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Title: TMEM16A channels : molecular physiology and pharmacological regulation
Author: Adomaviciene, Aiste
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2012
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Calcium-activated chloride channels (CaCCs) are a class of the ligand-gated channels involved in numerous cellular functions. In vascular smooth muscle, these ion channels couple agonist-induced calcium-release from the sarcoplasmic reticulum to membrane depolarisation and vasoconstriction. For this reason, CaCCs have been suggested as a potential molecular target to treat a range of vascular disorders. These ion channels, however, have not been yet explored as a drug target because their molecular identity has been elusive and their pharmacology has been restricted to compounds with low potency and poor specificity. The general aims of this work of thesis are: i) to define the molecular identity of CaCCs in vascular smooth muscle, ii) to investigate how the structural features of the identified channel relate to its functional properties and iii) to examine how drug binding modulates CaCC activity. The main findings are the following:1) By using RNA interference technology and patch-clamp analysis, the Tmem16A gene was found to encode for CaCCs in pulmonary artery smooth muscle. Furthermore, Tmem16A appeared to be expressed in other vascular smooth muscles suggesting that this ion channel may represent CaCCs in various vascular beds.2) To understand the physiology and pharmacology of TMEM16A channels it is of a fundamental importance to elucidate the molecular mechanisms by which channel gating and conductance are achieved. TMEM16A comprises eight putative transmembrane domains (TMs) with TM5 and TM6 flanking a putative re-entrant loop, which resembles the pore of other ion channels. Using a chimeric approach the role of this region was investigated. The re-entrant loop of TMEM16A was found to mediate a range of functional roles: it controlled the response of the channel to intracellular calcium, the permeation of anions and the expression of channels on the plasma membrane. Specifically, a non-canonical trafficking motif was identified within in a 38 amino acid region within the re-entrant loop.3) Drugs that modulate the function of TMEM16A channels are currently limited. The generic chloride channel blocker anthracene-9-carboxylic acid (A9C) was found to produce a bimodal effect on TMEM16A currents: low concentrations of A9C activated the channels, while doses higher than ~300 µM produced current inhibition. These two effects were mediated via A9C binding to two separate sites. Binding of A9C into the pore resulted in channel inhibition, while A9C binding to an extracellular site increased the open probability of the channel. To conclude, this work of thesis has revealed the molecular identity of CaCCs in vascular smooth muscle and elucidated the functional roles of the re-entrant loop of the TMEM16A channel protein. The identification of the activating and inhibiting A9C binding sites may help the development of selective blockers and activators of TMEM16A channels.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available