Backbone Mutations in Transmembrane Domains of a Ligand-Gated Ion Channel

England, Pamela M.; Zhang, Yinong; Dougherty, Dennis A.; Lester, Henry A.

doi:10.1016/s0092-8674(00)80962-9

Cited by 130 publications

(138 citation statements)

References 38 publications

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“…Dose-response studies for this construct (mouse ␣1 hemagglutinin:HIS, ␤, ␥, ␦) indicated that the EC 50 (25.5 M) and Hill coefficient (1.26) were near previously reported values for the wild-type nAChR (6). Xenopus laevis (Xenopus One, Dexter, MI) oocytes were surgically removed and injected with 1 ng of total cRNA as described (6). Average currents recorded from oocytes expressing nAChR 24-48 h after injection ranged from 2 to 10 A with 50 M ACh.…”

Section: Neurosciencesupporting

confidence: 68%

“…A polyhistidine tag (located at the C terminus) and a hemagglutinin epitope in the M3-M4 cytoplasmic loop (after D347) were engineered into the nAChR ␣1 subunit (the numbering of amino acid residues after D347 is thus 8 residues greater than in the wild-type protein). Dose-response studies for this construct (mouse ␣1 hemagglutinin:HIS, ␤, ␥, ␦) indicated that the EC 50 (25.5 M) and Hill coefficient (1.26) were near previously reported values for the wild-type nAChR (6). Xenopus laevis (Xenopus One, Dexter, MI) oocytes were surgically removed and injected with 1 ng of total cRNA as described (6).…”

Section: Neurosciencesupporting

confidence: 54%

“…We now have some understanding of the motions that occur during gating within the channel-lining domain (M2), and to some extent within the M1 domain (6)(7)(8). However, less is known of the transitions that occur in domains distal to the pore.…”

Section: Neurosciencementioning

confidence: 99%

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

Leite

Blanton

Shahgholi

et al. 2003

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

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We characterized the differential accessibility of the nicotinic acetylcholine receptor α1 subunit in the open, closed, and desensitized states by using electrophysiology-coordinated photolabeling by several lipophilic probes followed by mass spectrometric analysis. Voltage-clamped oocytes expressing receptors were preincubated with one of the lipophilic probes and were continually exposed to acetylcholine; UV irradiation was applied during 500-ms pulses to + 40 or to -140 mV (which produced closed or ≈50% open receptors, respectively). In the open state, there was specific probe incorporation within the N-terminal domain at residues that align with the β8–β9 loop of the acetylcholine-binding protein. In the closed state, probe incorporation was identified at several sites of the N-terminal domain within the conserved cysteine loop (residues 128–142), the cytoplasmic loop (M3–M4), and M4. The labeling pattern in the M4 region is consistent with previous results, further defining the lipid-exposed face of this transmembrane α-helix. These results show regions within the N-terminal domain that are involved in gating-dependent conformational shifts, confirm that the cysteine loop resides at or near the protein-membrane interface, and show that segments of the M3–M4 loop are near to the lipid bilayer

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Section: Neurosciencesupporting

confidence: 68%

Section: Neurosciencesupporting

confidence: 54%

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

Leite

Blanton

Shahgholi

et al. 2003

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

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“…The nAChR ␦ subunit extracellular part of M2 undergoes a pronounced change between closed and open conformations (27). Significant backbone deformation occurs at positions 13Ј, 16Ј, and 19Ј in nAChR ␣ subunit M2 during channel gating (28). If LGICs in the closed state assume a well ordered ␣-helix structure (1), then the ␤ 3 homopentamer may have undergone transition to a ␤-like conformation in this region on channel opening.…”

Section: Effect Of Sulfhydryl Modification On Nca Binding Of Single Cmentioning

confidence: 99%

Spontaneous Mobility of GABAA Receptor M2 Extracellular Half Relative to Noncompetitive Antagonist Action

Chen

Durkin

Casida

2006

Journal of Biological Chemistry

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The ␥-aminobutyric acid type A receptor ␤ 3 homopentamer is spontaneously open and highly sensitive to many noncompetitive antagonists(NCAs)andZn Ј to Glu 20 Ј) has a proposed NCA interaction site at S17ЈC in an ␣ 1 subunit (6) and a Zn 2ϩ site at His 17 Ј and Glu 20 Ј in the ␤ 3 subunit (5). Disulfide trapping experiments with heteromeric receptors (␣ 1 ␤ 1 or ␣ 1 ␤ 1 ␥ 2 ) have helped to define the structure and mobility of the extracellular half of M2. The following cases of spontaneous or induced disulfide bond formation have been observed (in M2 unless stated otherwise): ␣ 1 12ЈC with several Cys mutants in the intrasubunit M3 (7); ␥ 2 14ЈC with both ␣ 1 15ЈC and ␣ 1 16ЈC and also ␥ 2 13ЈC and ␣ 1 13ЈC (8); ␣-␤ or ␤-␤ spontaneous disulfide cross-linking at 17Ј and 20Ј (9); nonadjacent ␤-␤ disulfide bridging by ␤ 1 20ЈC (10). These relationships indicate that the M2 segments can undergo significant rotational and translational movement.The ␤ 3 homopentamer is ideal for defining the open state channel structure relative to NCA action because it is self-assembling, spontaneously open, and symmetrical with high sensitivities to NCAs and Zn 2ϩ (3,11,12). To further understand M2 structure and NCA action, here we have examined the role of the extracellular half of the M2 segments in NCA and Zn 2ϩ binding involving site-directed mutagenesis with Cys, Ser, and Ala replacements in M2 and M3 using two NCA radioligands. The structural aspects were studied with SCAM (substituted Cys accessibility method) and disulfide trapping (3, 13). This investigation establishes the conformational flexibility of the extracellular half of M2 segments in the ␤ 3 homopentamer and suggests that NCAs access their binding sites both directly, through the channel pore, and indirectly, through the interface of adjacent subunits. The relationships considered lead to a model of the ␤ 3 homopentameric GABA A receptor M2 segments relative to NCA action (shown in Fig. 1). 3 Positions in the M2 segment are identified by a system of index numbers in which the absolutely conserved basic residue at the N-terminal end of M2 is numbered 0Ј, i.e. GABA A R ␤ 3 Arg 250 . Residues toward the C terminus are numbered 1Ј, 2Ј, etc., and toward the N terminus are numbered Ϫ1Ј, Ϫ2Ј, etc. EXPERIMENTAL PROCEDURES Mutagenesis

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“…The kink may therefore act as a swivel enabling the outer half of M2 to move asynchronously with the inner half, where the gate is most likely positioned. Indeed, several lines of evidence suggest gating is mediated by a backbone rearrangement at this midpoint (7)(8)(9)(10). In addition, a rate equilibrium free energy state analysis shows that the M2-M3 domain is positioned midway along the agonist-induced "conformational wave" that extends from the ligand binding domain to the gate (11).…”

mentioning

confidence: 99%

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

Pless

Dibas

Lester

et al. 2007

Journal of Biological Chemistry

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155

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Models describing the structural changes mediating Cys loop receptor activation generally give little attention to the possibility that different agonists may promote activation via distinct M2 pore-lining domain structural rearrangements. We investigated this question by comparing the effects of different ligands on the conformation of the external portion of the homomeric ␣1 glycine receptor M2 domain. Conformational flexibility was assessed by tethering a rhodamine fluorophore to cysteines introduced at the 19 or 22 positions and monitoring fluorescence and current changes during channel activation. During glycine activation, fluorescence of the label attached to R19C increased by ϳ20%, and the emission peak shifted to lower wavelengths, consistent with a more hydrophobic fluorophore environment. In contrast, ivermectin activated the receptors without producing a fluorescence change. Although taurine and ␤-alanine were weak partial agonists at the ␣1R19C glycine receptor, they induced large fluorescence changes. Propofol, which drastically enhanced these currents, did not induce a glycine-like blue shift in the spectral emission peak. The inhibitors strychnine and picrotoxin elicited fluorescence and current changes as expected for a competitive antagonist and an open channel blocker, respectively. Glycine and taurine (or ␤-alanine) also produced an increase and a decrease, respectively, in the fluorescence of a label attached to the nearby L22C residue. Thus, results from two separate labeled residues support the conclusion that the glycine receptor M2 domain responds with distinct conformational changes to activation by different agonists. Glycine receptor (GlyR)4 chloride channels mediate inhibitory neurotransmission in the central nervous system (1). They comprise an assembly of five subunits that are each composed of a large N-terminal extracellular ligand-binding domain and four transmembrane ␣-helices (M1-M4). Cryo-electron microscopy images of the homologous Torpedo electropax nicotinic acetylcholine receptor (nAChR) transmembrane region reveals that the pore-lining M2 domains are kinked radially inward to form a central constriction at the membrane midpoint (2). There is currently a great deal of interest in understanding how M2 domains move to open the channel. Although the original model proposed a drastic rotation of M2 domains about their long axes (3), more recent evidence suggests that there is little if any rotation during receptor activation (4 -6). The precise nature of the structural change remains a matter for debate.The central pore kink is likely to introduce a degree of structural discontinuity because the hydrogen bonds responsible for maintaining ␣-helix rigidity are most likely broken. The kink may therefore act as a swivel enabling the outer half of M2 to move asynchronously with the inner half, where the gate is most likely positioned. Indeed, several lines of evidence suggest gating is mediated by a backbone rearrangement at this midpoint (7-10). In addition, a rate equilibrium fr...

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Backbone Mutations in Transmembrane Domains of a Ligand-Gated Ion Channel

Cited by 130 publications

References 38 publications

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

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

Spontaneous Mobility of GABAA Receptor M2 Extracellular Half Relative to Noncompetitive Antagonist Action

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

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