Calcium-activated chloride channels (CaCCs) are ubiquitously expressed in a plethora of cell types and, consequently, are involved in numerous cellular processes as diverse as epithelial secretion, regulation of cardiac excitability and smooth muscle contraction. Current pharmacology of CaCCs is limited to compounds with low potency and poor selectivity. The lack of knowledge surrounding the molecular identity of the CaCC has greatly hindered the development of more specific drugs and has impaired our understanding of the channel physiology and biophysics. The recent discovery that the TMEM16A gene codes for CaCCs has offered hope for new developments in these areas. CaCCs have been suggested as possible targets to treat a variety of conditions including asthma as well as pulmonary and systemic hypertension. Due to the ubiquitous expression of CaCCs and the ability of the channel to interact with a number of pharmacological compounds with diverse chemical structures however, it was hypothesised that TMEM16A could be a possible source for off-target drug effects and may represent a concern for safety pharmacology. The principal aim of this thesis was to assess the functional significance of TMEM16A in the cardiovascular system, as this is one of the major systems of concern for safety pharmacology and accounts for the largest number of post-market drug withdrawals. The main findings of this study can be summarised as follows: 1) RT-PCR analysis revealed a ubiquitous expression of TMEM16A in tissues of the rat and human cardiovascular systems, including systemic and pulmonary arteries as well as cardiac tissue. Analysis also revealed the presence of multiple TMEM16A splice variants in all rat tissues examined, in addition to a number of other TMEM16x family members. 2) Myography experiments using the "classical" CaCC blocker niflumic acid and newly identified TMEM16A blockers confirmed a functional role for TMEM16A in phenylephrine-induced vascular smooth muscle contraction. 3) The suitability of currently available Cl- channel blockers for use as pharmacological tools for TMEM16A research was assessed using conventional whole-cell patch clamp and high-throughput electrophysiology techniques to respectively compare their potencies and selectivity over other cardiovascular ion channels. Of the compounds tested, DIDS and T16Ainh-A01 appeared the most suitable blockers; however all compounds had a degree of non-selectivity, raising concerns for their use in functional studies. In conclusion, these findings provide evidence for the ubiquitous expression and functional significance of TMEM16A within the cardiovascular system and support the hypothesis that TMEM16A is a concern for safety pharmacology and should be included into future pre-clinical safety assays. The inadequacy of current inhibitors however highlights the urgency for the development of novel potent and selective channel modulators for future TMEM16A research.
|Date of Award||1 Aug 2015|
- The University of Manchester
|Supervisor||Alison Gurney (Supervisor) & Paolo Tammaro (Supervisor)|
- Calcium-activated chloride channel
- vascular smooth muscle