The effect of deoxygenation on whole-cell conductance of red blood cells from healthy individuals and patients with sickle cell disease

Abstract

Red blood cells from patients with sickle cell disease (SCD) exhibit increased electrogenic cation permeability, particularly following deoxygenation and hemoglobin (Hb) polymerisation. This cation permeability, termed P sickle , contributes to cellular dehydration and sickling, and its inhibition remains a major goal for SCD treatment. Nevertheless, its characteristics remain poorly defined, its molecular identity is unknown, and effective inhibitors have not been established. Here, patch-clamp methodology was used to record whole-cell currents in single red blood cells from healthy individuals and patients with SCD. Oxygenated normal red blood cells had a low membrane conductance, unaffected by deoxygenation. Oxygenated HbS cells had significantly increased conductance and, on deoxygenation, showed a further rise in membrane conductance. The deoxygenation-induced pathway was variable in magnitude. It had equal permeability to Na ؉ and K ؉ , but was less permeable to NMDG ؉ and Cl ؊ . Conductance to Ca 2؉ was also of a similar magnitude to that of monovalent cations. It was inhibited by DIDS (100 M), Zn 2؉ (100 M), and by Gd 3؉ (IC 50 IntroductionSickle cell disease (SCD) is caused by the presence in red blood cells of mutant hemoglobin, HbS (HbS-containing red blood cells are here called HbS cells, whereas normal HbA-containing red blood cells are called HbA cells). The reduced lifespan of HbS cells contributes to the prevailing anemia which characterizes the disease. 1 Furthermore, deoxygenated HbS polymerizes, distorting the red blood cell shape into a variety of elaborate patterns, including the eponymous sickle. Sickled cells participate in vascular occlusion and associated sequelae, including ischemia, organ dysfunction, pain, and, ultimately, death. 1 Although the molecular nature of the Hb defect underlying SCD is well established, 2,3 details of the pathophysiology are uncertain, and treatment remains largely supportive. 4 Dehydration of HbS cells, and particularly of certain HbS cell subpopulations, 5 markedly promotes polymerization by reducing the lag time to polymer formation. 6 Several membrane transport pathways promote solute loss, but a deoxygenation-induced cation permeability, called P sickle , is pre-eminent (reviewed by Joiner, 7 Gibson, 8 and Lew 9 ). P sickle increases cell membrane permeability to Ca 2ϩ (as well as to monovalent cations), thereby elevating cytosolic [Ca 2ϩ ]. Subsequent activation of the Ca 2ϩ -activated K ϩ channel (also known as the Gardos channel) mediates particularly rapid K ϩ loss, with Cl Ϫ following via separate pathways. 10 Inhibition of P sickle , which will reduce the propensity of cells to shrink, represents an immediate goal for SCD therapy. 7,9,11 Permeability studies on P sickle date from the seminal work of Tosteson and colleagues. 12 Subsequent radioactive tracer studies indicate that P sickle behaves like a conductive cation channel, lacking selectivity between alkali cations (including Na ϩ and K ϩ ), with moderate permeability to Ca 2ϩ and Mg 2ϩ , 1...

Mutations in the mechanotransduction protein PIEZO1 are associated with hereditary xerocytosis

“…42 Increased calcium levels mediated by P sickle lead to activation of the Gardos channel, a critical step in the dehydration of sickle erythrocytes. [43][44][45][46] A recent study shows similarities between erythrocyte nonspecific cation conductance pathways and P sickle and demonstrate sensitivity of the pathway to GsMTx4, indicating properties of a mechanosensitive ion channel. 47 PIEZO1 may serve as a sensor for cell swelling, as GsMTx4 blockade inhibits both mechanically activated currents and whole-cell regulatory volume decrease in NRK-49 cells.…”

In vitro Inhibitory Effects of Disodium Cromoglycate on Ionic Transports Involved in Sickle Cell Dehydration

“…[22][23][24] . It is well known that deoxygenation of RBC from SCD patients causes morphological sickling that in turn increases the membrane permeability, referred to as P sickle [6,7,9,13,25] , to monovalent (Na ) cations, with a secondary K + leak, this loss exceeding Na + gain. The K + loss causes cell dehydration, which in turn aggravates Hb S polymerization and sickling [8,[12][13][14] .…”

N-methyl-d-aspartate receptors in human erythroid precursor cells and in circulating red blood cells contribute to the intracellular calcium regulation

“…An increase in Ca 2ϩ "leak" and the concomitant elevation of intracellular Ca 2ϩ concentrations are typical for RBCs of patients with phosphofructokinase deficiency (75), thalassemia (7), and sickle cell disease (7,70). The importance of electrogenic Ca 2ϩ uptake pathway (P sickle ) in promoting sickle cell transformation upon deoxygenation has been acknowledged (8,70,72). The molecular identity of the nonselective cation channels, including those mediating P sickle , remains unknown.…”