Orai3 channel is the 2-APB-induced endoplasmic reticulum calcium leak.
We have studied in HeLa cells the molecular nature of the 2-APB induced ER Ca2+leak using synthetic Ca2+indicators that report changes in both the cytoplasmic ([Ca2+]i) and the luminal ER ([Ca2+]ER) Ca2+concentrations. We have tested the hypothesis that Orai channels participate in the 2-APB-induced ER Ca2+leak that was characterized in the companion paper. The expression of the dominant negative Orai1 E106A mutant, which has been reported to block the activity of all three types of Orai channels, inhibited the effect of 2-APB on the [Ca2+]ERbut did not decrease the ER Ca2+leak after thapsigargin (TG). Orai3 channel, but neither Orai1 nor Orai2, colocalizes with expressed IP3R and only Orai3 channel supported the 2-APB-induced ER Ca2+leak, while Orai1 and Orai2 inhibited this type of ER Ca2+leak. Decreasing the expression of Orai3 inhibited the 2-APB-induced ER Ca2+leak but did not modify the ER Ca2+leak revealed by inhibition of SERCA pumps with TG. However, reducing the expression of Orai3 channel resulted in larger [Ca2+]iresponse after TG but only when the ER store had been overloaded with Ca2+by eliminating the acidic internal Ca2+store with bafilomycin. These data suggest that Orai3 channel does not participate in the TG-revealed ER Ca2+leak but forms an ER Ca2+leak channel that is limiting the overloading with Ca2+of the ER store.
Department of Biochemistry, Cinvestav, Mexico City, 07000, Mexico.
Daniel
D
Leon-Aparicio
Department of Cell Biology and Development, Instituto de Fisiología Celular, UNAM, Mexico City, 04510, Mexico.
Jonathan
J
Pacheco
Department of Biochemistry, Cinvestav, Mexico City, 07000, Mexico.
Jesus
J
Chavez-Reyes
Department of Biochemistry, Cinvestav, Mexico City, 07000, Mexico.
Jose M
JM
Galindo
Department of Biochemistry, Cinvestav, Mexico City, 07000, Mexico.
Jesus
J
Valdes
Department of Cell Biology and Development, Instituto de Fisiología Celular, UNAM, Mexico City, 04510, Mexico.
Luis
L
Vaca
Department of Cell Biology and Development, Instituto de Fisiología Celular, UNAM, Mexico City, 04510, Mexico. Electronic address: aguerrero@cinvestav.mx.
Agustin
A
Guerrero-Hernandez
D001896:Boron Compounds / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology
D001896:Boron Compounds; D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; C522867:Orai3 protein, human; D019284:Thapsigargin; C109986:2-aminoethoxydiphenyl borate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium
2-APB; Endoplasmic reticulum calcium leak; Luminal calcium concentration; Orai3 channel; SERCA pump