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Title: Molecular pharmacology of native and exogenous vascular ion channels
Author: Ta, Chau My
ISNI:       0000 0004 6493 8698
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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Vascular Ca2+-activated chloride channels (CaCCs), are a class of ligand- and voltage-gated channels that couple agonist binding and inositol triphosphate (IP3) signalling to depolarisation of the cell membrane and smooth muscle contraction. The elucidation of the precise physiological role and the pharmacological profile of this channel has been accelerated by the recent discovery of the gene (TMEM16A) coding for these channels. Based on the rationale that depolarising membrane potential (Vm) causes contraction in vascular SMCs, the possibility of controlling vessel tone via the exogenous light-activated, depolarising Channelrhodopsin 2 (ChR2) channels was also investigated. The overall aim of this thesis is two folds: i) to define mechanisms of regulation of CaCCs by endogenous signalling molecules and pharmacological agents, and ii) to develop a new method of controlling vascular contraction by exogenous ion channels. The main findings of this thesis are: 1) Arteries (large conduit, systemic and pulmonary) obtained from mice lacking of one allele of TMEM16A gene (heterozygous knockouts) presented reduced response to α1-adrenergic receptor activation. This finding reinforced the view that TMEM16A proteins serve as key mechanism of modulation of artery tone. 2) Phosphatidylinositol 4,5-bisphosphate (PIP2), a lipid involved in the IP3 signalling cascade, is a key activator of native and cloned vascular TMEM16A channel. Furthermore, the closely related TMEM16B was inhibited by PIP2. These modulatory effect of PIP2 on these channels were especially pronounced in the physiological range of [Ca2+]i. 3) Anthracene-9-carboxylic acid (A9C), a general Cl- blocker, exhibited bimodal effects on TMEM16A-mediated currents. The inhibiting effect occurred via open-channel block mechanism while the activating effect was due to an increase in Po and a leftward shift in the steady-state activation curve. These mechanistic insights may help the design of novel drug (activators and inhibitors) that could be used to modulate blood vessel tone. 4) ChR2, a light-gated and non-selective cation channel, was introduced specifically into the SMC in mice (ChR2-SMC). Isolated arterial SMCs obtained from these mice showed light-dependent inward currents. ChR2-SMC artery rings also contracted in a light-dependent manner. The mechanism of lightinduced contraction involved voltage-gated Ca2+ channels (VGC) activation and Ca2+ influx. To conclude, this work of thesis has shed light on the functional role of TMEM16A in various vascular SMCs types. The regulation of TMEM16A by membrane lipid will lay the foundation for the identification of the PIP2 binding site on these channels, which could also be exploited as a site for pharmacological intervention. The study of A9C mechanisms of activation and inhibition may aid the development of selective blockers and activators for TMEM16A channels. Lastly, the control of vascular tone by ChR2 in mice can be used to control blood perfusion to organs and tissues for experimental purposes.
Supervisor: Tammaro, Paolo Sponsor: British Heart Foundation ; Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available