The molecular pharmacology and modulation of the proton-activated chloride channel PAC

Proton activated chloride (PAC) channels are activated in response to extracellular acidity, and mediate outwardly rectifying chloride currents. The channels are encoded by the TMEM206 (PACC1) gene and are expressed in a multitude of mammalian cell types, potentially being ubiquitous. Recent advances have been made in the understanding of the tertiary and quaternary structure and gating mechanisms of the PAC channel. However, elucidation of how the channel is regulated by synthetic drugs and biological factors is still in its infancy. Furthermore, the physiological and pathophysiogical roles that are played by the channel remain poorly defined. This thesis explores these unknown aspects of PAC channel pharmacology and molecular physiology, and the potential role that PAC may play in coupling pHe to contractility in vascular cells.
This thesis investigates these ideas as follows:
1) A systematic study was conducted into the magnitude of PAC currents in the presence of a range of Cl- channel modulators in HEK293T cells. Flufenamic Acid (FFA), a fenamate drug clinically used for non-steroidal anti-inflammatory therapy, was found to be a potent inhibitor of PAC. Electrophysiological analyses suggest that FFA is a gating modifier of PAC, inducing channel inhibition by reducing the open probability. FFA inhibition was found to be dependent on extracellular pH (pHe), with more potent inhibition being observed at the most acidic pHe. A PAC channel mutant with a single-point mutation within the proton-binding site, informed by computational docking, was found to be less sensitive to FFA block, indicating that PAC may interact within this region.
2) In vivo, cortical neurons express the acid-sensing Ovarian Cancer Gq- Protein Coupled Receptor (GqPCR) (OGR1), in addition to PAC. Both of these proteins activated by extracellular acidification during cerebral ischaemia. The possibility of functional modulation of PAC by OGR1 was therefore investigated. A combination of electrophysiology and heterologous expression of ORG1 and another GqPCR, the α1-adrenergic receptor, indicated that activation of these receptors did not affect PAC current amplitude. Furthermore, a detailed analysis of the effects of individual factors released during GqPCR activation, such as phosphatidyl-inositol(4,5)-bisphosphate (PIP2) and intracellular Ca2+ ([Ca2 and [Ca2+]i levels, and revealed that these factors do not interfere with PAC channel activity. In contrast, Ca2+ and PIP2 had profound effects on the amplitude of the heterologous Ca2+-activated Cl- Channel (CaCC) TMEM16A channel current, which was used a positive control in this part of the study. Collectively, the data suggest that PAC and OGR1 independently mediate the cellular response to extracellular acidity.
3) The role of Cl- channels in ischaemia was investigated further. TMEM16A is a key amplifier of pericyte contractility in in vitro models of cerebral ischaemia. Blockade of this channel was found to reduce capillary constriction and cell death in rat cortical slices. The data suggest that TMEM16A activation amplifies depolarization in these cells, leading to [Ca2+]i influx and subsequent contraction. This finding demonstrates the importance of TMEM16A channels in the control of microvascular tone. In contrast, myography and electrophysiological experiments, with focus on rat aorta, revealed that PAC is not a regulator of artery tone.
To conclude, the work presented in this thesis demonstrates that PAC is potently inhibited by FFA, and offers detailed insights into the underlying mode of action. Furthermore, it is demonstrated here that OGR1 and PAC mediate the cellular responses to extracellular acidity by independent mechanisms. The work also highlights a varied role of Cl- channels in the control of vascular tone. In summary, this project reveals new cellular mechanisms that underpin the physiological response to extracellular acidity, and suggests new therapeutic approaches for pharmacological modulation of these mechanisms.

Keywords: PAC
Proton-activated chloride channel
Ion channel
Chloride channel
ASOR
Vasculature
Patch-clamp electrophysiology
Advisor: Tammaro, P
Tucker, S