The ins and outs of Ca²⁺ signalling : regulation of Ca²⁺ entry and release in electrically non-excitable cells
University of Cambridge, 2005
Online
Hochschulschrift
Zugriff:
The work presented in this thesis demonstrates the complex regulation of Ca 2+ release and entry in non-excitable cells. Cells have access to a finite store of intracellular Ca 2+ and to an infinite pool within the extracellular space. Therefore Ca 2+ release and entry channels are kept under tight regulation to prevent cytosolic Ca 2+ reaching pathological levels. The principal investigations detailed here have been the modulation of inositol 1,4,5-trisphosphate (InsP 3 ) receptor (InsP 3 R) function by specialised proteins and the interplay between plasma membrane Ca 2+ entry channels. For the purpose of these studies I have used a combination of molecular biology and video imaging of live cells. The principal Ca 2+ release channels on the endoplasmic reticulum (ER) of non-excitable cells belong to the InsP 3 R gating can be regulated by Ca 2+ , either by acting on the receptor or through Ca 2+ -sensor proteins which modify InsP 3 R function. The experiments described in this thesis investigate the role of a novel family of endogenous EF-hand containing proteins, called neuronal Ca 2+ -binding proteins (CaBPs), which interact with InsP 3 Rs. It has been previously demonstrated that CaBPs activate InsP 3 Rs in the absence of InsP 3 . 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 InsP 3 -induced Ca 2+ release (IICR). Furthermore, although CaBP is structurally similar to the archetypal Ca 2+ -sensor protein calmodulin (CaM), these proteins have distinct effects and provide an additional level of InsP 3 R regulation. These data suggest that CaBPs can act as endogenous regulators of InsP 3 R activity, and may serve to tune the sensitivity of InsP 3 Rs to InsP 3 . The depletion of ER Ca 2+ stores results in the activation of plasma membrane Ca 2+ entry channels through an unknown mechanism. The Ca 2+ influx, termed store-operated Ca 2+ entry (SOCE), replenishes Ca 2+ stores and drives the Ca 2+ oscillations observed in response to many stimuli. However, it is becoming apparent that alternative Ca 2+ influx pathways can occur independently of the state of filling of the ER. These Ca 2+ 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 PLA 2 -mediated arachidonic acid production. The effects of CMZ could be inhibited by the PLA 2 inhibitor N-(p-amylcinnamoyl)anthranilic acid (ACA). Furthermore, the Ca 2+ entry activated by CMZ was mimicked by exogenous application of arachidonic acid. CMZ- and arachidonic acid-mediated Ca 2+ entry was pharmacologically distinct from SOCE. These data indicate that HeLa cells express two distinct Ca 2+ pathways that can operate independently of each other and which may be accountable for the control of different cellular processes.
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The ins and outs of Ca²⁺ signalling : regulation of Ca²⁺ entry and release in electrically non-excitable cells
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Autor/in / Beteiligte Person: | Holmes, Anthony Mark |
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Veröffentlichung: | University of Cambridge, 2005 |
Medientyp: | Hochschulschrift |
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