Limits...
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.

Show MeSH

Related in: MedlinePlus

Deoxygenation activates conductance and increases [Ca2+]i in red cells from SAD sickle mice.A. Representative current trace from an individual cell-attached patch on a SAD mouse erythrocyte, recorded first in oxygenated (upper trace, oxy, −Vp = −25 mV) and subsequently in deoxygenated conditions (lower trace, deoxy, −Vp = +75 mV). Symmetrical pipette and bath solutions contained (in mM) 150 Na methanesulfonate, 10 Na EDTA, and 10 Na HEPES, pH 7.4. B. Deoxygenation increased the NPo of 6 cell-attached patches recorded in symmetrical Na methanesulfate (*, p<0.05; n = 6). Substitution of pipette solution Na+ with NMDG blocked the deoxygenation-induced increase in NPo (n = 5). Values are means ± s.e.m, recorded at −Vp = −25 mV. C. Current-voltage relationship in a representative cell-attached patch on a SAD red cell exposed to deoxygenation with symmetrical Na methanesulfonate solutions in pipette and bath. Mean ± s.e.m. for fit of the amplitude histogram. D. Deoxygenation increases [Ca2+]i in SAD red cells but not in WT mouse red cells, in a manner inhibited by 1 µM GsMTx-4 and enhanced by 50 µM vanadate. Values are means ± s.e.m. of Fluo-3 fluorescence increase for (n) red cells from 3 mice studied in 8 experiments (SAD), from 1 mouse studied in 4 experiiments (WT and SAD + GsMTx-4) or from 1 mouse studied in 2 experiments (SAD + vanadate).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2806905&req=5

pone-0008732-g002: Deoxygenation activates conductance and increases [Ca2+]i in red cells from SAD sickle mice.A. Representative current trace from an individual cell-attached patch on a SAD mouse erythrocyte, recorded first in oxygenated (upper trace, oxy, −Vp = −25 mV) and subsequently in deoxygenated conditions (lower trace, deoxy, −Vp = +75 mV). Symmetrical pipette and bath solutions contained (in mM) 150 Na methanesulfonate, 10 Na EDTA, and 10 Na HEPES, pH 7.4. B. Deoxygenation increased the NPo of 6 cell-attached patches recorded in symmetrical Na methanesulfate (*, p<0.05; n = 6). Substitution of pipette solution Na+ with NMDG blocked the deoxygenation-induced increase in NPo (n = 5). Values are means ± s.e.m, recorded at −Vp = −25 mV. C. Current-voltage relationship in a representative cell-attached patch on a SAD red cell exposed to deoxygenation with symmetrical Na methanesulfonate solutions in pipette and bath. Mean ± s.e.m. for fit of the amplitude histogram. D. Deoxygenation increases [Ca2+]i in SAD red cells but not in WT mouse red cells, in a manner inhibited by 1 µM GsMTx-4 and enhanced by 50 µM vanadate. Values are means ± s.e.m. of Fluo-3 fluorescence increase for (n) red cells from 3 mice studied in 8 experiments (SAD), from 1 mouse studied in 4 experiiments (WT and SAD + GsMTx-4) or from 1 mouse studied in 2 experiments (SAD + vanadate).

Mentions: To minimize the contribution of anion currents to measured currents, and to enhance detection of monovalent cation currents, the effects of deoxygenation in SAD cells were studied in a Na methanesulfonate bath free of Ca2+. The pipette also contained Na methanesulfonate. The representative on-cell patch shown in Figure 2A was quiescent in room air, but upon deoxygenation exhibited channel activity with a chord conductance of 27 pS (between −Vp = +25 and −25 mV) and reversal potential (Erev) of +1 mV (Figure 2C). As shown in Figure 2B, deoxygenation increased mean NPo in SAD mouse red cell patches from 0.01±0.013 to 0.48±0.20 (n = 6; p<0.05). Patch duration under deoxygenation in these experiments was 8.4±1.8 min. Estimated single channel amplitude at −Vp = 25 mV was 0.70±0.14 pA (n = 6), with a calculated inward chord conductance of 28 pS (0 to −25 pS). With NMDG chloride rather than Na methanesulfonate in the pipette, room air patch NPo of 0.03 was unchanged by deoxygenation at 0.06 (n = 5, P = 0.28). The data demonstrate the activation by deoxygenation of nonspecific cation channel activity in the SAD mouse red cell membrane.


