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A chimera encoding the fusion of an acetylcholine-binding protein to an ion channel is stabilized in a state close to the desensitized form of ligand-gated ion channels

Abstract : To understand the mechanism of allosteric coupling between the ligand-binding domain and the ion channel of the Cys-loop ligand-gated ion channels (LGICs), we fused the soluble acetylcholine-binding protein (AChBP), which lacks an ion channel, to either the cationic serotonin type-3A ion channel (5HT(3A)) or the anionic glycine ion channel. Both linear chimeras expressed in HEK-293 cells display high affinity for the nicotinic agonist epibatidine (K(D) = 0.2-0.5 nM), but are not targeted to the cell surface. Only after substituting a ring of three loops located at the putative membrane side of the AChBP three-dimensional structure by the homologous residues of 5HT(3A), the resulting chimera AChBP(ring)/5HT(3A) (i) still displayed on intact cells an apparent high affinity for epibatidine, yet with a fourfold decrease (K(D) = 2.1 nM), (ii) displayed a high proportion of low affinity sites (11 +/- 7 microM) for the resting state stabilizing competitive antagonist alpha-bungarotoxin and (iii) was successfully targeted to the cell surface, as seen by immunofluorescence labelling. The AChBP(ring)/5HT(3A) chimera forms a pentameric structure, as revealed by sucrose gradient sedimentation. However, no whole-cell patch-clamp currents were detectable. Interestingly, binding assays with membrane fragments prepared from cells expressing AChBP(ring)/5HT(3A) showed a decrease in the apparent affinity for the agonists nicotine and epibatidine (5-fold), concomitant with an increase in the proportion of high-affinity sites (48 +/- 1 nM) for alpha-bungarotoxin. These results indicate that fusion of AChBP to an ion channel forms a pentameric receptor exposed to the cell surface and able to convert between discrete allosteric states, but stabilized in a high affinity state for epibatidine that likely corresponds to a desensitized form of LGICs. These artificial chimeras might offer a useful system to investigate signal transduction in LGICs.
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https://hal-pasteur.archives-ouvertes.fr/pasteur-00162535
Contributor : Yolande Meunier <>
Submitted on : Friday, July 13, 2007 - 4:17:28 PM
Last modification on : Wednesday, October 14, 2020 - 3:45:06 AM

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Thomas Grutter, Lia Prado de Carvalho, Virginie Dufresne, Antoine Taly, Markus Fischer, et al.. A chimera encoding the fusion of an acetylcholine-binding protein to an ion channel is stabilized in a state close to the desensitized form of ligand-gated ion channels. Comptes Rendus Biologies, Elsevier Masson, 2005, 328 (3), pp.223-34. ⟨10.1016/j.crvi.2004.11.004⟩. ⟨pasteur-00162535⟩

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