A gating mechanism proposed from a simulation of a human α7 nicotinic acetylcholine receptor

150

The nicotinic acetylcholine receptor (nAChR) is a cation-selective channel central to both neuronal and muscular processes and is considered the prototype for ligand-gated ion channels, motivating a structural determination effort that spanned several decades [Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution. J Mol Biol 346:967-989]. Purified nAChR must be reconstituted in a mixture containing cholesterol to function. Proposed modes of interaction between cholesterol and the protein range from specific binding to indirect membranemediated mechanisms. However, the underlying cause of nAChR sensitivity to cholesterol remains controversial, in part because the vast majority of functional studies were conducted before a medium resolution structure was reported. We show that the nAChR contains internal sites capable of containing cholesterol, whose occupation stabilizes the protein structure. We detect sites at the protein-lipid interface as conventionally predicted from functional data, as well as deeply buried sites that are not usually considered. Molecular dynamics simulations reveal that occupation of both superficial and deeply buried sites most effectively preserves the experimental structure; the structure collapses in the absence of bound cholesterol. In particular, we find that bound cholesterol directly supports contacts between the agonist-binding domain and the pore that are thought to be essential for activation of the receptor. These results likely apply to those other ion channels within the Cys-loop superfamily that depend on cholesterol, such as the GABA receptor.protein-lipid interaction ͉ ligand-gated ion channel ͉ cys loop receptor C ritical for memory and cognition, the nAChR is implicated in neurological disorders (1), addiction (2, 3), and the mechanism of anesthetics (4). Early efforts to reconstitute the nAChR noted that cholesterol as well as charged lipids (such as phosphatidic acid) are required for proper function (5). Proposed mechanisms for the role of cholesterol in nAChR function fall into two general types: indirect and direct. Indirect mechanisms are those in which a cholesterol-induced change in fluid or elastic properties of the lipid bilayer modulates the protein behavior. Although early studies (6, 7) indicated that current flow through the channel is insensitive to bilayer fluid properties, more recent studies (8, 9) have proposed that this result depends on the choice of order parameters. The direct mechanism requires cholesterol binding to the nAChR and effecting structural and/or dynamic changes. Proponents of this theory have determined that (i) cholesterol molecules in proximity to the protein exchange very slowly with the bulk lipid bilayer (10) (electron spin resonance difference spectroscopy); (ii) cholesterol accesses sites on the protein that cannot be occupied by phospholipids (11) (fluorescence quenching); and (iii) some cholesterol bound to the nAChR interacts with the phospholipids in the surrounding bilayer (12) (substit...

Section: Dockingsupporting

confidence: 51%

Section: Dockingmentioning

confidence: 98%

mentioning

confidence: 99%

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Embedded cholesterol in the nicotinic acetylcholine receptor

Brannigan

Hénin

Law

et al. 2008

150

“…This last conclusion is supported by several structural gating mechanisms (15,16,33). In particular, normal mode analysis recently proposed that the conformational transition that causes the opening of the ion pore is essentially a quaternary twist motion of the protein accompanied by discrete tertiary changes of each subunit (16).…”