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 conductance and increases [Ca2+]i in red cells from SAD sickle mice.A. Representative current trace from an individual cell-attached patch on a SAD mouse erythrocyte, recorded first in oxygenated (upper trace, oxy, −Vp = −25 mV) and subsequently in deoxygenated conditions (lower trace, deoxy, −Vp = +75 mV). Symmetrical pipette and bath solutions contained (in mM) 150 Na methanesulfonate, 10 Na EDTA, and 10 Na HEPES, pH 7.4. B. Deoxygenation increased the NPo of 6 cell-attached patches recorded in symmetrical Na methanesulfate (*, p<0.05; n = 6). Substitution of pipette solution Na+ with NMDG blocked the deoxygenation-induced increase in NPo (n = 5). Values are means ± s.e.m, recorded at −Vp = −25 mV. C. Current-voltage relationship in a representative cell-attached patch on a SAD red cell exposed to deoxygenation with symmetrical Na methanesulfonate solutions in pipette and bath. Mean ± s.e.m. for fit of the amplitude histogram. D. Deoxygenation increases [Ca2+]i in SAD red cells but not in WT mouse red cells, in a manner inhibited by 1 µM GsMTx-4 and enhanced by 50 µM vanadate. Values are means ± s.e.m. of Fluo-3 fluorescence increase for (n) red cells from 3 mice studied in 8 experiments (SAD), from 1 mouse studied in 4 experiiments (WT and SAD + GsMTx-4) or from 1 mouse studied in 2 experiments (SAD + vanadate).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0008732-g002: Deoxygenation activates conductance and increases [Ca2+]i in red cells from SAD sickle mice.A. Representative current trace from an individual cell-attached patch on a SAD mouse erythrocyte, recorded first in oxygenated (upper trace, oxy, −Vp = −25 mV) and subsequently in deoxygenated conditions (lower trace, deoxy, −Vp = +75 mV). Symmetrical pipette and bath solutions contained (in mM) 150 Na methanesulfonate, 10 Na EDTA, and 10 Na HEPES, pH 7.4. B. Deoxygenation increased the NPo of 6 cell-attached patches recorded in symmetrical Na methanesulfate (*, p<0.05; n = 6). Substitution of pipette solution Na+ with NMDG blocked the deoxygenation-induced increase in NPo (n = 5). Values are means ± s.e.m, recorded at −Vp = −25 mV. C. Current-voltage relationship in a representative cell-attached patch on a SAD red cell exposed to deoxygenation with symmetrical Na methanesulfonate solutions in pipette and bath. Mean ± s.e.m. for fit of the amplitude histogram. D. Deoxygenation increases [Ca2+]i in SAD red cells but not in WT mouse red cells, in a manner inhibited by 1 µM GsMTx-4 and enhanced by 50 µM vanadate. Values are means ± s.e.m. of Fluo-3 fluorescence increase for (n) red cells from 3 mice studied in 8 experiments (SAD), from 1 mouse studied in 4 experiiments (WT and SAD + GsMTx-4) or from 1 mouse studied in 2 experiments (SAD + vanadate).
Mentions: To minimize the contribution of anion currents to measured currents, and to enhance detection of monovalent cation currents, the effects of deoxygenation in SAD cells were studied in a Na methanesulfonate bath free of Ca2+. The pipette also contained Na methanesulfonate. The representative on-cell patch shown in Figure 2A was quiescent in room air, but upon deoxygenation exhibited channel activity with a chord conductance of 27 pS (between −Vp = +25 and −25 mV) and reversal potential (Erev) of +1 mV (Figure 2C). As shown in Figure 2B, deoxygenation increased mean NPo in SAD mouse red cell patches from 0.01±0.013 to 0.48±0.20 (n = 6; p<0.05). Patch duration under deoxygenation in these experiments was 8.4±1.8 min. Estimated single channel amplitude at −Vp = 25 mV was 0.70±0.14 pA (n = 6), with a calculated inward chord conductance of 28 pS (0 to −25 pS). With NMDG chloride rather than Na methanesulfonate in the pipette, room air patch NPo of 0.03 was unchanged by deoxygenation at 0.06 (n = 5, P = 0.28). The data demonstrate the activation by deoxygenation of nonspecific cation channel activity in the SAD mouse red cell membrane.

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