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Vol. 20, Issue 17, 3783-3791, September 1, 2009

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The Class II Phosphatidylinositol 3 kinase C2β Is Required for the Activation of the K⁺ Channel KCa3.1 and CD4 T-Cells

Shekhar Srivastava^*,, Lie Di^*,, Olga Zhdanova^,, Zhai Li^*,, Santosha Vardhana^,, Qi Wan^,, Ying Yan^||, Rajat Varma^¶, Jonathan Backer^||, Heike Wulff^#, Michael L. Dustin^,, and Edward Y. Skolnik^*,,

Division of Nephrology, *Departments of Pharmacology and Molecular Pathogenesis, The Helen L. and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute for Biomolecular Medicine, New York University Langone Medical Center, New York, NY 10016; ^¶T-Cell Biophysics Unit, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892; ^||Department of Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461; ^#Department of Pharmacology, University of California Davis, Davis, CA 95616

Submitted May 13, 2009; Revised June 19, 2009; Accepted June 30, 2009
Monitoring Editor: Carl-Henrik Heldin

The Ca²⁺-activated K⁺ channel KCa3.1 is required for Ca²⁺ influx and the subsequent activation of T-cells. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, directly phosphorylates and activates KCa3.1 and is required for the activation of human CD4 T lymphocytes. We now show that the class II phosphatidylinositol 3 kinase C2β (PI3K-C2β) is activated by the T-cell receptor (TCR) and functions upstream of NDPK-B to activate KCa3.1 channel activity. Decreased expression of PI3K-C2β by siRNA in human CD4 T-cells resulted in inhibition of KCa3.1 channel activity. The inhibition was due to decreased phosphatidylinositol 3-phosphate [PI(3)P] because dialyzing PI3K-C2β siRNA-treated T-cells with PI(3)P rescued KCa3.1 channel activity. Moreover, overexpression of PI3K-C2β in KCa3.1-transfected Jurkat T-cells led to increased TCR-stimulated activation of KCa3.1 and Ca²⁺ influx, whereas silencing of PI3K-C2β inhibited both responses. Using total internal reflection fluorescence microscopy and planar lipid bilayers, we found that PI3K-C2β colocalized with Zap70 and the TCR in peripheral microclusters in the immunological synapse. This is the first demonstration that a class II PI3K plays a critical role in T-cell activation.

Address correspondence to: Edward Skolnik (edward.skolnik{at}nyumc.org)