Control of calcium(2+) dynamics in T cells: A new role for the plasma membrane calcium(2+)-ATPase.

摘要

The amplitude and dynamic structure of Ca2+ signals in T cells play an important role in determining the efficiency and specificity of signal transduction. This dissertation explores the role of the PMCA in shaping Ca2+, signals in Jurkat human leukemic T cells. The introductory Chapter provides an overview of the mechanisms and functions of Ca2+ signals in T cells. The second chapter describes the materials and methods used in the experiments discussed herein. The third chapter examines the role of Ca2+ clearance mechanisms in shaping Ca2+ responses in Jurkat T cells. Single-cell voltage-clamp and calcium imaging experiments reveal that the PMCA is the primary extrusion mechanism in these cells and that its activity is slowly modulated by changes in [Ca2+]_i. As the primary Ca2+ clearance mechanism, PMCA activity and modulation play a major role in determining the spatiotemporal profile of Ca2+ signals in T cells. The fourth chapter discusses a novel Ca2+-clamp technique designed to quantitatively control the concentration of cytosolic calcium. The Ca 2+-clamp generates [Ca2+]_i steps ranging from 0.3–1.5μM with a risetime of a few seconds and duration >5 min. Using the Ca2+-clamp, PMCA activity was measured in single cells in order to characterize the Ca2+-dependence of PMCA modulation. The fifth and final Chapter examines the functional organization of PMCA and Ca2+-release activated Ca2+ (CRAC) channels in T cells. I provide the first evidence that there is a close functional coupling between PMCAs and CRAC channels. This dissertation thus demonstrates that the Plasma Membrane Ca2+-pump actively contributes to the complexity of Ca2+ signaling in T cells and constitutes an important part of a complex and highly interconnected Ca2+ signaling machine.