Conformation-dependent hydrophobic photolabeling of the nicotinic receptor: Electrophysiology-coordinated photochemistry and mass spectrometry

Leite, John F.; Blanton, Michael P.; Shahgholi, Mona; Dougherty, Dennis A.; Lester, Henry A.

doi:10.1073/pnas.2133028100

Cited by 50 publications

(54 citation statements)

References 60 publications

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“…By contrast, loops 2 and 7 appear to be located within close proximity of the pre-TM1 region only in the presence of GABA, suggesting that these domains move relative to one another during the open or desensitized state. It is of interest to note that a recent study that examined state-selective incorporation of hydrophobic probes in the nAChR identified loop 7 of the nAChR ␣ 1 subunit as a region that undergoes a conformational change during receptor activation (32). Based on these results, we propose that there are intramolecular interactions between loop 7 and the pre-TM1 region that are important for receptor activation.…”

Section: Double Mutant Experiments Reveal Asp 146 -Lys 215mentioning

confidence: 77%

Charged Residues in the β2 Subunit Involved in GABAA Receptor Activation

Kash¹,

Dizon

Trudell

et al. 2004

Journal of Biological Chemistry

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Fast synaptic inhibition in the mammalian central nervous system is mediated primarily via activation of the ␥-aminobutyric acid type A receptor (GABA A -R). Upon agonist binding, the receptor undergoes a structural transition from the closed to the open state. This transition, known as gating, is thought to be associated with a sequence of conformational changes originating at the agonist-binding site, ultimately resulting in opening of the channel. Using site-directed mutagenesis and several different GABA A -R agonists, we identified a number of highly conserved charged residues in the GABA A -R ␤ 2 subunit that appear to be involved in receptor activation. We then used charge reversal double mutants and disulfide trapping to investigate the interactions between these flexible loops within the ␤ 2 subunit. The results suggest that interactions between an acidic residue in loop 7 (Asp 146 ) and a basic residue in pretransmembrane domain-1 (Lys 215 ) are involved in coupling agonist binding to channel gating.Fast synaptic inhibition is a major determinant of network dynamics in the central nervous system (1). In the mammalian brain, this is mediated primarily via activation of the ␥-aminobutyric acid type A receptor (GABA A -R) 1 (2, 3). Each GABA A -R is composed of five subunits arranged around a central ion-conducting pore (4), with each subunit consisting of a large intracellular domain, four transmembrane domains (TM1-TM4), and a larger N-terminal extracellular domain (2). Experimental evidence suggests that the GABA-binding site lies within an asymmetric pocket formed at the interface between the ␣ and ␤ receptor subunits (5-8). The channel "gate" in the GABA A -R is believed to be formed by charged residues in the TM1-TM2 loop (9 -11). The binding of agonist triggers a complex structural transition that results in the opening of the gate, allowing ions to flow through the channel. The mechanisms by which this occurs remain poorly defined.The nature of the coupling between binding and channel opening in this receptor family has been recently investigated in several laboratories. Interactions between charged residues in the flexible loops 2 and 7 in the extracellular domain and those in the short linker between the second and third transmembrane domains (TM2-3L) of the GABA A -R ␣ 1 subunit have been implicated in the process of receptor activation (12). In addition, a recent report suggests that the pre-TM1 region is critical for receptor activation in the closely related serotonin (5-HT 3 ) receptor (13). In this study, we used site-directed mutagenesis and a number of GABA A -R agonists of varying efficacies to examine the contribution of the corresponding flexible loops in the GABA A -R ␤ 2 subunit to receptor activation. Based on previous studies (12-14) and the presence of highly conserved charged residues (see Fig. 1), we hypothesized that interactions between charged residues in these domains are crucial for coupling agonist binding to channel gating. We tested this hypothesis using a charge revers...

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Section: Double Mutant Experiments Reveal Asp 146 -Lys 215mentioning

confidence: 77%

Charged Residues in the β2 Subunit Involved in GABAA Receptor Activation

Kash¹,

Dizon

Trudell

et al. 2004

Journal of Biological Chemistry

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“…The conformational change of loop F during channel activation is further supported by increased photolabeling of loop F in the α1 subunit of nAChR in the open state, although the direction of the movement is not completely clear [53] . Mutation of a loop F residue (εD175N) of nAChR clearly influences channel gating, suggesting the importance of the loop F in channel activation [54] .…”

Section: Activation Mechanismmentioning

confidence: 99%

Allosteric activation mechanism of the cys-loop receptors

et al. 2009

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Binding of a neurotransmitter to its ionotropic receptor opens a distantly located ion channel, a process termed allosteric activation. Here we review recent advances in the molecular mechanism by which the cys-loop receptors are activated with emphasis on the best studied nicotinic acetylcholine receptors (nAChRs). With a combination of affinity labeling, mutagenesis, electrophysiology, kinetic modeling, electron microscopy (EM), and crystal structure analysis, the allosteric activation mechanism is emerging. Specifically, the binding domain and gating domain are interconnected by an allosteric activation network. Agonist binding induces conformational changes, resulting in the rotation of a β sheet of aminoterminal domain and outward movement of loop 2, loop F, and cys-loop, which are coupled to the M2-M3 linker to pull the channel to open. However, there are still some controversies about the movement of the channel-lining domain M2. Nine angstrom resolution EM structure of a nAChR imaged in the open state suggests that channel opening is the result of rotation of the M2 domain. In contrast, recent crystal structures of bacterial homologues of the cys-loop receptor family in apparently open state have implied an M2 tilting model with pore dilation and quaternary twist of the whole pentameric receptor. An elegant study of the nAChR using protonation scanning of M2 domain supports a similar pore dilation activation mechanism with minimal rotation of M2. This remains to be validated with other approaches including high resolution structure determination of the mammalian cys-loop receptors in the open state.

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“…Numerous MS-based covalent labeling experiments have been conducted on membrane proteins [28,29], e.g., for topologic investigations [30], as well as for monitoring conformational changes [29,[31][32][33][34] and binding interactions [35][36][37].…”

mentioning

confidence: 99%