Smelt, Charles L.C.; (2019) Allosteric modulation of pentameric ligand-gated ion channels. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The primary focus of this thesis is to examine the pharmacological properties of pentameric ligand-gated ion channels (pLGICs), also known as Cys-loop receptors. The family of human pLGICs includes nicotinic acetylcholine receptors (nAChRs), and receptors activated by 5-hydroxytryptamine, glycine, γ-aminobutyric acid (GABA). Signalling through pLGICs is facilitated through the binding of endogenous agonists to extracellular orthosteric binding sites. In addition, pLGICs can be modulated and activated via distinct allosteric binding sites. The pharmacological effects observed with these ‘allosteric’ compounds potentiation (a positive allosteric modulator, PAM) or inhibition (a negative allosteric modulator, NAM) of agonist-evoked responses as well as direct agonist activation (an allosteric agonist). In this thesis, a range of pharmacological, electrophysiological and molecular biological techniques have been employed to examine allosteric modulation and activation of vertebrate and invertebrate pLGICs expressed in native invertebrate neurons or Xenopus laevis oocytes. Using a revised structural model of the human α7 nAChR, a virtual screen identified several putative allosteric modulators. All of the 25 highest-ranked hits identified were inhibitors of one of four previously identified protein targets: carbonic anhydrase II (CAII), cyclin-dependent kinase 2 (CDK2), Na+-K+-Cl- cotransporter (NKCC) and DNA gyrase (DNAG). The top candidate for each category was DB04763, DB08122, DB00695 [furosemide] and DB00487 [pefloxacin], respectively. Using two-electrode voltage clamp electrophysiology, I showed that three of the compounds (DB04763, DB08122 and pefloxacin) were inhibitors of human α7, whereas furosemide was a potentiator of human α7. Site-directed mutagenesis (SDM) studies suggested that the compounds bind at the transmembrane intersubunit allosteric site of the α7 nAChR. Furosemide, identified in the virtual screen as a PAM of human α7, was reported to reduce seizures in rat models of autosomal dominant frontal lobe epilepsy (ADNFLE) but the mechanism of action was reported to be unknown (Yamada et al. 2013). These transgenic rat models harbour a mutation α4(S284L) within CHRNA4 gene, which encodes the α4 nAChR subunit. This mutation was identified in families with ADNFLE and in sporadic cases of nocturnal frontal lobe epilepsy (NFLE). I introduced the S284L mutation into human α4 and co-expressed with β2 resulting in a ten-fold shift for both α4β2 stoichiometries. This shift in agonist sensitivity has been thought to contribute to the epileptogenic phenotype. I observed that furosemide is an antagonist of ACh-evoked responses of human α4β2 nAChR. These data are indicative that furosemide may have a secondary antiepileptic mechanism through the inhibition of aberrant mutant α4(S284L)β2 nAChRs. Other work has focussed upon two types of anion-selective invertebrate receptors: glutamate-gated chloride channels (GluCl) and GABA-gated chloride channels (RDL) from pest and pollinator insect species. A population of ivermectin and spinosad resistant western flower thrips (WFT) was predicted to have a resistance-associated mutation within GluCl, the main target of ivermectin. I isolated GluCl from both resistant and susceptible WFT populations and found no resistant-associated mutation. Further studies are required to determine whether a mutation may be present in another target or the possible role of detoxifying enzymes. In order to investigate allosteric modulation of pollinator species, RDL and a GluCl was cloned from bumblebee (Bombus terrestris). I characterised a range of chloride channel antagonists (fipronil, picrotoxin, picrotin (PTN), and picrotoxinin (PNN)) on both bumblebee RDL and GluCl. Interestingly, picrotin, which was thought to be inactive on invertebrate Cys-loops, was an inhibitor of RDL; it was not able to inhibit agonist responses on GluCl, however. Indeed, I found that all the chloride channel compounds were RDL-selective, being more potent on RDL. Surprisingly, picrotoxin (an equimolar mix of PTN and PNN) was found to be more potent than the compounds alone, suggesting the compounds had a synergistic effect. SDM studies indicated that PTN and PNN may have different binding modes. These studies and the observed synergistic effect are indicative that these compounds may bind at an allosteric site distinct from the channel. I subsequently investigated the agonist and modulatory effects of monoterpenes on bumblebee RDL. Monoterpenes are phenolic compounds derived from plant essential oils (e.g thymol, menthol and cavacrol). Most compounds had a modulatory effect, but only some was able to activate the channel alone. I generated an in silico homology model of bumblebee RDL and identified a putative intersubunit allosteric binding site at the top of the transmembrane region, similar to that identified in human GABAA receptors. Finally, I observed that heterologously expressed Bombus terrestris RDL had a variable GABA sensitivity in Xenopus oocytes. A range of BtRDL GABA EC50 values, approximately between 3 – 30 μM, were observed depending on various conditions that affected receptor expression. The sensitivity to GABA was altered significantly by varying the amount of cRNA injected, the oocyte’s incubation time before functional analysis, co-expression with chaperones or the expression vector used to generate cRNA. A high level of expression resulted in large GABA-evoked responses (> 3 μA) that were highly sensitive to the activation by GABA, yielding a low EC50 (~ 3 - 10 μM). These data support the hypothesis that GABA-sensitivity of BtRDL is correlated to receptor surface expression.

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