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 Ca 2؉ 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 Ca 2؉ entry into the ER was evident even when intracellular flow of Ca 2؉ was in the direction of ER to cytosol due to the presence of caffeine. ER Ca 2؉ 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 Ca 2؉ found in the ER. Second, we transiently expressed an ER-targeted derivative of aequorin, which reports Ca 2؉ by luminescence. In both cases, the Ca 2؉ concentration in the ER increased in response to extracellular Ca 2؉ 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 Ca 2؉ uptake at very high Ca 2؉ concentrations. Second, ryanodine receptor blockers inhibited the appearance of cytosolic Ca 2؉ during CCE if added before Ca 2؉ 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 Ca 2؉ during CCE is the ER. Furthermore, kinetic data demonstrated that Ca 2؉ filled the ER before the cytosol during CCE. Our results suggest a plasma membrane Ca 2؉ channel and an ER Ca 2؉ channel joined in tandem, allowing Ca 2؉ to flow directly from the extracellular space to the ER. This explains CCE; any decrease in ER [Ca 2؉ ] relative to extracellular [Ca 2؉ ] would provide the gradient for refilling the ER through a mass-action mechanism.Ca 2ϩ is a critical regulator for a large number of cells, and it is known that for many, the key signaling event is the release of this ion from the ER 1 (1, 2). After release most of the Ca 2ϩ is re-sequestered to the ER through the CaATPase, although some is lost through the plasma membrane to the cell exterior. Maintaining the ER pool of Ca 2ϩ requires re-entry from outside the cell. Casteels and Droogmans (3) first reported a correlation between depletion of ER [Ca 2ϩ ] and entry of Ca 2ϩ from extracellular sources into the cell. This phenomenon was then extensively studied by Putney (4) and named "store-operated" or capacitative calcium entry (CCE).The mechanism for CCE remains unknown, although two general models have been proposed. One, borrowed from the known association of the skeletal muscle plasma membrane potential sensor (L channel) and ER protein (ryanodine receptor), posits a direct connection in which Ca 2ϩ depletion within the ER is sensed by an ER protein, transmitted by proteinprotein interaction to the plasma membrane protein, which then allows entry of Ca 2ϩ into the cytosol. A second, based on second messenger signaling systems such as that for cyclic AMP, posits a mess...

Membrane depolarization increases ryanodine sensitivity to Ca2+ release to the cytosol in L6 skeletal muscle cells: Implications for excitation–contraction coupling

“…We confirmed the dye method for SR Ca 2þ measurement by incorporating a Ca 2þ sensor protein into the SR by transfection. 40 Experiments in the present study further support this view. Figure 5 shows that the SR lumen of the L6 cells accumulates extracellular Ca 2þ even in the presence of both thapsigargin as well as high concentrations of Ry, so that no exchange with cytosolic Ca 2þ is possible.…”

“…In previous studies, we have provided evidence for a direct interaction of the DHPR and the RyR to explain SOCE. 11,12 We suggested that these two Ca 2þ channels together form a combined channel for the direct entry of Ca 2þ from the extracellular space to the SR lumen. As this process occurs in resting (polarized) conditions, the DHPR is static in location, directly facing (and occupying) an RyR.…”

Sub-second calcium coupling between outside medium and subplasmalemmal stores during overstimulation/depolarisation-induced ciliary beat reversal in Paramecium cells

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Intracellular Ca2+ Release Triggers Translocation of Membrane Marker FM1–43 from the Extracellular Leaflet of Plasma Membrane into Endoplasmic Reticulum in T Lymphocytes

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