title: A direct mass-action mechanism explains capacitative calcium entry in Jurkat and skeletal L6 muscle cells.
abstract: We examined capacitative calcium entry (CCE) in Jurkat and in L6 skeletal muscle cells. We found that extracellular Ca2+ can enter the endoplasmic reticulum (ER) of both cell types even in the presence of thapsigargin, which blocks entry into the ER from the cytosol through the CaATPase. Moreover, extracellular Ca2+ entry into the ER was evident even when intracellular flow of Ca2+ was in the direction of ER to cytosol due to the presence of caffeine. ER Ca2+ content was assessed by two separate means. First, we used the Mag-Fura fluorescent dye, which is sensitive only to the relatively high concentrations of Ca2+ found in the ER. Second, we transiently expressed an ER-targeted derivative of aequorin, which reports Ca2+ by luminescence. In both cases, the Ca2+ concentration in the ER increased in response to extracellular Ca2+ after the ER had been previously depleted despite blockade by thapsigargin. We found two differences between the Jurkat and L6 cells. L6, but not Jurkat cells, inhibited Ca2+ uptake at very high Ca2+ concentrations. Second, ryanodine receptor blockers inhibited the appearance of cytosolic Ca2+ during CCE if added before Ca2+ in both cases, but the L6 cells were much more sensitive to ryanodine. Both of these can be explained by the known difference in ryanodine receptors between these cell types. These findings imply that the origin of cytosolic Ca2+ during CCE is the ER. Furthermore, kinetic data demonstrated that Ca2+ filled the ER before the cytosol during CCE. Our results suggest a plasma membrane Ca2+ channel and an ER Ca2+ channel joined in tandem, allowing Ca2+ to flow directly from the extracellular space to the ER. This explains CCE; any decrease in ER [Ca2+] relative to extracellular [Ca2+] would provide the gradient for refilling the ER through a mass-action mechanism.
authors.affiliation: Department of Pharmaceutical Sciences, St. John's University, Jamaica, New York 11439, USA.
authors.forename: Bisni
authors.initials: B
authors.lastname: Narayanan
authors.forename: Mohammad N
authors.initials: MN
authors.lastname: Islam
authors.forename: Diana
authors.initials: D
authors.lastname: Bartelt
authors.forename: Raymond S
authors.initials: RS
authors.lastname: Ochs
mesh_terms: D000331:Aequorin / Q000235:genetics; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002460:Cell Line; D002614:Chelating Agents; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives*; D015870:Gene Expression; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D008163:Luminescent Measurements; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal / Q000378:metabolism*; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000502:physiology; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection
chemical_list: D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D019837:Ryanodine Receptor Calcium Release Channel; C072323:Mag-Fura-2-acetoxymethyl ester; D002110:Caffeine; D000331:Aequorin; D019284:Thapsigargin; D007811:Lanthanum; D000252:Calcium-Transporting ATPases; D002118:Calcium; D016257:Fura-2