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Hypoxia activates a Ca2+-permeable cation conductance sensitive to carbon monoxide and to GsMTx-4 in human and mouse sickle erythrocytes.

Vandorpe DH, Xu C, Shmukler BE, Otterbein LE, Trudel M, Sachs F, Gottlieb PA, Brugnara C, Alper SL - PLoS ONE (2010)

Bottom Line: Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation.Deoxygenation-induced elevation of [Ca(2+)](i) in mouse sickle erythrocytes did not require KCa3.1 activity.Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Vascular Medicine Unit, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca(2+)] ([Ca(2+)](i)) and subsequent activation of K(Ca) 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration.

Methodology/principal findings: We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca(2+)- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 microM), dipyridamole (100 microM), DIDS (100 microM), and carbon monoxide (25 ppm pretreatment). Deoxygenation also elevates sickle erythrocyte [Ca(2+)](i), in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca(2+)](i) in mouse sickle erythrocytes did not require KCa3.1 activity.

Conclusions/significance: The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca(2+)-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca(2+) for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.

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Deoxygenation activates a Ca2+-permeable conductance in human SS cells.A. Representative current traces from a cell-attached patch on an individual human SS erythrocyte recorded before (oxy) and after onset of deoxygenation (deoxy). Pipette solution contained (in mM) 100 CaCl2, 10 Na HEPES, pH 7.4. Bath solution contained (in mM) 150 Na methanesulfonate, 10 Na HEPES, pH 7.4. Holding potential was −Vp = −25 mV. Open states at right are derived from the open state histogram (not shown). B. Current-voltage relationship derived from the deoxygenated currents measured in the patch of panel A. Mean ± s.e.m. for fit of the amplitude histogram. C. The low NPo of inward single channel activity of human SS cells recorded in the on-cell configuration with Ca2+ in the pipet is increased by deoxygenation (*, P<0.02). The deoxygenation-induced increase in NPo is prevented by inclusion of 1 µM GsMTx-4 in the pipette. Values are means ± s.e.m. (n = 4–5), recorded at −Vp = −25 mV.
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pone-0008732-g005: Deoxygenation activates a Ca2+-permeable conductance in human SS cells.A. Representative current traces from a cell-attached patch on an individual human SS erythrocyte recorded before (oxy) and after onset of deoxygenation (deoxy). Pipette solution contained (in mM) 100 CaCl2, 10 Na HEPES, pH 7.4. Bath solution contained (in mM) 150 Na methanesulfonate, 10 Na HEPES, pH 7.4. Holding potential was −Vp = −25 mV. Open states at right are derived from the open state histogram (not shown). B. Current-voltage relationship derived from the deoxygenated currents measured in the patch of panel A. Mean ± s.e.m. for fit of the amplitude histogram. C. The low NPo of inward single channel activity of human SS cells recorded in the on-cell configuration with Ca2+ in the pipet is increased by deoxygenation (*, P<0.02). The deoxygenation-induced increase in NPo is prevented by inclusion of 1 µM GsMTx-4 in the pipette. Values are means ± s.e.m. (n = 4–5), recorded at −Vp = −25 mV.

Mentions: Ca2+ entry into deoxygenated SS cells via Psickle is believed to be a major trigger of SS cell dehydration via KCa3.1. However, in a previous report [16] deoxygenation did not increase whole cell currents recorded in human SS cells in symmetrical Ca2+ solutions. We therefore sought electrophysiological evidence of deoxygenation-activated Ca2+ permeation in cell-attached patches with CaCl2 in the pipette and Na methanesulfonate in the bath. As shown in Figures 5A and 5C, deoxygenation increased patch conductance. Patch NPo was not different from zero in room air, but increased to 0.90±0.23 upon deoxygenation (P<0.05; n = 5). Mean deoxygenation time prior to activation of conductance was 21±10 sec. The induced current displayed moderate inward rectification in these conditions, with amplitude of 0.64±0.089 pA at −Vp =  −25 mV (n = 5), corresponding to a 25 pS chord conductance. The I-V curve of the Figure 5A patch, with Erev of +7 mV (Figure 5B) was consistent with an inward Ca2+ current and a substantial fraction of outward K+ current through a nonspecific cation conductance. The calculated Erev(Cl−), −18 mV for ∼100 mM [Cl−]i and more negative values as [Cl−]i falls with increasing time in the methanesulfonate bath, suggests that the contribution of Cl− permeability to the observed currents is a minor one.


