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


RESULTS
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
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.
Vandorpe
David H
DH
Molecular and Vascular Medicine Unit, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.
Xu
Chang
C
Shmukler
Boris E
BE
Otterbein
Leo E
LE
Trudel
Marie
M
Sachs
Frederick
F
Gottlieb
Philip A
PA
Brugnara
Carlo
C
Alper
Seth L
SL
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000755:Anemia, Sickle Cell / Q000097:blood; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002248:Carbon Monoxide / Q000633:toxicity*; D015687:Cell Hypoxia; D004176:Dipyridamole / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D004913:Erythrocytes, Abnormal / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000633:toxicity*; D013111:Spider Venoms / Q000633:toxicity*
D015220:Calcium Channels; D036341:Intercellular Signaling Peptides and Proteins; C411249:MTx4 protein, Grammostola spatulata; D010455:Peptides; D013111:Spider Venoms; D004176:Dipyridamole; D002248:Carbon Monoxide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium