Title:
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Functional characterisation of expressed ɑ9ɑ10 nicotinic
acetylcholine receptor channels
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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.
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