A conserved arginine with non‐conserved function is a key determinant of agonist selectivity in α7 nicotinic ACh receptors

Abstract

The α7 and α4β2* ("*" denotes possibly assembly with another subunit) nicotinic acetylcholine receptors (nAChRs) are the most abundant nAChRs in the mammalian brain. These receptors are the most targeted nAChRs in drug discovery programmes for brain disorders. However, the development of subtype-specific agonists remains challenging due to the high degree of sequence homology and conservation of function in nAChRs. We have developed C(10) variants of cytisine, a partial agonist of α4β2 nAChR that has been used for smoking cessation. The C(10) methyl analogue used in this study displays negligible affinity for α7 nAChR, while retaining high affinity for α4β2 nAChR. Experimental Approach: The structural underpinning of the selectivity of 10-methylcytisine for α7 and α4β2 nAChRs was investigated using molecular dynamic simulations, mutagenesis and whole-cell and single-channel current recordings. Key Results: We identified a conserved arginine in the β3 strand that exhibits a nonconserved function in nAChRs. In α4β2 nAChR, the arginine forms a salt bridge with an aspartate residue in loop B that is necessary for receptor expression, whereas in α7 nAChR, this residue is not stabilised by electrostatic interactions, making its side chain highly mobile. This lack of constrain produces steric clashes with agonists and affects the dynamics of residues involved in agonist binding and the coupling network. Conclusion and Implications: We conclude that the high mobility of the β3-strand arginine in the α7 nAChR influences agonist binding and possibly gating network and desensitisation. The findings have implications for rational design of subtypeselective nAChR agents.

A Functional Interaction Between Y674-R685 Region of the SARS-CoV-2 Spike Protein and the Human α7 Nicotinic Receptor

“…Xenopus care and housing followed the UK Home Office code of practice guidelines for the species. Stage V and VI Xenopus oocytes were prepared as previously described [ 28 ] and then injected with 2–6 ng of human α7 subunit cDNA into the nucleus in a volume of 23.0 nL, using a Nanoject Automatic Oocyte Injector (Drummond, Broomall, USA). To favour the expression of α7, its cDNA was co-injected with chaperone NACHO cDNA (GenBank accession no BC050273.1) at a ratio of 1 α7: 0.01 NACHO [ 21 , 29 ].…”

A Functional Interaction Between the SARS-CoV-2 Spike Protein and the Human α7 Nicotinic Receptor

“…Injected oocytes were incubated until use at 18ºC in a solution (OR2) containing 82 mM NaCl, 2 mM KCl, 2 mM MgCl2, 5 mM HEPES; pH 7.5, supplemented with 0.1 mg/mL streptomycin, 1000 U/mL Penicillin and 100 μg/mL amikacin. Oocytes were used for electrophysiological recordings one to two days after injection [27,28].…”

“…Xenopus care and housing followed the UK Home Office code of practice guidelines for the species. Stage V and VI Xenopus oocytes were prepared as previously described [27], and then injected with 2-6 ng of human α7 subunit cDNA into the nucleus in a volume of 23.0 nL, using a Nanoject Automatic Oocyte Injector (Drummond, Broomall, USA). To favour the expression of α7, its cDNA was coinjected with chaperone NACHO cDNA (GenBank accession no BC050273.1) [21,28] at a ratio of 1 α7: 0.01 NACHO.…”

“…Currents were recorded using an automated platform equipped with standard two electrode voltage-clamp configuration (HiClamp; Multi Channel Systems, Reutlignen, Germany). This system differs from standard electrophysiology and other automated platforms because, instead of applying the compound in the perfusion, the oocyte is moved into a well from a 96-well microtiter plate containing 230 l of the desired solution, as previously described [27]. Experiments were carried out only if the resting potential of the impaled oocytes was greater than -10 mV and the total holding current less than 0.2 A.…”

“…The peak of the ACh responses were normalised to the responses elicited by 1 mM ACh. For both set of studies, the normalised data were fit with the Hill equation using GraphPad software version 5, as previously described [27]. Data are expressed as means ± SEM from 5-6 oocytes obtained from at least three different batches of oocytes (N).…”