Nicotinic Receptor Binding Site Probed with Unnatural Amino Acid Incorporation in Intact Cells

Nowak, Mark W.; Kearney, Patrick C.; Sampson, Jeffrey R.; Saks, Margaret E.; Labarca, Cesar; Silverman, Scott; Zhong, Wenge; Thorson, Jon S.; Abelson, John; Davidson, Norman; Schultz, Peter G.; Dougherty, Dennis A.; Lester, Henry A.

doi:10.1126/science.7716551

Cited by 234 publications

(177 citation statements)

References 39 publications

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“…We can compare the above free energy losses consequent to mutations with estimates of the relative strengths, specifically, of cation-π interactions. Fluorination of the aromatic ring, which decreases cation-π forces, increases the EC 50 for ACh at TrpB and TyrC2 but not at TyrA (12,13,16). That cation-π interactions are large at TrpB and TyrC2 is in agreement with our free energy estimates.…”

Section: Discussionsupporting

confidence: 82%

“…1B). These residues were previously identified in AChRs (4)(5)(6)(7)(8), and many studies of the functional consequences of mutations confirm that all four contribute to both the agonist binding and channel gating phases of AChR activation (9)(10)(11)(12)(13)(14)(15)(16). In these experiments, the roles of the aromatic residues in AChR activation were assayed by measuring mutation-induced changes in binding affinities, dose-response EC 50 values of whole-cell currents, or activation rate constants estimated from single-channel currents.…”

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confidence: 99%

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Sources of energy for gating by neurotransmitters in acetylcholine receptor channels

Purohit

Bruhova

Auerbach

2012

Proc. Natl. Acad. Sci. U.S.A.

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Nicotinic acetylcholine receptors (AChRs) mediate signaling in the central and peripheral nervous systems. The AChR gating conformational change is powered by a low-to high-affinity change for neurotransmitters at two transmitter binding sites. We estimated (from single-channel currents) the components of energy for gating arising from binding site aromatic residues in the α-subunit. All mutations reduced the energy (TyrC1>>TrpB≈TyrC2>TyrA), with TyrC1 providing ∼40% of the total. Considered one at a time, the fractional energy contributions from the aromatic rings were TrpB ∼35%, TyrC1 ∼28%, TyrC2 ∼28%, and TyrA ∼10%. Together, TrpB, TyrC1, and TyrC2 comprise an "aromatic triad" that provides much of the total energy from the transmitter for gating. Analysis of mutant pairs suggests that the energy contributions from some residues are nearly independent. Mutations of TyrC1 cause particularly large energy reductions because they remove two favorable and approximately equal interactions between the aromatic ring and the quaternary amine of the agonist and between the hydroxyl and αLysβ7.A llosteric proteins receive energy from the environment at sensors and transfer it, via a global conformational change, to an effector site that controls the protein's functional output. In ion channels, the sensor energy can be chemical, electrical, mechanical, or thermal and the effector is a "gate" that regulates the ionic conductance of the pore. The nicotinic acetylcholine receptor (AChR) is a pentameric ligand-gated ion channel that mediates fast chemical transmission at vertebrate neuromuscular synapses. Agonist molecules, including the neurotransmitter ACh, bind at two sites that can undergo a low-to high-affinity conformational change that increases the probability of the protein adopting an active, ion-permeable shape. In this report, we describe some of the key sources of energy at the transmitter binding sites that serve to promote the AChR gating isomerization.The adult neuromuscular AChR is composed of four different but homologous subunits (1, 2) (α1 2 βδε; Fig. 1A). Each subunit has a β-barrel and four transmembrane α-helices. The five β-barrels form the extracellular domain, and the five sets of helices comprise the transmembrane domain. The two transmitter binding sites are in the extracellular domain at the α-δ and α-ε subunit interfaces. Each of these sites is ∼55 Å from the gate region in the transmembrane domain.The extracellular domain of the AChR α1-subunit has 59% of its sequence conserved with the Lymnaea stagnalis acetylcholine binding protein (AChBP). In the X-ray structures of AChBPs, the primary subunit of the ligand binding site (α-subunit in AChRs) has four conserved aromatic residues in three different loops: TyrA, TrpB, TyrC1, and TyrC2 (3) (Fig. 1B). These residues were previously identified in AChRs (4-8), and many studies of the functional consequences of mutations confirm that all four contribute to both the agonist binding and channel gating phases of AChR activation (9-16). In these experim...

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Section: Discussionsupporting

confidence: 82%

mentioning

confidence: 99%

Sources of energy for gating by neurotransmitters in acetylcholine receptor channels

Purohit

Bruhova

Auerbach

2012

Proc. Natl. Acad. Sci. U.S.A.

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“…The first sitedirected amino acid incorporation methods relied on chemically acylated tRNAs, which were added to the in vitro translation systems (Noren et al, 1989). Subsequently, through microinjection of aminoacylated tRNAs, membrane proteins could be tagged in vivo with unnatural amino acids in Xenopus oocytes (Nowak et al, 1995). The next step was to make this system even more simple and effective by selecting for aminoacyl-tRNA synthetases, which could aminoacylate the tRNAs in vivo.…”

Section: Site-directed Incorporation Of Unnatural Amino Acidsmentioning

confidence: 99%

Gelatinase-mediated migration and invasion of cancer cells

Björklund

Koivunen

2005

Biochimica et Biophysica Acta (BBA) - Reviews on Cancer

465

609

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“…modified tRNAs containing unnatural amino acids) [256]. This is an approach that has been used successfully to examine the role of amino acids located at the agonist binding site [256,257] and within the ion channel pore [258].…”

Section: Page 16 Of 40mentioning

confidence: 99%

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

Millar

2009

Biochemical Pharmacology

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Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop family of neurotransmitter-gated ion channels, a family that also includes receptors for γ-aminobutyric acid, glycine and 5-hydroxytryptamine. In humans, nAChRs have been implicated in several neurological and psychiatric disorders and are major targets for pharmaceutical drug discovery. In addition, nAChRs are important targets for neuroactive pesticides in insects and in other invertebrates. Historically, nAChRs have been one of the most intensively studied families of neurotransmitter receptors. They were the first neurotransmitter receptors to be biochemically purified and the first to be characterized by molecular cloning and heterologous expression. Although much has been learnt from studies of native nAChRs, the expression of recombinant nAChRs has provided dramatic advances in the characterization of these important receptors. This review will provide a brief history of the characterization of nAChRs by heterologous expression. It will focus, in particular, upon studies of recombinant nAChRs, work that has been conducted by many hundreds of scientists during a period of almost 30 years since the molecular cloning of nAChR subunits in the early 1980's.

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Nicotinic Receptor Binding Site Probed with Unnatural Amino Acid Incorporation in Intact Cells

Cited by 234 publications

References 39 publications

Sources of energy for gating by neurotransmitters in acetylcholine receptor channels

Sources of energy for gating by neurotransmitters in acetylcholine receptor channels

Gelatinase-mediated migration and invasion of cancer cells

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

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