Acetylcholine receptor M3 domain: stereochemical and volume contributions to channel gating

Wang, Hailong; Milone, Margherita; Ohno, Kinji; Shen, Xing Ming; Tsujino, Akira; Batocchi, Anna Paola; Tonali, P.; Brengman, Joan M.; Engel, Andrew G.; Sine, Steven M.

doi:10.1038/6326

Cited by 116 publications

(86 citation statements)

References 29 publications

(19 reference statements)

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“…Any of these changes may reduce the energy barrier for channel opening and, thus, facilitate the transition from a closed to an open state. Mutations at Val-385 of the ␣ subunit of muscle type nACh receptors revealed that both volume and stereochemistry contribute to channel gating but not to binding affinity (39). The observation that some 5-HT 3A receptor mutants, such as R222F and R222I, exhibits spontaneous channel opening (14) is consistent with the notion that Arg-222 mutations can reduce the energy barrier for channel opening.…”

Section: Discussionsupporting

confidence: 72%

Arginine 222 in the Pre-transmembrane Domain 1 of 5-HT3A Receptors Links Agonist Binding to Channel Gating

Zhang

Stewart

et al. 2003

Journal of Biological Chemistry

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Ligand-gated ion channels are integral membrane proteins that mediate fast synaptic transmission. Molecular biological techniques have been extensively used for determining the structure-function relationships of ligand-gated ion channels. However, the transduction mechanisms that link agonist binding to channel gating remain poorly understood. Arginine 222 (Arg-222), located at the distal end of the extracellular N-terminal domain immediately preceding the first transmembrane domain (TM1), is conserved in all 5-HT 3A receptors and ␣7-nicotinic acetylcholine receptors that have been cloned. To elucidate the possible role of Arg-222 in the function of 5-HT 3A receptors, we mutated the arginine residue to alanine (Ala) and expressed both the wildtype and the mutant receptor in human embryonic kidney 293 cells. Functional studies of expressed wild-type and mutant receptors revealed that the R222A mutation increased the apparent potency of the full agonist, serotonin (5-HT), and the partial agonist, 2-Me-5-HT, 5-and 12-fold, respectively. In addition, the mutation increased the efficacy of 2-Me-5-HT and converted it from a partial agonist to a full agonist. Furthermore, this mutation also converted the 5-HT 3 receptor antagonist/ very weak partial agonist, apomorphine, to a potent agonist. Kinetic analysis revealed that the R222A mutation increased the rate of receptor activation and desensitization but did not affect rate of deactivation. The results suggest that the pre-TM1 amino acid residue Arg-222 may be involved in the transduction mechanism linking agonist binding to channel gating in 5-HT 3A receptors.In the nervous system, serotonin type 3 (5-HT 3 ) 1 receptors can mediate fast excitatory synaptic transmission and modulate neurotransmitter release (1). To date two 5-HT 3 receptor subunits have been identified: 5-HT 3A and 5-HT 3B (2, 3). The 5-HT 3A receptor subunits can form functional channels homomerically (2), whereas the 5-HT 3B receptor subunits are nonfunctional when expressed alone (3). However, the 5-HT 3B receptor subunits can form heteromeric channels with the 5-HT 3A receptor subunits, which results in modified biophysical characteristics compared with homomerically expressed 5-HT 3A receptor subunits (3). 5-HT 3A and 5-HT 3B receptor subunits also have different distribution patterns in the nervous system. The 5-HT 3A receptor subunits are expressed in both central and peripheral neurons, whereas the 5-HT 3B receptor subunits are restricted to peripheral neurons (4). This suggests that homomeric 5-HT 3A receptors play a dominant role in 5-HT 3 receptor-mediated responses in the central nervous system. 5-HT 3 receptors belong to a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine (nACh) receptors, glycine receptors, and ␥-aminobutyric acid type A receptors (5). The subunits in this superfamily are thought to assemble as pentamers with each subunit containing a large extracellular N-terminal domain, four transmembrane domains (TM1-TM4), a large intracellular loop be...

