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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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Related in: MedlinePlus

Deoxygenation reversibly activates a conductance in red cells from SAD sickle mice.A. Representative current trace from an individual cell-attached patch on a SAD sickle mouse erythrocyte before deoxygenation (upper trace, oxy), 1 min post-deoxygenation (middle trace, deoxy), and 4 min post-reoxygenation (lower trace, reoxy); −Vp  = −50 mV. Symmetric pipette and bath solutions contained (in mM) 140 NaCl, 4 KCl, 1CaCl2, 1 MgCl2, 10 Na HEPES, pH 7.4. B. Seal resistance was maintained during deoxygenation in all 9 cells, and during reoxygenation in 6 of the 9 cells (*, p<0.01 vs. oxy; p =  N.S. vs. re-oxy, ANOVA). C. Summary of patches such as in panel A, showing that the increased NPo (product of the number of single channels and the channel open probability) observed after deoxygenation was reversible (without change in seal resistance) upon reoxygenation (*, p<0.02 vs. oxy; p<0.05 vs. reoxy, ANOVA). Values are means ± s.e.m.
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pone-0008732-g001: Deoxygenation reversibly activates a conductance in red cells from SAD sickle mice.A. Representative current trace from an individual cell-attached patch on a SAD sickle mouse erythrocyte before deoxygenation (upper trace, oxy), 1 min post-deoxygenation (middle trace, deoxy), and 4 min post-reoxygenation (lower trace, reoxy); −Vp  = −50 mV. Symmetric pipette and bath solutions contained (in mM) 140 NaCl, 4 KCl, 1CaCl2, 1 MgCl2, 10 Na HEPES, pH 7.4. B. Seal resistance was maintained during deoxygenation in all 9 cells, and during reoxygenation in 6 of the 9 cells (*, p<0.01 vs. oxy; p =  N.S. vs. re-oxy, ANOVA). C. Summary of patches such as in panel A, showing that the increased NPo (product of the number of single channels and the channel open probability) observed after deoxygenation was reversible (without change in seal resistance) upon reoxygenation (*, p<0.02 vs. oxy; p<0.05 vs. reoxy, ANOVA). Values are means ± s.e.m.

Mentions: The on-cell patch records of Figure 1A, with NaCl in both pipette and bath, show that deoxygenation activates noisy channel activity in the SAD sickle mouse red cell membrane. Deoxygenation increased SAD red cell patch NPo from the room air value of 0.01±0.01 to 0.65±0.22 (n = 9). Subsequent reoxygenation decreased nPo to 0.06±0.03 in those patches that survived (n = 6; Figure 1C). Calculated chord conductance was 20 pS (between −Vp = 0 and −50 mV; n = 9). Visual inspection of current traces indicated onset of increased activity at 7.5±3.1 sec after deoxygenation (n = 9; measured from the bath solution change artifact). The nearly complete cessation of channel activity upon re-oxygenation was accompanied by maintenance of a stable giga-ohm seal (Figure 1B). The time to steady-state recovery of quiescence after re-oxygenation was 57±20 sec (n = 6). Thus, the noisy nature of the deoxygenation-induced conductance in on-cell patches did not reflect deoxygenation-induced leak due to loss of the original tight seal.


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 reversibly activates a conductance in red cells from SAD sickle mice.A. Representative current trace from an individual cell-attached patch on a SAD sickle mouse erythrocyte before deoxygenation (upper trace, oxy), 1 min post-deoxygenation (middle trace, deoxy), and 4 min post-reoxygenation (lower trace, reoxy); −Vp  = −50 mV. Symmetric pipette and bath solutions contained (in mM) 140 NaCl, 4 KCl, 1CaCl2, 1 MgCl2, 10 Na HEPES, pH 7.4. B. Seal resistance was maintained during deoxygenation in all 9 cells, and during reoxygenation in 6 of the 9 cells (*, p<0.01 vs. oxy; p =  N.S. vs. re-oxy, ANOVA). C. Summary of patches such as in panel A, showing that the increased NPo (product of the number of single channels and the channel open probability) observed after deoxygenation was reversible (without change in seal resistance) upon reoxygenation (*, p<0.02 vs. oxy; p<0.05 vs. reoxy, ANOVA). Values are means ± s.e.m.
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Related In: Results  -  Collection

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pone-0008732-g001: Deoxygenation reversibly activates a conductance in red cells from SAD sickle mice.A. Representative current trace from an individual cell-attached patch on a SAD sickle mouse erythrocyte before deoxygenation (upper trace, oxy), 1 min post-deoxygenation (middle trace, deoxy), and 4 min post-reoxygenation (lower trace, reoxy); −Vp  = −50 mV. Symmetric pipette and bath solutions contained (in mM) 140 NaCl, 4 KCl, 1CaCl2, 1 MgCl2, 10 Na HEPES, pH 7.4. B. Seal resistance was maintained during deoxygenation in all 9 cells, and during reoxygenation in 6 of the 9 cells (*, p<0.01 vs. oxy; p =  N.S. vs. re-oxy, ANOVA). C. Summary of patches such as in panel A, showing that the increased NPo (product of the number of single channels and the channel open probability) observed after deoxygenation was reversible (without change in seal resistance) upon reoxygenation (*, p<0.02 vs. oxy; p<0.05 vs. reoxy, ANOVA). Values are means ± s.e.m.
Mentions: The on-cell patch records of Figure 1A, with NaCl in both pipette and bath, show that deoxygenation activates noisy channel activity in the SAD sickle mouse red cell membrane. Deoxygenation increased SAD red cell patch NPo from the room air value of 0.01±0.01 to 0.65±0.22 (n = 9). Subsequent reoxygenation decreased nPo to 0.06±0.03 in those patches that survived (n = 6; Figure 1C). Calculated chord conductance was 20 pS (between −Vp = 0 and −50 mV; n = 9). Visual inspection of current traces indicated onset of increased activity at 7.5±3.1 sec after deoxygenation (n = 9; measured from the bath solution change artifact). The nearly complete cessation of channel activity upon re-oxygenation was accompanied by maintenance of a stable giga-ohm seal (Figure 1B). The time to steady-state recovery of quiescence after re-oxygenation was 57±20 sec (n = 6). Thus, the noisy nature of the deoxygenation-induced conductance in on-cell patches did not reflect deoxygenation-induced leak due to loss of the original tight seal.

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