Functional Domains of the Nicotinic Acetylcholine Receptor a

261

149

mentioning

confidence: 98%

d-Tubocurarine binding sites are located at alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor.

Pedersen

Cohen²

1990

261

149

“…A 4.6-Å structure (1) shows five Ϸ160-Å-long rod-shaped subunits surrounding a central channel. From the extracellular side the subunits have a counterclockwise order of ␣␥␣␦␤ (2,3). Each subunit has a large extracellular Nterminal or ligand-binding domain followed by four transmembrane regions, M1-M4 (4).…”

mentioning

confidence: 99%

A fluorophore attached to nicotinic acetylcholine receptor βM2 detects productive binding of agonist to the αδ site

Dahan

Dibas

Petersson

et al. 2004

110

To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the ␤19 position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by (⌬F͞F) Ϸ10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose-response relations for ⌬F agreed well with those for epibatidine-induced currents, but were shifted Ϸ100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the ␣␥-binding site than to the ␣␦ site and (ii) ACh binds with reverse-site selectivity, these data suggest that ⌬F monitors an event linked to binding specifically at the ␣␦-subunit interface. In experiments with flash-applied agonists, the earliest detectable ⌬F occurs within milliseconds, i.e., during activation. T he muscle nicotinic acetylcholine receptor (nAChR) is a well studied member of the Cys-loop family of neurotransmittergated ion channels. A 4.6-Å structure (1) shows five Ϸ160-Å-long rod-shaped subunits surrounding a central channel. From the extracellular side the subunits have a counterclockwise order of ␣␥␣␦␤ (2, 3). Each subunit has a large extracellular Nterminal or ligand-binding domain followed by four transmembrane regions, M1-M4 (4). The structure of the extracellular ligand-binding sites, at the ␣␥ and ␣␦ interfaces, resembles that of a homologous molluscan ACh-binding protein (3). Numerous biochemical and electrophysiological experiments indicate that M2 lines the channel (5).The nAChR exists in at least four distinct, interconvertible conformational states: resting, open, fast-onset-desensitized, and slow-onset-desensitized (6). The open and the fast-onsetdesensitized states presumably have moderate affinity for ACh, are metastable (on millisecond time scales), and are present in low concentrations at equilibrium. The supralinear doseresponse relation (Hill coefficient Ͼ1) suggests that the open state of the channel is much more likely to be associated with the presence of two bound agonist molecules than with a single bound agonist (7). In the prevailing kinetic scheme, receptors in the resting state bind two agonist molecules, isomerize to the open state, and in the continued presence of agonist, desensitize.After removal of agonist, the agonist-receptor complex dissociates and the channel closes within milliseconds; but desensitized receptors isomerize more slowly to the resting state (tens of milliseconds to hundreds of seconds). Thermodynamic considerations suggest that the resting state has low affinity for agonist, whereas the slow-onset-desensitized state is the most stable state in the presence of agonist because of its high affinity.Kinetic analyses of single-channel and macroscopic function suggests that in the resting state, the affinity of ACh for the two sites differs by a factor of...

“…1B), or 1.9 x 109 AChR molecules per oocyte. For the 250-kDa multisubunit AChR, the minimum center-to-center distance between channels is 9-10 nm (19,(22)(23)(24)(25). The channels in closely packed areas (Fig.…”

Section: Methodsmentioning

confidence: 99%

Atomic force microscopy of cloned nicotinic acetylcholine receptor expressed in Xenopus oocytes.

Lal

Yu²

1993

The nicotinic acetylcholine receptor (AChR) was expressed in Xenopus oocytes from in vitro transcribed mRNA and was imaged by atomic force microscopy. A characteristic pentameric structure of AChR was readily observed on the extracellular face of the cell membrane, with a central pore surrounded by protruding AChR subunits. These structures were seen only in mRNA-injected oocytes that also gave acetylcholine-induced membrane currents. The size of individual AChR channels, the angles between subunits, and the interchannel spacing were all compatible with the current model of AChR. In addition, localized patches of microscopic AChR clustering were observed, with packing density approaching that at the neuromuscular junction. These findings show the potential of studying cloned membrane proteins in oocytes for both their surface topography and their structurefunction relationship in native membrane without the need for crystallization.A large number of membrane proteins, including receptors, ion channels, and transporters, have been molecularly cloned, allowing characterization of their function. Based on the primary amino acid sequence of these proteins, their secondary structure and topology in the membrane have been proposed. However, their quaternary structure is mostly unknown. This is mainly due to the lack of a suitable method for three-dimensional imaging of membrane proteins: the existing techniques such as x-ray diffraction require crystallization, which is very difficult, if not impossible, for integral membrane proteins.Recently developed scanning probe microscopy, especially scanning tunneling microscopy (STM) and atomic force microscopy (AFM), can obtain three-dimensional images with nanometer resolution under physiological conditions (1-3). Images ofproteins, membranes, and nucleic acids have been obtained with . However, potential pitfalls of STM imaging of biological specimens exist (7), and image formation mechanisms are not known for relatively nonconducting biological materials of thickness greater than a few nanometers (2). This is because STM imaging relies on the probability of electron tunneling through the energy barrier, and such probability decreases exponentially with distance. AFM, on the other hand, is well suited for biological materials because it does not require electron-conductive materials, and has been used on many biological specimens (5,(8)(9)(10)(11)(12)(13)(14)(15), including macromolecules in noncrystalline form. However, membrane macromolecules studied by AFM were either purified or reconstituted in lipid vesicles. Such manipulations may alter the native topography of proteins due to either purification damage or altered lipid environment. In an attempt to image proteins in the intact cell membrane, we utilized Xenopus oocytes and expressed acetylcholine receptor (AChR) subunits from cDNA clones. We report here the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in a...