Hypoxia activates a Ca2+-permeable cation conductance sensitive to carbon monoxide and to GsMTx-4 in human and mouse sickle erythrocytes.

Vandorpe DH, Xu C, Shmukler BE, Otterbein LE, Trudel M, Sachs F, Gottlieb PA, Brugnara C, Alper SL - PLoS ONE (2010)

Deoxygenation activates a Ca2+-permeable conductance in human SS cells.A. Representative current traces from a cell-attached patch on an individual human SS erythrocyte recorded before (oxy) and after onset of deoxygenation (deoxy). Pipette solution contained (in mM) 100 CaCl2, 10 Na HEPES, pH 7.4. Bath solution contained (in mM) 150 Na methanesulfonate, 10 Na HEPES, pH 7.4. Holding potential was −Vp = −25 mV. Open states at right are derived from the open state histogram (not shown). B. Current-voltage relationship derived from the deoxygenated currents measured in the patch of panel A. Mean ± s.e.m. for fit of the amplitude histogram. C. The low NPo of inward single channel activity of human SS cells recorded in the on-cell configuration with Ca2+ in the pipet is increased by deoxygenation (*, P<0.02). The deoxygenation-induced increase in NPo is prevented by inclusion of 1 µM GsMTx-4 in the pipette. Values are means ± s.e.m. (n = 4–5), recorded at −Vp = −25 mV.
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Related In: Results  -  Collection

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pone-0008732-g005: Deoxygenation activates a Ca2+-permeable conductance in human SS cells.A. Representative current traces from a cell-attached patch on an individual human SS erythrocyte recorded before (oxy) and after onset of deoxygenation (deoxy). Pipette solution contained (in mM) 100 CaCl2, 10 Na HEPES, pH 7.4. Bath solution contained (in mM) 150 Na methanesulfonate, 10 Na HEPES, pH 7.4. Holding potential was −Vp = −25 mV. Open states at right are derived from the open state histogram (not shown). B. Current-voltage relationship derived from the deoxygenated currents measured in the patch of panel A. Mean ± s.e.m. for fit of the amplitude histogram. C. The low NPo of inward single channel activity of human SS cells recorded in the on-cell configuration with Ca2+ in the pipet is increased by deoxygenation (*, P<0.02). The deoxygenation-induced increase in NPo is prevented by inclusion of 1 µM GsMTx-4 in the pipette. Values are means ± s.e.m. (n = 4–5), recorded at −Vp = −25 mV.
Mentions: Ca2+ entry into deoxygenated SS cells via Psickle is believed to be a major trigger of SS cell dehydration via KCa3.1. However, in a previous report [16] deoxygenation did not increase whole cell currents recorded in human SS cells in symmetrical Ca2+ solutions. We therefore sought electrophysiological evidence of deoxygenation-activated Ca2+ permeation in cell-attached patches with CaCl2 in the pipette and Na methanesulfonate in the bath. As shown in Figures 5A and 5C, deoxygenation increased patch conductance. Patch NPo was not different from zero in room air, but increased to 0.90±0.23 upon deoxygenation (P<0.05; n = 5). Mean deoxygenation time prior to activation of conductance was 21±10 sec. The induced current displayed moderate inward rectification in these conditions, with amplitude of 0.64±0.089 pA at −Vp =  −25 mV (n = 5), corresponding to a 25 pS chord conductance. The I-V curve of the Figure 5A patch, with Erev of +7 mV (Figure 5B) was consistent with an inward Ca2+ current and a substantial fraction of outward K+ current through a nonspecific cation conductance. The calculated Erev(Cl−), −18 mV for ∼100 mM [Cl−]i and more negative values as [Cl−]i falls with increasing time in the methanesulfonate bath, suggests that the contribution of Cl− permeability to the observed currents is a minor one.

Bottom Line: Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation.Deoxygenation-induced elevation of [Ca(2+)](i) in mouse sickle erythrocytes did not require KCa3.1 activity.Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Vascular Medicine Unit, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca(2+)] ([Ca(2+)](i)) and subsequent activation of K(Ca) 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration.

Methodology/principal findings: We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca(2+)- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 microM), dipyridamole (100 microM), DIDS (100 microM), and carbon monoxide (25 ppm pretreatment). Deoxygenation also elevates sickle erythrocyte [Ca(2+)](i), in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca(2+)](i) in mouse sickle erythrocytes did not require KCa3.1 activity.

Conclusions/significance: The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca(2+)-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca(2+) for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.

Show MeSH
Related in: MedlinePlus