Understanding the mode of action of TRPC1/4/5 ion channel modulators

Calcium ions are critical for cellular signalling and impact almost all aspects of cellular life. TRPC channels are non-selective cation channels permeable to both Na+ and Ca2+. TRPC channels are ubiquitously expressed in mammalian tissue and are linked to a wide range of pathological and physiological mechanisms. TRPC proteins can form both homo-and hetero-tetrameric channels. The natural composition and heteromerization of TRPC channels is poorly understood. However, they have been reported to be particularly promiscuous in the formation of heteromers. Over recent years numerous modulators of TRPC1/4/5 channels have been published; among them (–)-Engelrin A and Pico145 have emerged as particularly potent and specific activators and inhibitors of TRPC1/4/5 channels, respectively. However there still lacks evidence around the mode of action of these modulators and the ability to differentiate between TRPC1, TRPC4 and TRPC5 homomers and heteromers. This thesis explores approaches to unravel the mechanism of action of TRPC1/4/5 modulators. Firstly, recently published TRPC1/4/5 modulators, along with the pathology and advancements in structural information are reviewed in Chapter 1. Secondly, current approaches towards chemical labelling of target proteins is explored in Section 1.6 of Chapter 1. A range of cellular and biochemical techniques have been used in this thesis to unravel the mechanism of action of ion channel modulators and these have been briefly explained in Chapter 2. Chapter 3 details the development of novel TRPC5 modulators and investigation of the mechanism of action through calcium recording, electrophysiology and cyclic voltammetry experiments. Findings from these experiments suggest that the synthetic flavonol-based TRPC5 modulators act directly on the channel. The work in Chapter 4 details the use of photoaffinity probes based on the TRPC1/4/5 channel inhibitor, Pico145, to indicate a direct interaction with TRPC5. The work in this chapter identified that a known TRPC5 channel activator can distinguish between the closely related TRPC4 and TRPC5 proteins (~70% sequence identity). Structure activity relationships were explored on a series of TRPC5 inhibitors in Chapter 5. Overall this thesis demonstrates how multiple approaches can be used to unravel the mechanism of action of ion channel modulators in a synergistic manner.