Sanders, Victoria R; (2022) Pharmacological characterisation of pentameric ligand-gated ion channels. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis primarily focuses on adding to the knowledge surrounding the pharmacological characteristics of pentameric ligand-gated ion channels, that are involved in neurotransmission in both vertebrate and invertebrate species. Employing a range of electrophysiological, computational, and molecular biology techniques, this thesis aimed to give insight into multiple pLGICs and more specifically the allosteric modulation of this superfamily of receptors. When focusing on human nicotinic receptors (nAChRs), an example of vertebrate pLGICs, this thesis details the effects of four compounds which were identified as potential allosteric modulators of alpha7 nAChRs in a virtual screening of a drug database. Of these four, furosemide was identified as a positive allosteric modulator of alpha7 nAChRs, potentiating the responses of endogenous ligands through a binding site distinct from that of the endogenous ligand. The remaining three were shown to be negative allosteric modulators, eliciting non-competitive inhibition of endogenous ligand responses. Two of the four compounds, pefloxacin and furosemide, were selected for further experimentation on alpha4beta2 nAChRs. In addition, analogues of these compounds were also tested to demonstrate the different pharmacological profiles of structurally similar compounds. In silico docking of compounds into alpha4beta2 nAChR structures and site-directed mutagenesis studies gave insight into a putative binding site for these compounds within the transmembrane domain of these receptors. This thesis also focused on two anion-selective invertebrate pLGICs: glutamate-gated chloride channels (GluCl) and GABA-gated chloride channels (RDL) cloned from Bombus terrestris. When characterising BtRDL there was an apparent change in GABA-affinity with expression levels. In addition, receptor populations with differing GABA-affinities had differing affinities for non-competitive inhibitors, picrotin and picrotoxinin. A selective allosteric agonist, okaramine-B, for BtGluCl was also characterised, showing agonist effects through a putative binding site that is distinct from that of glutamate as determined by in silico docking studies.

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