The Lipid-Facing M4 Helix of α4β2 nACh and 5-HT3A Receptors

Mesoy, Susanne 

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Pentameric ligand-gated ion channels (pLGICs) are expressed throughout the human nervous system, and contribute to a range of muscle, gut, and neurological functions. Elucidating their mechanism of action and how it might be modulated would improve our understanding of the nervous system, and contribute to building tools to treat diseases arising from dysregulated pLGICs.

The outermost lipid-facing transmembrane helix (M4) and the lipids surrounding it have recently emerged as important factors in pLGIC function. To investigate the role of the M4 helix in cation-selective mammalian pLGICs, I studied the effects of mutations in the M4 helices of the 5-HT3A and α4β2 nACh receptors. I used a membrane potential-sensitive fluorescent dye, two-electrode voltage clamp and manual patch-clamp for functional characterisation of mutant receptors in HEK293 cells and Xenopus oocytes, and radioligand binding and immunofluorescence to assess ligand binding and receptor expression.

I show that 1 out of 28 alanine mutations in the 5-HT3AR M4 and 8 out of28 double alanine mutations in the α4β2 nAChR abolish receptor function in HEK cells without ablating ligand binding, indicating that the M4 helices of these cation-selective pLGICs are involved in, and can modulate, receptor function. I explored the mechanism of action of these key M4 residues by characterising prospective interaction partners, and identified a potential chain of interactions going from the outermost M4 helix all the way to the channel pore.

I also show that eight of the nine 5-HT3AR and α4β2 nAChR mutants that showed ligand binding but no receptor function in HEK cells, showed WT-like function when expressed in Xenopus oocytes. In addition, M4 mutations that altered the function of receptors expressed in HEK cells had different effects on receptors expressed in oocytes. Together this shows that the role of the M4 helix in cation-selective pLGIC function depends on the expression system.

For comparison, I investigated another peripheral helix in the 5-HT3AR; the N-terminal helix, which rests above the extracellular domain of mammalian pLGICs, and showed that it is important for correct receptor expression.

Overall, this work shows that the M4 helix of cation-selective pLGICs is an attractive target for receptor modulation by small-molecule binding, as this helix is both accessible, poorly conserved between pLGICs, and intimately involved in receptor function. It has also laid the groundwork for further understanding the functional mechanism of pLGICs, especially the interactions of the M4 helices and with the rest of the transmembrane helical bundle. Finally, it has highlighted the dependency on the expression system of both pLGIC function and of the role of the M4 helix, and emphasises the need to understand the native environment of these receptors and how that modulates function.

Lummis, Sarah
pLGIC, Cys-loop receptor, 5-HT3, Serotonin receptor, nicotinic acetylcholine receptor
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge