Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, PC178

Poster Communications

Evidence for a streptomycin-sensitive transport pathway in human sickle erythrocytes

S. Dalibalta1, J. A. Browning1, J. S. Gibson2, D. Rees3, C. Ellory1

1. Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom. 2. Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom. 3. Molecular Haematology, King's College London School of Medicine, London, United Kingdom.

  • Figure 1. Representative data showing percentage of lysed deoxygenated HbS cells in 60 min with inhibition by streptomycin

  • Figure 2. Representative I-V current measurement in HbS cells, before and after addition of streptomycin

HbS erythrocytes from individuals with sickle cell disease (SCD) have altered membrane transport compared to HbA erythrocytes from healthy individuals (Brugnara, 2004). In particular, HbS cells show increased permeability to cations upon deoxygenation. This pathway, termed Psickle, contributes to erythrocyte shrinkage (Tosteson et al.,1952), accelerating sickling. The identity of Psickle is elusive. We have demonstrated electrophysiologically that whole-cell conductance of HbS cells was higher than in HbA cells, and potentiated by deoxygenation. This pathway shares properties with Psickle (Browning et al., 2007). Deoxygenated HbS cells, unlike HbA cells, are permeable to certain organic osmolytes, including sucrose, and this pathway is inhibited by dipyrimadole and DIDS (Ellory et al., 2008). Here we show that streptomycin, an inhibitor of stretch-activated channels (Shen et al., 2003), inhibits both these pathways. HbS and HbA cells were collected from volunteers with full ethical consent. Lysis experiments were performed at 37oC (Ellory et al., 2008). Briefly, streptomycin (0.1 to 10 mM) was added to erythrocytes (haematocrit 4%) suspended in buffered sucrose solution (300 mM) and the suspension maintained deoxygenated for 60 min. Lysis was determined by measuring haemoglobin release optically. Erythrocyte membrane currents were recorded at room temperature (Browning et al., 2007), using conventional whole-cell patch clamp techniques. I-V relationships were obtained by applying a series of 300 ms test potentials from -80 to +80 mV in 10 mV increments from a holding potential of 0 mV. Currents were analysed by averaging the current values of the final 50 ms evoked by each test potential. In control experiments, about 25% of HbS cells lysed after 60 min in sucrose. About 60% of lysis was inhibited by streptomycin, IC50 0.13 ± 0.06 mM (n = 3) (Figure 1). Whole-cell current measurements were compared between HbA and HbS cells using Na+-containing bath and pipette solutions. A representative I-V curve is shown for HbS cells before and after the addition of 50 µM streptomycin (Figure 2). HbS cells demonstrated considerably larger whole-cell currents (p < 0.05) than HbA cells, although both had a reversal potential of approximately 0 mV. Application of streptomycin (50 µM) to the bath inhibited 74% of membrane current at +80 mV in HbS cells, compared to only 24% in HbA cells (not shown). Results show that streptomycin can inhibit an electrophysiological conductance, thought to be Psickle, in addition to a selective permeability pathway in HbS cells. We hypothesise that this transport pathway may represent Psickle. Further studies can help elucidate the molecular identity of Psickle, which can have important therapeutic implications for SCD patients.

Where applicable, experiments conform with Society ethical requirements