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Title: The ins and outs of Ca²⁺ signalling : regulation of Ca²⁺ entry and release in electrically non-excitable cells
Author: Holmes, Anthony Mark
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2005
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The work presented in this thesis demonstrates the complex regulation of Ca2+ release and entry in non-excitable cells. Cells have access to a finite store of intracellular Ca2+ and to an infinite pool within the extracellular space. Therefore Ca2+ release and entry channels are kept under tight regulation to prevent cytosolic Ca2+ reaching pathological levels. The principal investigations detailed here have been the modulation of inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) function by specialised proteins and the interplay between plasma membrane Ca2+ entry channels. For the purpose of these studies I have used a combination of molecular biology and video imaging of live cells. The principal Ca2+ release channels on the endoplasmic reticulum (ER) of non-excitable cells belong to the InsP3R gating can be regulated by Ca2+, either by acting on the receptor or through Ca2+-sensor proteins which modify InsP3R function. The experiments described in this thesis investigate the role of a novel family of endogenous EF-hand containing proteins, called neuronal Ca2+-binding proteins (CaBPs), which interact with InsP3Rs. It has been previously demonstrated that CaBPs activate InsP3Rs in the absence of InsP3. However, work presented here shows an opposite effect. Using a heterologous expression system in COS-7 cells, and microinjection of recombinant CaBP in Xenopus oocytes, it is demonstrated that CaBP interacts with and inhibits InsP3-induced Ca2+ release (IICR). Furthermore, although CaBP is structurally similar to the archetypal Ca2+-sensor protein calmodulin (CaM), these proteins have distinct effects and provide an additional level of InsP3R regulation. These data suggest that CaBPs can act as endogenous regulators of InsP3R activity, and may serve to tune the sensitivity of InsP3Rs to InsP3. The depletion of ER Ca2+ stores results in the activation of plasma membrane Ca2+ entry channels through an unknown mechanism. The Ca2+ influx, termed store-operated Ca2+ entry (SOCE), replenishes Ca2+ stores and drives the Ca2+ oscillations observed in response to many stimuli. However, it is becoming apparent that alternative Ca2+ influx pathways can occur independently of the state of filling of the ER. These Ca2+ entry routes are therefore termed non-SOCE. The data presented in this thesis suggests that the CaM antagonist calmidazolium (CMZ) activates a non-SOCE pathway via PLA2-mediated arachidonic acid production. The effects of CMZ could be inhibited by the PLA2 inhibitor N-(p-amylcinnamoyl)anthranilic acid (ACA). Furthermore, the Ca2+ entry activated by CMZ was mimicked by exogenous application of arachidonic acid. CMZ- and arachidonic acid-mediated Ca2+ entry was pharmacologically distinct from SOCE. These data indicate that HeLa cells express two distinct Ca2+ pathways that can operate independently of each other and which may be accountable for the control of different cellular processes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available