Conformational changes in extracellular loop 2 associated with signal transduction in the glycine receptor

Cederholm, Jennie M. E.; Absalom, Nathan L.; Sugiharto, Silas; Griffith, Renate; Schofield, Peter R.; Lewis, T.

doi:10.1111/j.1471-4159.2010.07021.x

Cited by 3 publications

(8 citation statements)

References 34 publications

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“…6, C-E). Although we initially thought that A52E or A52N might compete with this salt bridge, we found that the 180°o rientation of the side chain of Ala52 versus Glu53 in a ␤ strand was preserved, a result that agrees with recent cysteine substitutions in loop 2 by Cederholm et al (2010). We suggest that these mutations at Ala52 predominantly affect interactions within loop 2 (Fig.…”

Section: Position 52 and Ethanol Sensitivitysupporting

confidence: 80%

“…This addition did not alter agonist sensitivity. Prior studies indicate that this lack of effect cannot be attributed to the inaccessibility of MTSES to position 52 (Crawford et al, 2007Cederholm et al, 2010). Moreover, binding a neutral MTS reagent to a cysteine substitution at position 53 did not alter the sensitivity of the mutant receptor to agonist .…”

Section: Discussionmentioning

confidence: 85%

“…We tried several models in which these four residues were distributed into the aligned structure; we found that grouping all four residues together into the loop after Ala82 in 3EAM produced the best structure without serious van der Waals overlaps. The resulting alignment (Bertaccini et al, 2010) is essentially identical to that described by Cederholm et al (2010) (Baenziger and Corringer, 2011).…”

Section: Methodsmentioning

confidence: 81%

“…We used charged MTS reagents in combination with cysteine substitutions as an alternative approach to introducing charged mutations for assessing the effect of charge at position 52 on sensitivity to glycine and ethanol. Previous work found that positions 52 and 53 are accessible to and capable of binding MTS reagents (Crawford et al, 2007Cederholm et al, 2010). Oocytes expressing WT or A52C GlyRs were exposed to negatively charged 2-sulfonatoethyl MTS sodium salt (MTSES) (10 mM) for 2 min to saturate the substituted cysteine residues with covalent disulfide bonds to the reagent.…”

Section: Methodsmentioning

confidence: 99%

“…Our interest in the role of loop 2 in GlyR function was increased by investigations that showed a key role for loop 2 in the gating process (Cederholm et al, 2010). Cederholm et al prepared cysteine mutations of each residue in loop 2 and then measured the rate of activation of these mutants by MTS reagents.…”

Section: Introductionmentioning

confidence: 99%

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Charge and Geometry of Residues in the Loop 2 β Hairpin Differentially Affect Agonist and Ethanol Sensitivity in Glycine Receptors

Perkins

Trudell²,

Asatryan³

et al. 2012

J Pharmacol Exp Ther

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Recent studies highlighted the importance of loop 2 of ␣1 glycine receptors (GlyRs) in the propagation of ligand-binding energy to the channel gate. Mutations that changed polarity at position 52 in the ␤ hairpin of loop 2 significantly affected sensitivity to ethanol. The present study extends the investigation to charged residues. We found that substituting alanine with the negative glutamate at position 52 (A52E) significantly left-shifted the glycine concentration response curve and increased sensitivity to ethanol, whereas the negative aspartate substitution (A52D) significantly right-shifted the glycine EC 50 but did not affect ethanol sensitivity. It is noteworthy that the uncharged glutamine at position 52 (A52Q) caused only a small right shift of the glycine EC 50 while increasing ethanol sensitivity as much as A52E. In contrast, the shorter uncharged asparagine (A52N) caused the greatest right shift of glycine EC 50 and reduced ethanol sensitivity to half of wild type. Collectively, these findings suggest that charge interactions determined by the specific geometry of the amino acid at position 52 (e.g., the 1-Å chain length difference between aspartate and glutamate) play differential roles in receptor sensitivity to agonist and ethanol. We interpret these results in terms of a new homology model of GlyR based on a prokaryotic ion channel and propose that these mutations form salt bridges to residues across the ␤ hairpin (A52E-R59 and A52N-D57). We hypothesize that these electrostatic interactions distort loop 2, thereby changing agonist activation and ethanol modulation. This knowledge will help to define the key physical-chemical parameters that cause the actions of ethanol in GlyRs.