Section: Discussionmentioning

confidence: 62%

Molecular tuning of fast gating in pentameric ligand-gated ion channels

Grütter

Carvalho

Dufresne

et al. 2005

110

Neurotransmitters such as acetylcholine (ACh) and glycine mediate fast synaptic neurotransmission by activating pentameric ligandgated ion channels (LGICs). These receptors are allosteric transmembrane proteins that rapidly convert chemical messages into electrical signals. Neurotransmitters activate LGICs by interacting with an extracellular agonist-binding domain (ECD), triggering a tertiary͞quaternary conformational change in the protein that results in the fast opening of an ion pore domain (IPD). However, the molecular mechanism that determines the fast opening of LGICs remains elusive. Here, we show by combining whole-cell and single-channel recordings of recombinant chimeras between the ECD of ␣7 nicotinic receptor (nAChR) and the IPD of the glycine receptor (GlyR) that only two GlyR amino acid residues of loop 7 (Cys-loop) from the ECD and at most five ␣7 nAChR amino acid residues of the M2-M3 loop (2-3L) from the IPD control the fast activation rates of the ␣7͞Gly chimera and WT GlyR. Mutual interactions of these residues at a critical pivot point between the agonist-binding site and the ion channel fine-tune the intrinsic opening and closing rates of the receptor through stabilization of the transition state of activation. These data provide a structural basis for the fast opening of pentameric LGICs.allosteric proteins ͉ chimeric receptor ͉ Cys-loop receptor ͉ transition state P entameric ligand-gated ion channels (LGICs), such as the cationic nicotinic acetylcholine receptor (nAChR) and the anionic glycine receptor (GlyR), mediate fast excitatory or inhibitory chemical neurotransmission between neurons (1-6). A unique feature of these receptors is that they activate the ion channel, a process known as gating, in less than a ms. For nicotinic receptors, a detailed single-channel analysis has recently established a speed limit for the opening of the ion channel in the s time range (7). Perturbations of this rapid transmission pathway by natural mutants lead to severe diseases such as congenital myasthenic syndromes (8), hereditary hyperekplexia (9), or epileptic disorders (10).Pentameric LGICs, or Cys-loop receptors, are composed of five homologous subunits, sharing a common structural organization, arranged (pseudo)symmetrically around the central ion pore (1, 2). All subunits are made of two distinct topological domains: the extracellular (ECD) and the ion pore domains (IPD). First, the ECD is folded into a twisted ␤-sandwich core, as revealed by x-ray crystallographic studies of the mollusk acetylcholine-binding protein (AChBP), a soluble pentameric protein homologous to the extracellular domain of LGICs (11-14). Second, electron microscopy images of Torpedo nAChR at 4-Å resolution revealed that the four transmembrane segments (M1 to M4) of the IPD are folded into ␣-helices joined by linking loops of variable lengths (15). By combining these structural data, we built a 3D model of the full ␣7 nAChR (16). In this model, the coupling zone located at the interface between the two domains is framed by f...

“…allostery ͉ spontaneous opening ͉ ligand-gated ion channel ͉ ligand binding N icotinic AChRs (nAChRs) are a well studied prototype for ligand-gated ion channels (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). nAChRs are pentamers consisting of either homo or hetero subunits.…”

mentioning

confidence: 99%

Spontaneous conformational change and toxin binding in α7 acetylcholine receptor: Insight into channel activation and inhibition

Tjong

Zhou

2008

Nicotinic AChRs (nAChRs) represent a paradigm for ligand-gated ion channels. Despite intensive studies over many years, our understanding of the mechanisms of activation and inhibition for nAChRs is still incomplete. Here, we present molecular dynamics (MD) simulations of the ␣7 nAChR ligand-binding domain, both in apo form and in ␣-Cobratoxin-bound form, starting from the respective homology models built on crystal structures of the acetylcholine-binding protein. The toxin-bound form was relatively stable, and its structure was validated by calculating mutational effects on the toxin-binding affinity. However, in the apo form, one subunit spontaneously moved away from the conformation of the other four subunits. This motion resembles what has been proposed for leading to channel opening. At the top, the C loop and the adjacent ␤7-␤8 loop swing downward and inward, whereas at the bottom, the F loop and the C terminus of ␤10 swing in the opposite direction. These swings appear to tilt the whole subunit clockwise. The resulting changes in solvent accessibility show strong correlation with experimental results by the substituted cysteine accessibility method upon addition of acetylcholine. Our MD simulation results suggest a mechanistic model in which the apo form, although predominantly sampling the ''closed'' state, can make excursions into the ''open'' state. The open state has high affinity for agonists, leading to channel activation, whereas the closed state upon distortion has high affinity for antagonists, leading to inhibition.allostery ͉ spontaneous opening ͉ ligand-gated ion channel ͉ ligand binding N icotinic AChRs (nAChRs) are a well studied prototype for ligand-gated ion channels (1-19). nAChRs are pentamers consisting of either homo or hetero subunits. The N-terminal extracellular regions of the subunits make up the ligand-binding domain (LBD), with two or five binding sites in hetero and homo pentamers, respectively. The central regions form the transmembrane domain (TMD), which is an ion channel selective for cations such as Na ϩ , K ϩ , and Ca 2ϩ [supporting information (SI) Fig. S1]. A central issue is the mechanisms of channel activation by agonists and inhibition by antagonists. How is agonist/ antagonist binding transmitted to trigger channel opening/ closing? What are the necessary conformational changes? Experimental and computational studies together have begun to provide mechanistic insights at the atomic level. Here, we present a molecular dynamics (MD) simulation study of the ␣7 nAChR LBD (consisting of five ␣-type subunits), both in apo form and in ␣-Cobratoxin-bound form. Our simulation results are in broad agreement with a wealth of experimental data. Combining the present work with experimental data and previous computational results, we propose a mechanistic model in which the apo form, although predominantly sampling the ''closed'' state, can make excursions into the ''open'' state. Details of the conformational change leading to channel activation/inhibition are presented.A number ...