title: The conductance of red blood cells from sickle cell patients: ion selectivity and inhibitors.
abstract: The abnormally high cation permeability in red blood cells (RBCs) from patients with sickle cell disease (SCD) occupies a central role in pathogenesis. Sickle RBC properties are notably heterogeneous, however, thus limiting conventional flux techniques that necessarily average out the behaviour of millions of cells. Here we use the whole-cell patch configuration to characterise the permeability of single RBCs from patients with SCD in more detail. A non-specific cation conductance was reversibly induced upon deoxygenation and was permeable to both univalent (Na+, K+, Rb+) and also divalent (Ca2+, Mg2+) cations. It was sensitive to the tarantula spider toxin GsMTx-4. Mn2+ caused partial, reversible inhibition. The aromatic aldehyde o-vanillin also irreversibly inhibited the deoxygenation-induced conductance, partially at 1mM and almost completely at 5mM. Nifedipine, amiloride and ethylisopropylamiloride were ineffective. In oxygenated RBCs, the current was pH sensitive showing a marked increase as pH fell from 7.4 to 6, with no change apparent when pH was raised from 7.4 to 8. The effects of acidification and deoxygenation together were not additive. Many features of this deoxygenation-induced conductance (non-specificity for cations, permeability toCa2+ andMg2+, pH sensitivity, reversibility, partial inhibition by DIDS and Mn2+) are shared with the flux pathway sometimes referred to as Psickle. Sensitivity to GsMTx-4 indicates its possible identity as a stretch-activated channel. Sensitivity to o-vanillin implies that activation requires HbS polymerisation but since the conductance was observed in whole-cell patches, results suggest that bulk intracellular Hb is not involved; rather a membrane-bound subfraction is responsible for channel activation. The ability to record P(sickle)-like activity in single RBCs will facilitate further studies and eventual molecular identification of the pathway involved.
authors.affiliation: Department of Physiology, Anatomy & Genetics, Oxford, UK.
authors.forename: Y-L
authors.initials: YL
authors.lastname: Ma
authors.forename: D C
authors.initials: DC
authors.lastname: Rees
authors.forename: J S
authors.initials: JS
authors.lastname: Gibson
authors.forename: J C
authors.initials: JC
authors.lastname: Ellory
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000755:Anemia, Sickle Cell / Q000097:blood*; D001547:Benzaldehydes / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004553:Electric Conductivity; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D006451:Hemoglobin, Sickle / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D036341:Intercellular Signaling Peptides and Proteins; D017136:Ion Transport; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008564:Membrane Potentials; D049990:Membrane Transport Modulators / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D055503:Protein Multimerization; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D013111:Spider Venoms / Q000494:pharmacology; D013997:Time Factors
chemical_list: D001547:Benzaldehydes; D006451:Hemoglobin, Sickle; D036341:Intercellular Signaling Peptides and Proteins; C411249:MTx4 protein, Grammostola spatulata; D049990:Membrane Transport Modulators; D010455:Peptides; D013111:Spider Venoms; C060843:2-vanillin; D008345:Manganese; D012964:Sodium; D008274:Magnesium; D012413:Rubidium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D011188:Potassium; D010100:Oxygen; D002118:Calcium