Fleet, Amanda; (1997) Calcium signalling and buffering: The role of annexin VI. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Intracellular free calcium concentrations and their changes are important since calcium is a ubiquitous second messenger in eukaryotic cells. Raised intracellular calcium concentrations have been implicated in cell division, gene regulation, exocytosis and many other cell functions. The magnitude of the free calcium concentration change observed after stimulation depends on two things: the amount of calcium influx into the cytoplasm, and the extent to which this calcium is buffered by cell constituents. Calcium influx into the cytoplasm occurs in two ways: release of stored calcium from intracellular organelles such as the endoplasmic reticulum, and calcium entry into the cell across the cell membrane from the extracellular environment. A431 cells, a human epithelial cell line, respond to epidermal growth factor (EGF) with two phases, an initial rise in intracellular free calcium concentration which is followed by a maintained plateau of raised intracellular free calcium. The initial, peak calcium response is due to calcium release from internal stores, whereas the maintained calcium change is from calcium entry into the cell across the plasmalemma. A431 cells transfected with the protein annexin VI, a member of a family of proteins that bind to phospholipids and calcium, grow more slowly than wild type A431 (wtA431) cells and have a reduced calcium response to EGF. The hypothesis that annexin VI was acting as a calcium buffer was tested and it was found that there was no difference in calcium buffering capacity between wild type and transfected (annexin VI+) cells. Annexin VI+ cells have a pattern of response to EGF that is distinct from the wild type cells in displaying no sustained calcium elevation following stimulation. The hypothesis suggested from the results of these experiments is that calcium influx across the cell membrane is inhibited by annexin VI, thereby removing the maintained calcium increase within the cells following stimulation. The prolonged raised calcium might be necessary for cell division and cell regulation.

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