) is also called the "Gardos" pathway since Dr. George Gardos was the first to report the effect of calcium on potassium permeability of human red cells [77]. The Gardos channel is activated by micromolar concentrations of calcium (k50 = 0.3 to 2 micromol/L). Each human red cell carries approximately 120 ± 36 channels per cell [78]. The Gardos channel is inhibited by charybdotoxin, clotrimazole, other imidazole antimycotics, and senicapoc [79-81].
The molecular structure of the Gardos channel has been determined. This channel is the prototype of the intermediate conductance calcium-gated potassium channels (IK channels) and is now designated as KCNN4 [82-84]. No other type of potassium channel has been demonstrated in human erythrocytes.
In addition to its role in sickle cell anemia, the Gardos channel appears to play an important role in protecting from lysis erythrocytes affected by cytoskeletal abnormalities, compensating for their reduced surface area/volume ratio [85].
Role in sickle erythrocytes — The Gardos channel, as well as the K-Cl cotransporter mentioned above, plays a major role in the dehydration of sickle red cells [47]. Deoxygenation and sickling increase calcium permeability, resulting in activation of the Gardos channel, potassium loss, and dehydration [86-88]. In vitro studies suggest that the majority of irreversibly sickled cells, which contribute to vaso-occlusion, are markedly dehydrated (hyperdense), a process that seems more dependent upon the Gardos channel [47,51].
The observation that senicapoc is a specific inhibitor of the Gardos channel has provided the opportunity for testing the physiologic and pathophysiologic role of this pathway in vivo [80,81,89]. The potential clinical efficacy of this approach is discussed separately.
Modulation of the Gardos channel by arginine and potentially by nitric oxide (NO) has been demonstrated in transgenic sickle cell mice [90]. Chemokines and endothelins have also been shown to activate the Gardos channel in vitro and may play a role in the dehydration of sickle erythrocytes in vivo [91,92].
Role in dehydrated stomatocytosis — Mutations in the Gardos channel have been identified in several families affected by dehydrated stomatocytosis. Increased sensitivity of these mutated channels to calcium leads to pronounced dehydration of the red cells and hemolysis [93-95]. (See "Hereditary stomatocytosis (HSt) and hereditary xerocytosis (HX)".)
Voltage-gated channel — When the membrane potential of human red cells becomes inside-positive, a voltage-activated movement of sodium, potassium, and calcium can be demonstrated [96,97]. This voltage-activated, nonselective cation movement may take place via the Na+-K+-ATPase pump, since it can be demonstrated in proteoliposomes containing reconstituted shark ATPase [98].
Water channels — Water movement passively follows that of cations and anions or is induced by changes in tonicity of the environment surrounding the red cells. The transport of water across the red cell membrane can occur at a much faster rate via water channels. The red cell integral membrane protein CHIP28 has been shown to mediate water transport [99]. This protein is now called aquaporin-1 and is the prototype of the aquaporin family of water transporters (figure 1) [3]. The Colton blood group antigen polymorphism is a single amino acid substitution in an extracellular domain of aquaporin-1 [100]. (See "Red blood cell antigens and antibodies", section on 'Colton blood group system'.)
A few families have been identified in which members are deficient in aquaporin-1 and are Colton null [101,102]. Surprisingly, affected patients are clinically normal except for a modest reduction in urinary concentrating ability [102].
Cation channels — Transient receptor potential channels of canonical type (TRPC) 6 are present in human erythrocytes [103]. These channels mediate movement of cations such as Na and K and are responsible for the leak of Ca into the red cell. They may become active in the terminal phase of the erythrocyte lifetime and provide the pathway for entry of Ca and initiation of eryptosis (apoptosis-like death of erythrocytes) [104].
Mechanosensitive channels — Piezo proteins are the pore-forming subunits of channels that are capable of generating electrical currents in response to mechanical (eg, tension) stimuli, suggesting that these proteins play an important role in maintaining red cell volume homeostasis [105]. Mutations in the piezo1 gene have been shown to be associated with hereditary xerocytosis and/or dehydrated stomatocytosis [106,107]. At present, the physiological function of piezo1 in human red cells is not known. Studies in sickle erythrocytes have shown that the cation permeability associated with sickling (Psickle) is mediated by piezo1 [108].
Red cell aging, ion transport, and content — Red cell aging is associated with a progressive increase in cell density due to loss of K+ and water. However, in the terminal phase of the red cell lifespan, dense erythrocytes gain a substantial amount of Na+ and swell. Using density gradient centrifugation, they can be found in the lightest fraction, together with the reticulocytes. This stage most likely represents a terminal phase immediately preceding the removal of both normal and sickle erythrocytes from the circulation [109,110].
SUMMARY — The water, cation, and anion contents of red cells are continuously regulated by the activity and interactions of several ion transport systems (figure 1). The importance of these systems is indicated by the various diseases occurring when red cell ion transport is impaired (table 1). The following systems are involved:
●Water content – Water can move across the red cell membrane by passive diffusion and also via the specific water channel aquaporin-1. These allow the red cell to be in osmotic equilibrium with the plasma. As a result, the water content of the human red cell is determined by the intracellular sodium, potassium, and Cl content and the osmolality of the plasma. (See 'Water content' above and 'Water channels' above.)
●Cation content – The low sodium, low calcium, and high potassium milieu of the red cell is maintained by the energy-driven Na-K-ATPase and Ca-ATPase pumps, and by the passive gradient-driven Na-K-Cl transport, K-Cl cotransport, Na-H exchanger, Na-Mg exchanger, Ca-activated potassium (Gardos) channels and possibly the mechanosensitive Piezo-1 channel.
●Anion content – The AE1 anion exchanger (band 3) primarily functions as a chloride-bicarbonate exchanger. Its physiological function to facilitate CO2 transport from tissues to alveoli may be related to its ability to bind deoxyhemoglobin, but not oxyhemoglobin, with high affinity. (See 'Anion content' above and 'Anion exchanger (band 3)' above.)
ACKNOWLEDGMENT — We are saddened by the death of Stanley L Schrier, MD, who passed away in August 2019. The editors at UpToDate gratefully acknowledge Dr. Schrier's role as Section Editor on this topic, his tenure as the founding Editor-in-Chief for UpToDate in Hematology, and his dedicated and longstanding involvement with the UpToDate program.
54 : Oral magnesium supplements reduce erythrocyte dehydration in patients with sickle cell disease.
72 : Cell-specific mitotic defect and dyserythropoiesis associated with erythroid band 3 deficiency.
