Conformational Changes in the Nicotinic Acetylcholine Receptor during Gating and Desensitization

Abstract

The nicotinic acetylcholine receptor (nAChR) is a member of the important Cys-loop ligand-gated ion channel superfamily that modulates neuronal excitability. After responding to their agonists, their actions are terminated either by removal of ligand or by fast and slow desensitization, processes that play an important role in modulating the duration of conducting states and hence of integrated neuronal behavior. We monitored structural changes occurring during fast and slow desensitization in the transmembrane domain of the Torpedo nAChR using time-resolved photolabeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-iodophenyl) diazirine (TID). After channel opening, TID photolabels a residue on the δ-subunit's M2-M3 loop and a cluster of four residues on δM1 and δM2, defining an open state pocket [Arevalo, E. et al. (2005) J. Biol. Chem. 280, 13631-13640]. We now find that photolabeling of this pocket persists during the transition to the fast desensitized state, decreasing only with the transition to the slow desensitized state. In contrast, photoincorporation in the channel lumen at the conserved 9′ leucines on the second transmembrane helix (M2-9′) decreased successively during the resting to open and open to fast desensitized state transitions, implying that the local conformation is different in each state, a conclusion consistent with the hypothesis that there are separate gates for channel opening and desensitization. Thus, although during fast desensitization there is a conformation change in the channel lumen at the level of M2-9′, there is none in the regions of the δ-subunit's M2-M3 loop and the interior of its M1-M4 helix bundle until slow desensitization occurs.The Cys-loop ligand-gated ion channel superfamily, which includes nicotinic acetylcholine receptors (nAChRs), 5-hydroxytryptamine type 3 (5-HT 3 ) receptors, γ-aminobutyric acid type A (GABA A ) receptors, and glycine receptors, has been studied intensively. These channels modulate synaptic and extrasynaptic neuronal excitability in response to their agonists and their actions are terminated either by removal of ligand or by desensitization, a process that plays an important role in modulating the duration of ligand-gated conducting states and hence of integrated neuronal behavior (1-3). A primary challenge is not only to understand how agonist binding in the extracellular ligand-binding domain (LBD) triggers a conformation *To whom correspondence may be addressed: Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, 32 Fruit St., Boston. MA 02114. Tel.: 617-726-8985, Fax: 617-724-8644, k_miller@helix.mgh.harvard.edu. IHY and DCC contributed equally to this work. Supporting InformationSupporting information is available showing HPLC purification of labeled peptides and a molecular model. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 January 12. Published in final ...

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