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.

Figure 1. The effect of oxygen tension on whole-cell currents recorded in red blood cells (RBCs) from patients with sickle cell disease (SCD)

Currents were measured in RBCs from homozygous (HbSS) SCD patients at holding potentials −100 to +100 mV, as described in the Methods. The main cations in pipette and extracellular solutions were K⁺ and Na⁺, respectively. Solutions were nominally Ca²⁺ free and any contaminant Ca²⁺ was removed by inclusion of EGTA (2 mm). Measurements were carried out in the same RBC first under oxygenated conditions (20% O₂), then when deoxygenated (O₂ <1%), after which the cell was subsequently reoxygenated (20%). Traces represent means ± SEM for 5 RBCs.

Figure 2. The effect of oxygen tension and inhibitors on whole-cell Ca²⁺ currents recorded in red blood cells (RBCs) from patients with sickle cell disease (SCD)

Currents were measured in RBCs from homozygous (HbSS) SCD patients at holding potentials −100 to +100 mV, as described in the Methods. In this case, the main cation in both pipette and extracellular solutions was Ca²⁺ (82 mm). The absence of K⁺ prevented any current passing through the Gardos channel. Measurements were carried out in the same RBC first under oxygenated conditions (20% O₂), after which the perfusate was switched to deoxygenated saline (O₂ <1%), in the absence and then presence of Gd³⁺ (50 μm) or streptomycin (100 μm). Traces represent means ± SEM for 6 RBCs.

Figure 3. The effect of oxygen tension and inhibitors on whole-cell Mg²⁺ currents recorded in red blood cells (RBCs) from patients with sickle cell disease (SCD)

Currents were measured in RBCs from homozygous (HbSS) SCD patients at holding potentials −100 to +100 mV, as described in the Methods. In this case, the main cation in both pipette and extracellular solutions was Mg²⁺ (82 mm). The absence of K⁺ prevented any current passing through the Gardos channel. Measurements were carried out in the same RBC first under oxygenated conditions (20% O₂), after which the perfusate was switched to deoxygenated saline (O₂ <1%), in the absence and then presence of Gd³⁺ (50 μm) or streptomycin (100 μm). A, voltage clamp protocol; B, single representative experiment showing currents from a single RBC; C and D, means ± SEM for 4–8 RBCs.

Figure 4. The effect of extracellular cation substitution on whole-cell currents recorded in red blood cells (RBCs) from patients with sickle cell disease (SCD)

Currents were measured in RBCs from homozygous (HbSS) SCD patients at holding potentials −100 to +100 mV, as described in the Methods. The main cation in the pipette was K⁺, with contaminant Ca²⁺ removed by inclusion of EGTA (2 mm). The initial extracellular cation was Na⁺. Measurements were carried out in the same RBC first under oxygenated conditions (20% O₂), after which the cell was deoxygenated (O₂ <1%). Na⁺ was then substituted with Rb⁺ whilst maintaining deoxygenated conditions. Traces represent means ± SEM for 5 RBCs.

Figure 5. The effect of tarantula spider toxin (GsMTx-4) on deoxygenation-induced whole-cell currents recorded in red blood cells (RBCs) from patients with sickle cell disease (SCD)

Currents were measured in RBCs from homozygous (HbSS) SCD patients at holding potentials −100 to +100 mV, as described in the Methods. The main cations in pipette and extracellular solutions were K⁺ and Na⁺, respectively. Solutions were nominally Ca²⁺ free and any contaminant Ca²⁺ was removed by inclusion of EGTA (2 mm). Measurements were carried out in the same RBC first under oxygenated conditions (20% O₂), after which the cell was deoxygenated (O₂ <1%), first in the absence and then the presence of GsMTx-4 (1 μm). Traces represent means ± SEM for 5 RBCs.

Figure 6. The effect of Mn²⁺ on deoxygenation-induced whole-cell currents recorded in red blood cells (RBCs) from patients with sickle cell disease (SCD)

Currents were measured in RBCs from homozygous (HbSS) SCD patients at holding potentials −100 to +100 mV, as described in the Methods. The main cations in pipette and extracellular solutions were K⁺ and Na⁺, respectively. Solutions were nominally Ca²⁺ free and any contaminant Ca²⁺ was removed by inclusion of EGTA (2 mm). Measurements were carried out in the same RBC first under oxygenated conditions (20% O₂), after which the cell was deoxygenated (O₂ <1%), first in the absence and then the presence of Mn²⁺ (100 or 200 μm). Traces represent means ± SEM for 3–8 RBCs.

Figure 7. The effect of cation channel blockers on deoxygenation-induced whole-cell currents recorded in red blood cells (RBCs) from patients with sickle cell disease (SCD)

Currents were measured in RBCs from homozygous (HbSS) SCD patients at holding potentials −100 to +100 mV, as described in the Methods. The main cations in pipette and extracellular solutions were K⁺ and Na⁺, respectively. Solutions were nominally Ca²⁺ free and any contaminant Ca²⁺ was removed by inclusion of EGTA (2 mm). Measurements were carried out in the same RBC first under oxygenated conditions (20% O₂), after which the cell was deoxygenated (O₂ <1%), first in the absence and then the presence of inhibitors: A, nifedipine (50 μm), n = 4; B, amiloride (10, 100 and 1000 μm), n = 3–5; C, ethylisopropylamiloride (EIPA; 10, 100 and 1000 μm), n = 4. All traces represent means ± SEM for 3–5 RBCs.

Figure 8. The effect of o-vanillin on deoxygenation-induced whole-cell currents recorded in red blood cells (RBCs) from patients with sickle cell disease (SCD)

Currents were measured in RBCs from homozygous (HbSS) SCD patients at holding potentials −100 to +100 mV, as described in the Methods. The main cations in pipette and extracellular solutions were K⁺ and Na⁺, respectively. Solutions were nominally Ca²⁺ free and any contaminant Ca²⁺ was removed by inclusion of EGTA (2 mm). RBCs were pre-incubated for 30 min in the absence or presence if o-vanillin after which currents were recorded first in oxygenated cells (20% O₂) and then under deoxygenated conditions (O₂ <1%): A, 1 mm and (B) 5 mmo-vanillin. Traces represent means ± SEM for 4 or 5 RBCs.

Figure 9. The effect of pH and oxygen tension on whole whole-cell currents recorded in red blood cells (RBCs) from patients with sickle cell disease (SCD)

Currents were measured in RBCs from homozygous (HbSS) SCD patients at holding potentials −100 to +100 mV, as described in the Methods. The main cations in pipette and extracellular solutions were K⁺ and Na⁺, respectively. Solutions were nominally Ca²⁺ free and any contaminant Ca²⁺ was removed by inclusion of EGTA (2 mm). A, in the same RBC, currents were recorded under oxygenated conditions (20% O₂) sequentially at 3 different extracellular pHs: pH 7.4, 6 and 8.2 (n = 7). B, in the same RBC, currents were recorded first under oxygenated conditions (20% O₂), pH 7.4, after which the cell was deoxygenated (O₂ <1%), and measurements repeated sequentially at pH 7.4, 6.0 and 8.2 (n = 3).