Storage lesion of red blood cells (RBCs) is a well-recognized process characterized by complex morphological and functional changes.1–3 Those changes deteriorate the life-saving quality of stored blood with a reported increase in mortality for some categories of patients receiving “old” versus “new” blood.4 The importance of RBC cation gradients (K+ and Na+) dissipation in the process of storage lesion has been recently highlighted.2 Here we report a previously unrecognized nonselective cation channel in human RBCs (patch-clamp) activated whenever extracellular Ca2+ is removed and very likely contributing to the cation gradients dissipation when opened. In view of the existence of such a channel the use of non-Ca2+-chelating anticoagulants like heparin, preventing channel opening, can reduce cation gradients dissipation and help limit and delay RBCs storage lesion.
Blood was obtained from healthy donors after giving an informed consent in compliance with the ethical requirements of all involved institutions. Further details on the experimental methods are provided in the supplemental material (Supplemental Digital Content, https://links.lww.com/HS/A17).
Experiments were performed with a chip-based planar patch-clamp setup (NPC-16 Patchliner, Nanion Technologies, Munich, Germany). Using a Cs+-based internal solution and a tetraethylammonium chloride (TEACl)-based external solution, application of 2 mM CaCl2 in the external solution, followed by a CaCl2-free external solution, leads to an increase in the membrane conductance (Fig. 1A). The I/V curve of the Ca2+-blocked current (Fig. 1B) showing a significant outward part and a negative reversal potential, was largely compatible with the blocked current being an outward cation current (carried by Cs+ as the predominant ion in the internal solution). However, a contribution by an inward anion conductance could not be excluded. Both DIDS-sensitive and DIDS-insensitive Cl− currents were reported in human RBCs.5 Thus to examine the probability that the Ca2+-blocked current might possibly be carried by anions we substituted Cl− in the external solution with impermeant anions. The Ca2+ block persisted (Fig. 1C and D) pointing to the cationic nature of the blocked current.
It should be noted that due to the high ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) in the internal solution, all the above measurements were done in the absence of intracellular Ca2+-dependent functions, including the absence of possible Ca2+-activated Cl− channels similar to those in mice.6
We further characterized the Calcium-inhibited Cation Channel (CiCC) and examined if the current was blocked by Ca2+ also in physiological conditions (Fig. 2). Using a K+-based internal and a Na+-based external solutions, we applied successively 2 mM CaCl2, 0 mM CaCl2, 2 mM CaCl2, and 20 mM CaCl2-external solutions. Similar to the experiments with the Cs+- and TEACl-based solutions, the current increased after changing from a 2 mM CaCl2 to a 0 mM CaCl2 external solution (Fig. 2A–C). However, the current failed to decrease significantly when the 0 mM CaCl2 solution was changed again to a 2 mM CaCl2 solution (Fig. 2A–C). It was only after the 2 mM CaCl2 was replaced by a 20 mM CaCl2-external solution that the current significantly decreased (Fig. 2A–C).
The I/V curves for the Ca2+-blocked current in Cs+- and tetraethylammonium (TEA)-based solutions (Fig. 1B and D) and in physiological solutions (Fig. 2D) revealed that the Ca2+-blocked channel is a voltage-independent nonselective cation channel conducting Cs+ as well as Na+ and K+. It has a slight preference for K+ over Na+ as in physiological solutions the I/V curve of the blocked current, rather than crossing the x-axis at 0 mV, is shifted to potentials closer to the K+ reversal potential and the outward current carried by K+ is bigger than the inward current carried by Na+.
Knowing the role of divalent cations and especially Ca2+ in the formation and maintenance of gigaseals, the observed increase in conductance after changing a Ca2+ containing with a Ca2+-free external solution, could be mistaken for a worsening of the gigaseal and the appearance of leak currents. This might explain why the channel was not reported so far.
A channel blocked by extracellular Ca2+ is not without a precedent. Nonselective monovalent cation currents blocked by extracellular Ca2+ have been described in epithelial cells of frog skin7 and toad urinary bladder7 as well as in chicken and rabbit intestine8 with a suggested function in volume homeostasis.7 Trying to speculate on the identity of the reported channel made us consider the Piezo channels. Piezo1 has been reported in RBCs9 and has an important role in volume regulation with mutations of the channel causative for hereditary xerocytosis characterized by decreased RBC cation content and cell dehydration.10 However, the mechanical (stretch- and pressure-induced) activation of the channel11 and its recently discovered voltage sensitivity12 argue against such an assumption.
Going beyond electrophysiological experiments we were able to give further evidence for the existence of the CiCC by detecting cation gradients dissipation in RBCs of blood anticoagulated with a Ca2+-chelating anticoagulant (ethylenediaminetetraacetic acid [EDTA]) versus a non-Ca2+-chelating anticoagulant (heparin) (Fig. 3). We observed a decrease in the Na+ plasma concentration (Fig. 3B) and an increase in the K+ plasma concentration (Fig. 3C) in freshly drawn blood from healthy donors collected in EDTA versus heparin vacutainers consistent with the existence of a Ca2+-blocked nonselective cation channel which, when opened in the presence of a Ca2+ chelator, generates/contributes to the observed ion imbalance. Furthermore, the estimated flux of monovalent cations trough the channel which is 4.3 mM/min (details of calculation to be found in Supplemental Material, Supplemental Digital Content, https://links.lww.com/HS/A17) is in full agreement, both in time and magnitude, with the cation imbalance measured by us minutes after blood withdrawal (Fig. 3).
A channel in the membrane of RBCs being opened or unblocked by the removal of Ca2+ may have significant consequences. Blood for transfusion and for a variety of laboratory tests is all supplied with anticoagulants. The most commonly used anticoagulants at present are heparin, the tripotassium or trisodium salts of EDTA, and trisodium citrate (or the citrate-based citrate phosphate dextrose-adenine [CPDA]).13 While heparin interferes with coagulation by forming a complex with antithrombin III in the plasma to prevent thrombin formation, CPDA and especially EDTA act by chelating Ca2+ which is essential for the initiation and advancing of coagulation.13 Ca2+ removal reduces proteolysis and is further beneficial as it is believed to help suppress Gardos channel activity and maintain RBCs hydration as well as for preventing ATP consumption by the Ca2+ ATPase and calpain activation in stored cells. Taking into consideration CiCC, however, the benefits of Ca2+ removal should be reconsidered.
RBCs dissipation of ion gradients and increases in osmotic fragility, decreases in glucose and increases in lactate levels, and decreases in pH over time occurring in stored blood1 are only a small part of the detrimental RBCs storage lesion.1–3 Although there is a wide, evidence-reinforced, consensus that during storage ATP is decreased and reactive oxygen species are increased with a consequent RBC membrane oxidation and disruption of the cytoskeleton, aggregation of band 3 and release of vesicles,2 the sequence of changes and a possible trigger of the storage lesion are not quite clear. The cation leak, especially that present immediately,2 supported also by our results, could be a generator and an important contributor to various aspects of storage lesion such as early morphological changes, increases in scrambling and phosphatidylserine exposure; vesiculation and oxidation.2 In RBCs collected or stored in solutions supplemented with Ca2+ chelators, the cation gradient dissipation may be helped and enhanced by the opening of the CiCC. In view of the importance of the reduction of the cation leak for delaying and limiting storage lesion, we consider essential to draw the attention to CiCC. Although our description is limited to functional evidence and is lacking a molecular identification of the channel, the consequences from its existence could be easily prevented by control of extracellular Ca2+.
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