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

Abstract

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...

Conformational Variability of the Glycine Receptor M2 Domain in Response to Activation by Different Agonists

“…2, C and D). The blue shift in the MTSR emission peak is characteristic of an increase in hydrophobicity in the fluorophore environment, most likely reflecting a movement of the fluorophore to a more hydrophobic environment in the open state (15). As expected, MTSR-incubated oocytes expressing ␣1WT GlyRs did not show ⌬F or spectral shifts (Fig.…”

“…Finally, Dahan et al (15) investigated acetylcholine-and epibatidine-mediated ⌬F and ⌬I changes at muscle (␣␤␥␦) nAChRs via a rhodamine label attached to the ␤ subunit 19Ј position. They concluded that ⌬F reported a conformational change at the ␣␦ subunit interface.…”

“…However, it has been suggested on the basis of substituted cysteine accessibility studies that the pore blocker, picrotoxin, can change the conformation of the outer GlyR pore region suggesting in turn that it may interact allosterically with agonistinduced conformational changes (14). Our aim is to investigate the conformational changes induced by agonists, antagonists, and allosteric modulators by covalently labeling different residues near the extracellular M2 boundary with the sulfhydrylreactive fluorophores, methanethiosulfonate-rhodamine (MTSR) and tetramethylrhodamine-maleimide (TMRM), and simultaneously measuring current changes and fluorescence changes (15,16). This approach has been employed so far in only a few studies on Cys loop receptors (15,17,18).…”

“…Our aim is to investigate the conformational changes induced by agonists, antagonists, and allosteric modulators by covalently labeling different residues near the extracellular M2 boundary with the sulfhydrylreactive fluorophores, methanethiosulfonate-rhodamine (MTSR) and tetramethylrhodamine-maleimide (TMRM), and simultaneously measuring current changes and fluorescence changes (15,16). This approach has been employed so far in only a few studies on Cys loop receptors (15,17,18). A particular advantage of the GlyR is that it displays little or no desensitization thereby facilitating the quantitation of ligand-induced fluorescence spectral shifts.…”

Functional asymmetry of transmembrane segments in nicotinic acetylcholine receptors

“…The conformational changes are not necessarily the same for all subunits (Dahan et al 2004;Grosman and Auerbach 2000) and structural data from electron microscopy from Torpedo californica nAChR propose that rotational movements of the a subunits promote channel gating (Miyazawa et al 2003;Unwin 2005).…”