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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 elevates [Ca2+]i in SAD sickle red cells in the absence of Kcnn4/KCa3.1/IK1 “Gardos channel”.Fluo-3-loaded SAD red cells were subjected to deoxygenation at t = 0. Whereas red cells with normal mouse hemoglobin that lacked KCa3.1 [IK1(−/−)] showed no change in [Ca2+]i, SAD red cells lacking KCa3.1 [SAD/IK1(−/−)] responded to deoxygenation with a substantial increase in [Ca2+]i that later fell to a sustained value ∼50% of peak levels. Values are means ± s.e.m. for (n) red cells from two mice studied in two experiments.
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pone-0008732-g008: Deoxygenation elevates [Ca2+]i in SAD sickle red cells in the absence of Kcnn4/KCa3.1/IK1 “Gardos channel”.Fluo-3-loaded SAD red cells were subjected to deoxygenation at t = 0. Whereas red cells with normal mouse hemoglobin that lacked KCa3.1 [IK1(−/−)] showed no change in [Ca2+]i, SAD red cells lacking KCa3.1 [SAD/IK1(−/−)] responded to deoxygenation with a substantial increase in [Ca2+]i that later fell to a sustained value ∼50% of peak levels. Values are means ± s.e.m. for (n) red cells from two mice studied in two experiments.

Mentions: Deoxygenation-induced elevation of [Ca2+]i might require or be sustained by membrane hyperpolarization caused by KCa3.1 activation. Moreover, the elevated Fluo-3 fluorescence intensity suggestive of elevated [Ca2+]i could be influenced by KCa3.1-mediated cell shrinkage of later onset. However, the hypoxia-induced 17% increase in Fluo-3 fluorescence intensity within 2–3 min in human SS red cells (Fig. 4B) is greater than can be simply explained by SS discocyte shrinkage in Cl− medium containing 1 mM Ca2+ during 3 min anoxia at 37°C (Figure 6 in [35]). We nonetheless examined the response to deoxygenation of red cells from SAD/kcnn4−/− mice genetically lacking the Kcnn4/IK1/KCa3.1 K+ channel. The normal MCV reduction elicited in wildtype mouse red cells by exposure to A23187 in Ca2+-containing medium [39] was abolished in red cells from both kcnn4−/− mice [6] and from SAD/kcnn4−/− mice (Shmukler and Alper, data not shown). The absence of KCa3.1 in otherwise normal mouse red cells had no effect on the lack of deoxygenation-sensitive [Ca2+]i elevation. In contrast, SAD/kcnn4−/− red cells elevated [Ca2+]i in response to deoxygenation (Figure 8), as did SAD red cells (Figure 2D) with intact Gardos channel activity [39], [6]. However, shortly after achieving peak [Ca2+]i, SAD/kcnn4−/− red cells exhibited a fall in [Ca2+]i to a value (Figure 8) approximately 50% that of SAD cells (Figure 2D). This lower plateau value may represent reduced driving force for Ca2+ entry through the Psickle-like deoxygenation-induced permeability pathway in the less hyperpolarized state of cells lacking KCa3.1.


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 elevates [Ca2+]i in SAD sickle red cells in the absence of Kcnn4/KCa3.1/IK1 “Gardos channel”.Fluo-3-loaded SAD red cells were subjected to deoxygenation at t = 0. Whereas red cells with normal mouse hemoglobin that lacked KCa3.1 [IK1(−/−)] showed no change in [Ca2+]i, SAD red cells lacking KCa3.1 [SAD/IK1(−/−)] responded to deoxygenation with a substantial increase in [Ca2+]i that later fell to a sustained value ∼50% of peak levels. Values are means ± s.e.m. for (n) red cells from two mice studied in two experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2806905&req=5

pone-0008732-g008: Deoxygenation elevates [Ca2+]i in SAD sickle red cells in the absence of Kcnn4/KCa3.1/IK1 “Gardos channel”.Fluo-3-loaded SAD red cells were subjected to deoxygenation at t = 0. Whereas red cells with normal mouse hemoglobin that lacked KCa3.1 [IK1(−/−)] showed no change in [Ca2+]i, SAD red cells lacking KCa3.1 [SAD/IK1(−/−)] responded to deoxygenation with a substantial increase in [Ca2+]i that later fell to a sustained value ∼50% of peak levels. Values are means ± s.e.m. for (n) red cells from two mice studied in two experiments.
Mentions: Deoxygenation-induced elevation of [Ca2+]i might require or be sustained by membrane hyperpolarization caused by KCa3.1 activation. Moreover, the elevated Fluo-3 fluorescence intensity suggestive of elevated [Ca2+]i could be influenced by KCa3.1-mediated cell shrinkage of later onset. However, the hypoxia-induced 17% increase in Fluo-3 fluorescence intensity within 2–3 min in human SS red cells (Fig. 4B) is greater than can be simply explained by SS discocyte shrinkage in Cl− medium containing 1 mM Ca2+ during 3 min anoxia at 37°C (Figure 6 in [35]). We nonetheless examined the response to deoxygenation of red cells from SAD/kcnn4−/− mice genetically lacking the Kcnn4/IK1/KCa3.1 K+ channel. The normal MCV reduction elicited in wildtype mouse red cells by exposure to A23187 in Ca2+-containing medium [39] was abolished in red cells from both kcnn4−/− mice [6] and from SAD/kcnn4−/− mice (Shmukler and Alper, data not shown). The absence of KCa3.1 in otherwise normal mouse red cells had no effect on the lack of deoxygenation-sensitive [Ca2+]i elevation. In contrast, SAD/kcnn4−/− red cells elevated [Ca2+]i in response to deoxygenation (Figure 8), as did SAD red cells (Figure 2D) with intact Gardos channel activity [39], [6]. However, shortly after achieving peak [Ca2+]i, SAD/kcnn4−/− red cells exhibited a fall in [Ca2+]i to a value (Figure 8) approximately 50% that of SAD cells (Figure 2D). This lower plateau value may represent reduced driving force for Ca2+ entry through the Psickle-like deoxygenation-induced permeability pathway in the less hyperpolarized state of cells lacking KCa3.1.

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