Abstract
B cell activation requires sustained elevation of cytoplasmic free calcium, achieved by influx through store-operated calcium (SOC) channels. The molecular identity of these channels is not known. Ectopic expression of the raft-associated tetraspan protein CD20 in Chinese hamster ovary cells introduced a novel SOC entry pathway that was permeable to strontium as well as to calcium. The activity of this SOC pathway was abolished by deletion of a cytoplasmic sequence in CD20 essential for its efficient raft localization. Strontium-permeable SOC channels were detected in B cells, and B cell receptor-stimulated influx was significantly reduced by downregulation of CD20 expression using short interfering RNA and also by cholesterol depletion. This is the first evidence that raft-associated CD20 constitutes a component of a SOC entry pathway activated by the B cell receptor.
Highlights
The function of CD20, a tetraspan transmembrane protein expressed in B lymphocytes, is not yet fully elucidated, electrophysiological evidence of calcium channel activity has been reported
In this report we demonstrate that CD20, when expressed in Chinese hamster ovary (CHO) cells, dramatically enhanced storeoperated calcium (SOC) entry through strontiumpermeable ion channels, providing the first evidence that CD20 is a component of a SOC entry pathway
Novel SOC Entry in CD20-expressing CHO Cells—CHO cells expressing exogenous CD20 or control CHO cells transfected with vector alone were loaded with the calcium-sensitive dye, fura-2, and the fluorescence values in a field of Ͼ20 cells were recorded during the perfusion of various reagents
Summary
Cell Lines and Culture—Ramos, BJAB, and Molt-4 cells were maintained in RMPI, 10% fetal bovine serum, and CHO cells in ␣-minimum Eagle’s medium, 10% fetal bovine serum. Molt-4 T cells expressing CD20 or CD20 deletion constructs were described previously [14]. Cholera toxin conjugated to horseradish peroxidase was purchased from Sigma. CaCl2 and SrCl2 were added to the buffer at 1 mM final concentration as indicated. Ramos and BJAB cells were grown in normal culture medium and adhered to poly-lysinecoated cover slips by low speed centrifugation on the day of the experiment. Cell Stimulation and Pretreatment Conditions—ATP and thapsigargin (Tg) were used at 100 M and 1 M final concentration, respectively. SKF96365 was added to the EGTA buffer at 50 M final concentration and maintained for about 9 min. Flow Cytometry—Transfected BJAB or CHO cells were incubated with B1 anti-CD20 monoclonal antibody followed by fluorescein isothiocyanate-conjugated anti-mouse IgG. The images were acquired and analyzed using a Fluor-S MAX MultiImager and Quantity One software, respectively (Bio-Rad)
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