Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC194
Deoxygenation-induced Mg2+ currents in red blood cells from sickle cell patients
Y. Ma1, S. Dalibalta1, S. Morris1, D. C. Rees2, J. S. Gibson3, C. Ellory1
1. Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom. 2. Molecular Haematology, King's College Hospital, London, United Kingdom. 3. Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
Sickle cell disease (SCD) patients show chronic anaemia and acute ischaemic episodes. Their red blood cells (RBCs) have increased ion permeability, particularly when deoxygenated (1). This abnormality contributes to pathogenesis by causing solute loss and RBC shrinkage. Consequent elevation of [HbS] markedly encourages its polymerisation, RBC sickling and altered rheology. RBCs show high [Ca2+]i and reduced [Mg2+]i (1). Mg2+ depletion may increase activity of the KCl cotransporter which mediates coupled KCl efflux. Abnormal divalent cation permeability of RBCs is therefore important in pathogenesis. Deoxygenation is also associated with activation of an as yet unidentified cation channel (2), Psickle, permeable to Ca2+ and Mg2+ and which may participate in perturbation of these cations. Our current work uses electrophysiological methods to characterise RBC permeability to these ions. Routine discarded RBC samples were obtained from SCD patients (HbSS) using EDTA as anticoagulant. RBCs were washed in saline comprising (in mM): NaCl 145; MOPS 10 and glucose 5, pH 7.4. Membrane conductance was ascertained using whole-cell patch clamp, as described previously (3), in fully oxygenated (O2 20%) or deoxygenated (O2 <1%) conditions. Pipette and bath solutions contained Mg2+ as the predominant cation (82mM). Flow perfusion was used to alter bath solutions and oxygenation. In some experiments, Gd3+ or streptomycin, both promiscuous stretch-activated channel blockers, were added to the bath. In symmetrical Mg2+ solutions (Fig 1), RBCs showed considerable Mg2+ conductance which increased on deoxygenation: at -90mV, currents were -79.3±23.0 and -304.2±68.8pA (n=10) at high and low O2, respectively (p<0.05). Currents were similar in magnitude to those recorded in symmetrical Na+ or K+ solutions. They were markedly inhibited by Gd3+ and much reduced by streptomycin (Fig 1): at -90mV, with Gd3+ (50µM) the current fell to -35.2±15.9pA (n=4) and with streptomycin (100µM) the current was -92.4±32.42.9pA (n=5), both p<0.05 cf no inhibitor. Similar results were obtained in Ca2+ solutions. Findings indicate elevated permeability of sickle cells to Mg2+ and Ca2+. Currents appear to be the electrical manifestation of Psickle via stretch-activated channels, with elevated activity following membrane distortion by deoxygenation-induced HbS polymers. These electrical pathways could be involved in the altered Mg2+ and Ca2+ homeostasis observed in RBCs from SCD patients. Future work will be aimed at their further characterisation, mechanism of activation and identification of potential inhibitors amenable to clinical use.
Where applicable, experiments conform with Society ethical requirements