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Functional characterisation of expressed ɑ9ɑ10 nicotinic acetylcholine receptor channels

The primary aim of this project was to investigate receptor and ion channel coupling in a heterologous expression system, the GH4C1 cell line. The primary focus was the interaction between the a9alO nicotinic acetylcholine receptor (nAChR) and the small conductance calcium (Ca2+) -sensitive potassium (K+) channel, subtype 2 (SK2). In both inner hair cells (rnCs) and outer hair' cells (OHCs), the a9alO nAChR and the SK2 channel are spatially and functionally associated, or 'coupled'. Both the a9alO nAChR and SK2 were transiently expressed in vitro and functionally evaluated. The channels were then coexpressed and the interaction between the two was examined electrophysiologically. Channel blockers, receptor antagonists and an intracellular Ca2+ chelator were used, in conjunction with electrophysiological methods. No evidence of the interaction between the a9alO nAChR and the SK2 channel was found. Prior to the investigation of the coupling interaction, the endogenous currents of the GH4C1 cells were also assessed. Other experiments were aimed at recapitulating the interaction of an L-type Ca2+ channel (LTCC) and the SK2 channel in vitro, which also occurs in neonatal IHCs. These channels were not found to associate, in the cell line. Additionally, the direct effect of ryanodine upon the SK2 channel was assessed in Human embryonic kidney (HEK) 293 cells, and found to have no effect.

Identiferoai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:652042
Date January 2013
CreatorsBere Harding, Court Edmund de la
PublisherUniversity of Bristol
Source SetsEthos UK
Detected LanguageEnglish
TypeElectronic Thesis or Dissertation

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