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Section: Discussionsupporting

confidence: 72%

Arginine 222 in the Pre-transmembrane Domain 1 of 5-HT3A Receptors Links Agonist Binding to Channel Gating

Zhang

Stewart

et al. 2003

Journal of Biological Chemistry

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“…Also, the non-functionality of two of these mutations (␣M282W and ␣V285W) agree with the previous ␣M3 Torpedo study (32), thus suggesting that ␣Met-282 and ␣Val-285 are critical constraint positions in both AChR species. Furthermore, previous mutations at the ␣Val-285 position revealed that changes in stereochemistry and volume at this site affect the ion channel function of the AChR (23,32,33).…”

Section: Discussionmentioning

confidence: 99%

“…MFVAI (blue font) and MLFTMIF*V*IS*S*II (highlighted in turquoise) were studied using tryptophan-scanning mutagenesis and patch clamp (32). V (violet font and italic) and F (blue font and italic) were studied using site-directed mutagenesis and patch clamp (23,31). F and S were labeled as lipid-exposed using photolabeling affinity (10).…”

mentioning

confidence: 99%

Tryptophan-scanning Mutagenesis in the αM3 Transmembrane Domain of the Muscle-type Acetylcholine Receptor

Otero-Cruz

Baéz-Pagán

Caraballo-González

et al. 2007

Journal of Biological Chemistry

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“…Electrophysiological studies were performed in BOSC cells (20) using the cell-attached patch-clamp method essentially as described previously (21). For electrophysiological studies, BOSC cells (20), a variant of the HEK293 cell line, were transfected with human wild-type or mutant nAChR subunit cDNAs using calcium phosphate precipitation.…”

Section: Methodsmentioning

confidence: 99%

An Ion Selectivity Filter in the Extracellular Domain of Cys-loop Receptors Reveals Determinants for Ion Conductance

Hansen

Wang

Taylor

et al. 2008

Journal of Biological Chemistry

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Ion selectivity defines two major classes of Cys-loop receptors. Receptors that selectively translocate cations are excitatory and include vertebrate nAChRs 3 and 5-HT 3 receptors, whereas receptors that selectively translocate anions are inhibitory and include ␥-aminobutyric acid and glycine receptors. A molecular basis for ion selectivity was first proposed based on conserved rings of charged residues and the observation that mutations of these residues in the nAChR influence conductance and selectivity (supplemental Fig. S1) (1). Subsequent studies have focused on reversing selectivity (2, 3) and comparing determinants of ion conductance in nicotinic receptors with those in other Cys-loop receptors (4). These studies described ion selectivity filters in transmembrane-spanning domains using mutagenesis and electrophysiological techniques. More recently, mutations of residues in a channel cytoplasmic region altered conductance in 5-HT 3A receptors (5), suggesting that other domains form vestibules leading into the channel that may influence ion conductance and selectivity. In Cys-loop receptors, a large N-terminal domain encloses a vestibule that extends from the constricted ion pore extracellularly by 60 Å. Structural and computational studies have suggested that regions within the N-terminal domain contribute to ion conductance and selectivity (6, 7), but direct experimental evidence is lacking.Also lacking is a chemical description of ion selectivity and conductance in Cys-loop receptors at the atomic level. Cryoelectron microscopy applied to the nAChR from Torpedo provided structural information at a resolution of 4 Å (6, 8), but single ions and most amino acid side chains could not be resolved. Currently, our understanding of ion translocation through channels comes from studies on voltage-gated ion channels (9 -11), where non-hydrated ions are coordinated in a pore lined with partial charges of carbonyl groups of the protein backbone, and single ions pass processionally in a linear chain through the channel. Functional studies suggest a fundamentally different mechanism of ion translocation in Cys-loop receptors. First, hydrophobic ␣-helices line the pore, and ions remain hydrated as they pass. Second, ions are coordinated by fully charged amino acid side chains in multiple locations along the ion translocation pathway. Third, the diameter of the channel pore is larger in the Cys-loop family of receptors. Herein, we describe a novel ion selectivity filter stemming from the ␤-sheets of the extracellular ligand-binding domain of nAChRs and provide a high resolution atomic structure of ion coordination in the water-soluble AChBP. Using the low resolution structure of the nAChR transmembrane domain (8), we show spatial and charge similarities between the ␤-sheet filter and ␣-helical filters of the transmembrane domain. EXPERIMENTAL PROCEDURESA gene chemically synthesized from oligonucleotides encoding the soluble Ac_AChBP was expressed in HEK293S cells lacking the N-acetylglucosaminyltransferase I gene (GnTI ...

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Acetylcholine receptor M3 domain: stereochemical and volume contributions to channel gating

Cited by 116 publications

References 29 publications

Arginine 222 in the Pre-transmembrane Domain 1 of 5-HT3A Receptors Links Agonist Binding to Channel Gating

Arginine 222 in the Pre-transmembrane Domain 1 of 5-HT3A Receptors Links Agonist Binding to Channel Gating

Tryptophan-scanning Mutagenesis in the αM3 Transmembrane Domain of the Muscle-type Acetylcholine Receptor

An Ion Selectivity Filter in the Extracellular Domain of Cys-loop Receptors Reveals Determinants for Ion Conductance

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