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J. Biol. Chem., Vol. 278, Issue 45, 44188-44196, November 7, 2003

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A Direct Mass-action Mechanism Explains Capacitative Calcium Entry in Jurkat and Skeletal L6 Muscle Cells^*

Bisni Narayanan, Mohammad N. Islam, Diana Bartelt, and Raymond S. Ochs¶

From the Departments of Pharmaceutical Sciences and Biological Sciences, St. John's University, Jamaica, New York 11439

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.

Received for publication, June 19, 2003 , and in revised form, August 26, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Pharmaceutical Sciences, School of Pharmacy, St. John's University, 8000 Utopia Pkwy., Jamaica, NY 11439. Tel.: 718-990-1678; Fax: 718-990-1936; E-mail: ochsr{at}stjohns.edu.

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