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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 increases whole cell currents in human and SAD mouse sickle red cells.A. Capacitance-normalized whole cell currents in nystatin-permeabilized patches on intact SAD mouse red cells recorded first in room air (oxy) and then in deoxygenated conditions (deoxy; *, p = 0.016, Wilcoxon; n = 7). B. Capacitance-normalized currents in nystatin-permeabilized patches on intact human SS cells recorded first in room air (oxy) and then in deoxygenated conditions (deoxy; *, p = 0.031, Wilcoxon; n = 6). In both cell types, symmetric pipette and bath solutions contained 150 mM Na methanesulfonate. Values are means ± s.e.m.
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pone-0008732-g007: Deoxygenation increases whole cell currents in human and SAD mouse sickle red cells.A. Capacitance-normalized whole cell currents in nystatin-permeabilized patches on intact SAD mouse red cells recorded first in room air (oxy) and then in deoxygenated conditions (deoxy; *, p = 0.016, Wilcoxon; n = 7). B. Capacitance-normalized currents in nystatin-permeabilized patches on intact human SS cells recorded first in room air (oxy) and then in deoxygenated conditions (deoxy; *, p = 0.031, Wilcoxon; n = 6). In both cell types, symmetric pipette and bath solutions contained 150 mM Na methanesulfonate. Values are means ± s.e.m.

Mentions: Browning et al. showed that deoxygenation-activated currents recorded from human SS cells in conventional whole cell configuration were larger than currents recorded from different SS cells in room air [16]. To extend our on-cell patch results, we examined whole cell cation currents using the nystatin-permeabilized patch configuration in individual cells monitored sequentially in room air followed by deoxygenation, with symmetric Na methanesulfonate in the pipette and the bath. SAD red cells (Figure 7A) increased inward current at −100 mV holding potential from −31±9 nA in room air to −173±82 nA after deoxygenation (n = 7, p = 0.016). Human SS cells (Figure 7B) increased inward current at −100 mV from −48±22 nA in room air to −145±48 nA after deoxygenation (n = 6 p = 0.031). In both cell types, deoxygenation-induced currents were ohmic.


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 increases whole cell currents in human and SAD mouse sickle red cells.A. Capacitance-normalized whole cell currents in nystatin-permeabilized patches on intact SAD mouse red cells recorded first in room air (oxy) and then in deoxygenated conditions (deoxy; *, p = 0.016, Wilcoxon; n = 7). B. Capacitance-normalized currents in nystatin-permeabilized patches on intact human SS cells recorded first in room air (oxy) and then in deoxygenated conditions (deoxy; *, p = 0.031, Wilcoxon; n = 6). In both cell types, symmetric pipette and bath solutions contained 150 mM Na methanesulfonate. Values are means ± s.e.m.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2806905&req=5

pone-0008732-g007: Deoxygenation increases whole cell currents in human and SAD mouse sickle red cells.A. Capacitance-normalized whole cell currents in nystatin-permeabilized patches on intact SAD mouse red cells recorded first in room air (oxy) and then in deoxygenated conditions (deoxy; *, p = 0.016, Wilcoxon; n = 7). B. Capacitance-normalized currents in nystatin-permeabilized patches on intact human SS cells recorded first in room air (oxy) and then in deoxygenated conditions (deoxy; *, p = 0.031, Wilcoxon; n = 6). In both cell types, symmetric pipette and bath solutions contained 150 mM Na methanesulfonate. Values are means ± s.e.m.
Mentions: Browning et al. showed that deoxygenation-activated currents recorded from human SS cells in conventional whole cell configuration were larger than currents recorded from different SS cells in room air [16]. To extend our on-cell patch results, we examined whole cell cation currents using the nystatin-permeabilized patch configuration in individual cells monitored sequentially in room air followed by deoxygenation, with symmetric Na methanesulfonate in the pipette and the bath. SAD red cells (Figure 7A) increased inward current at −100 mV holding potential from −31±9 nA in room air to −173±82 nA after deoxygenation (n = 7, p = 0.016). Human SS cells (Figure 7B) increased inward current at −100 mV from −48±22 nA in room air to −145±48 nA after deoxygenation (n = 6 p = 0.031). In both cell types, deoxygenation-induced currents were ohmic.

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