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Title: Role of membrane PIP2 hydrolysis and depletion in receptor-induced inhibition of potassium M(Kv7)channels
Author: Hughes, Simon Anthony
ISNI:       0000 0004 2680 3119
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2008
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The M-current is a low-threshold and voltage gated potassium current with key roles in regulating neuronal excitability. M-channels have been found to be widely distributed throughout the central and peripheral nervous systems where channel abnormalities are known to contribute to several channelopathies. The channel can be regulated via Gq/11-coupled receptors such as the M1 muscarinic receptor which couples to phospholipase C (PLC) which acts to hydrolyze membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). Efforts to elucidate the mechanisms surrounding the modulation of this channel have lead to a number of suggested signal transducers including membrane PIP2 and calcium. The first part of this study attempts to confirm and clarify the involvement of PIP2 in M-channel regulation by expressing the pleckstrin homology domain of phospholipase C tagged with green fluorescent protein (GFPPLCa- PH) in cells taken from rat superior cervical ganglia (SCG) in combination with voltage-clamp membrane current recording. Upon stimulation by the muscarinic agonist oxotremorine-M (oxo-M), the probe is seen to translocate from the membrane, bound primarily to PIP2, and bind the hydrolysis product cytosolic inositol 1.4,5- trisphosphate (IP3). Using novel techniques I was able to estimate resting membrane [PIP2] and predict changes in mechanism components during PIP2 hydrolysis. Fluorescence changes also showed a close temporal and concentration-dependent correlation with muscarinic M-current inhibition, consistent with the PIP2 gating hypothesis. Further clarification was then achieved by over-expressing phosphatidylinositol-4-phosphate-5-kinase (PI4(5)K), an enzyme involved in PIP2 synthesis. The activation of the B2-bradykinin receptor, another Gq/11-coupled receptor, has been suggested to act via IP3-induced Ca2+ release with concurrent up-regulation of PIP2 synthesis. In this situation calculations involving GFP-PLCa-PH break down due to the probes affinity for IP3. Hence the second part of this study involved using a probe which solely binds PIP2, the yellow fluorescence tagged C-terminus of the transcription factor tubby, mutated to reduce affinity for PIP2 and thus allow translocation (tubby-R332H-YFP). This probe, in conjunction with M-current measurements using further voltage clamp techniques, highlighted oxo-M as primarily acting via PIP2 depletion and bradykinin chiefly via IP3 and calcium, confirming my initial hypotheses.
Supervisor: Not available Sponsor: Not available
Qualification Name: Not available Qualification Level: Doctoral
EThOS ID:  DOI: Not available