Engineering aequorin as an indicator of calcium signals near the BK channel

Reviriego Santos, Pablo 2009. Engineering aequorin as an indicator of calcium signals near the BK channel. PhD Thesis, Cardiff University.

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Abstract

The BK channel is a large conductance calcium-activated voltage- dependent potassium channel. This channel plays a key role as a negative feedback mechanism of membrane excitability and cellular Ca2+. There is substantial evidence suggesting that the Ca2+ activation of the BK channel is regulated by localised Ca2+ release from intracellular stores. The aim of the work presented in this thesis was to develop a novel method of measuring the local Ca2+ concentration controlling the BK channel activation. The p2 subunit, an auxiliary protein of the BK channel, was extracted from MG63 cells and cloned. Subsequently, the aequorin sequence was attached to its C-terminus using splicing by overlapping extension. The recombinant protein retained the features of the native proteins emitting light in response to Ca2+ and showed correct targeting to the ceil membrane. The resultant light emission of the new protein was diminished in comparison to the native aequorin. The p2-Aequorin and a cytosolic Luciferase-aequorin were successfully transfected in a HEK293 cell line which stably express the BK channel a subunit. The expression of the aequorin constructs in HEK293 cells in suspension revealed the presence of intracellular mechanosensitive Ca2+ channels. The main finding of this thesis was that the Ca2+ affecting the BK channel is regulated independently of cytosolic Ca2+ in HEK293 cells. Stimulation with agonists such as carbachol, ATP and cyclopiazonic acid demonstrated clear differences in the magnitude of BK channel microdomain and cytosolic Ca2+ signals. Short term exposure to caffeine induced a significant decrease in the Ca2+ signals near the channel. The addition of extracellular Ca2+ led to large Ca2+ transients close to the BK channel suggesting a store-operated Ca2+ mechanism. The Ca2+ effects produced by carbachol, ATP, caffeine and cyclopizaonic acid indicate a coupling between IP3-induced Ca2+ release from the ER and Ca2+- activation of the BK channel.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Pharmacy
Subjects:	R Medicine > RS Pharmacy and materia medica
ISBN:	9781303189289
Date of First Compliant Deposit:	30 March 2016
Last Modified:	03 Dec 2014 15:54
URI:	https://orca.cardiff.ac.uk/id/eprint/55830

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