Cooper, Sandra T.; (1998) Host cell-specific folding of the neuronal nicotinic acetylcholine receptor α7 and α8 subunits. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The α7 and α8 nicotinic acetylcholine receptor (nAChR) subunits when expressed in Xenopus oocytes, form functional homo-oligomeric ion channels which are potently inhibited by the nicotinic antagonist α-bungarotoxin (αBTX). However, expression of the rat α7 and chick α8 subunits in several mammalian cell lines demonstrates that the folding of α7 and α8 into a conformation able to bind αBTX or conformation- sensitive antibodies, is critically dependent on the host cell type. In all cell lines, production of α7 (~58 kDa) or α8 (~57 kDa) subunit protein was verified by metabolic labelling and immunoprecipitation using subunit-specific antibodies which recognise linear epitopes. In contrast, for both α7 and α8 subunits, antibodies which recognise conformation-dependent extracellular epitopes fail to detect subunit protein in cell lines which fail to produce nicotinic radioligand binding, implying that these subunits are misfolded. The α7 subunit was expressed in a panel of nine mammalian cell lines from neuronal (N1E-115, NCB-20, Neuro2A, NG108-15, PC12, SH-SY5Y) and non-neuronal (HEK293, CHO, COS-7) origins. However, elevated levels of αBTX-binding could be detected in only two (PC12 and SH-SY5Y) of the nine cell lines examined. The α8 subunit was expressed in three cell lines (HEK293, GH4C1, SH-SY5Y) and readily formed αBTX-binding sites in a polyclonal SH-SY5Y-α8 cell line and when transiently expressed in GH4C1 cells. Saturation binding revealed that α8 nAChRs expressed in transfected GH4C1-α8 cells bind epibatidine with high affinity (KD=0.24 ± 0.4 nM) although no specific αBTX or epibatidine binding could be detected in transfected HEK293-α8 cells. HEK293 cells which fail to correctly fold the α7 and α8 nAChR subunits form functional cell surface rat muscle nAChRs and homo-oligomeric 5-HT3 receptors. In addition, chimaeric subunits encoding the N-terminal region of α7 or α8 and the C-terminal domain of the 5-HT3 receptor were expressed very efficiently in all cell lines examined. These results implicate the C-terminal domain of α7 and α8 in the cell-type specific folding and further chimeras need to be generated to more precisely determine which region mediates this effect.

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