Alternate α4β2 nicotinic acetylcholine receptors of defined subunit composition and arrangement

Mirko Moroni, Anna Lisa Carbone, Isabel Bermudez
Oxford Brookes Universities, Oxford, United Kingdom

The α4β2 nicotinic acetylcholine receptor (nAChR) is the most abundant nAChR in the brain where it forms the high-affinity binding site for nicotine. α4β2 nAChRs belong to the Cys-loop family of ligand gated ion channels. When expressed in heterologous systems α4 and β2 subunits produce a mixture of two alternate stoichiometries, (α4)2(β2)3, and (α4)3(β2)2, that differ in functional pharmacology, desensitisation, single channel properties and sensitivity to chronic exposure to nicotine. In order to facilitate studies of each of these receptor forms, we have concatenated the five subunits of the α4β2 receptor using two different subunit orders to yield functional (α4)2(β2)3, and (α4)3(β2)2 nAChRs.

Concatenated (α4)2(β2)3, and (α4)3(β2)2 nAChRs were constructed using standard molecular biological procedures. The subunit order chosen for the (α4)3(β2)2 receptor was β_α_β_α_α Whilst the subunit order adopted for the (α4)2(β2)3 receptor was β_α_β_α_β. The signal peptide was removed from the second, third, fourth and fifth subunits. AGS oligonucleotides were used to link the subunits and the subunits-linkers were assembled as pentamers using standard ligation procedures. The concatenated constructs were cloned into the expression vector pCI (Promega, UK) and then expressed in Xenopus oocytes using cytoplasmic injection of capped RNAs. Expression levels and functional properties of the concatenated receptors were assessed by two-electrode voltage voltage-clamp recordings.

Robust functional expression of both constructs was achieved in Xenopus oocytes. Currents elicited 72 hours after microinjection of β_α_β_α_α and β_α_β_α_β amounted to about 70% and 44% respectively compared to currents elicited by non-linked subunits. Concentration response curves with the agonist ACh indicated that subunit concatenation had no significant effect (p < 0.05, student’s t tests) on agonist potency: linked (α4)2(β2)3 EC50 2.4 (2.1 – 2.7) μM (n = 4); non-linked (α4)2(β2)3 EC50 2.8 (2.1 - 3.7) μM (n = 5); linked (α4)3(β2)2 EC50 119 (79 – 183) (n = 4), non-linked (α4)3(β2)2 EC50 89 (73 – 109) μM (n = 6). The sensitivity of the linked receptor to the antagonist dihydro-beta-erythroidine was also similar to that of the non-linked receptors: (α4)2(β2)3 IC50 16 (11-17 nM (n = 4); non-linked (α4)2(β2)3 IC50 17 (14-20) nM (n = 5); linked (α4)3(β2)2 IC50 0.42 (0.36-0.44) (n = 4) μM, non-linked (α4)3(β2)2 IC50 0.31 (0.23-0.35) μM (n = 6).

The ability to express defined stoichiometries of the α4β2 nAChR that display similar properties to their non-linked counterparts should facilitate the development of stoichiometry-selective compounds as well as detailed studies of the functional structure of the alternate forms of the α4β2 nAChR.

Acknowledgement. MM and ALC contributed equally to the work and were supported by Oxford Brookes University PhD studentships. We thank Lucia Sivillotti, Paul Groot-Kormelink and Erwin Sigel for helpful advice.