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Section: Position 52 and Ethanol Sensitivitysupporting

confidence: 80%

Section: Discussionmentioning

confidence: 85%

Section: Methodsmentioning

confidence: 81%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Charge and Geometry of Residues in the Loop 2 β Hairpin Differentially Affect Agonist and Ethanol Sensitivity in Glycine Receptors

Perkins

Trudell²,

Asatryan³

et al. 2012

J Pharmacol Exp Ther

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Contributions of Conserved Residues at the Gating Interface of Glycine Receptors

Pless

Leung

Galpin

et al. 2011

Journal of Biological Chemistry

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Glycine receptors (GlyRs) are chloride channels that mediate fast inhibitory neurotransmission and are members of the pentameric ligand-gated ion channel (pLGIC) family. The interface between the ligand binding domain and the transmembrane domain of pLGICs has been proposed to be crucial for channel gating and is lined by a number of charged and aromatic side chains that are highly conserved among different pLGICs. However, little is known about specific interactions between these residues that are likely to be important for gating in ␣1 GlyRs. Here we use the introduction of cysteine pairs and the in vivo nonsense suppression method to incorporate unnatural amino acids to probe the electrostatic and hydrophobic contributions of five highly conserved side chains near the interface, Glu-53, Phe-145, Asp-148, Phe-187, and Arg-218. Our results suggest a salt bridge between Asp-148 in loop 7 and Arg-218 in the pre-M1 domain that is crucial for channel gating. We further propose that Phe-145 and Phe-187 play important roles in stabilizing this interaction by providing a hydrophobic environment. In contrast to the equivalent residues in loop 2 of other pLGICs, the negative charge at Glu-53 ␣1 GlyRs is not crucial for normal channel function. These findings help decipher the GlyR gating pathway and show that distinct residue interaction patterns exist in different pLGICs. Furthermore, a salt bridge between Asp-148 and Arg-218 would provide a possible mechanistic explanation for the pathophysiologically relevant hyperekplexia, or startle disease, mutant Arg-218 3 Gln. The glycine receptor (GlyR)2 chloride channel is a member of the Cys-loop receptor family, a subfamily of the pentameric ligand-gated ion channel (pLGIC) superfamily (1). GlyRs mediate fast inhibitory neurotransmission in the nervous system, and recent studies have provided a wealth of insight into the structure and function of the GlyR and other pLGICs. One of the more studied regions, the N-terminal ligand binding domain (LBD), is composed of a 10-strand ␤-sheet sandwich interconnected by 9 loops and the ligand binding pocket situated at the interface between adjacent subunits (2-8). The transmembrane domain (TMD) contains four ␣-helical segments (M1-M4), including the pore-lining M2 helices.How is the binding of an agonist molecule in the LBD communicated to the channel gate in the TMD, almost 60 Å away? This question has gained considerable attention in the past (9 -11), and previous studies have identified a number of charged residues likely to couple the LBD to the TMD via electrostatic interactions in different pLGICs (12)(13)(14)(15)(16)(17). A number of critical residues have been identified at the interface of LBD and TMD of ␣1 GlyRs (18 -22), but evidence for direct electrostatic interactions is thus far missing. The interface of LBD and TMD in ␣1 GlyRs is of interest not only because of its proposed role in channel gating but also because inherited mutations of side chains near this interface cause hyperekplexia or startle disease (23): Ala-52 ...

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TCR Nanoclusters as the Framework for Transmission of Conformational Changes and Cooperativity

Blanco¹,

Alarcón²

2012

Front. Immun.

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Increasing evidence favors the notion that, before triggering, the T cell antigen receptor (TCR) forms nanometer-scale oligomers that are called nanoclusters. The organization of the TCR in pre-existing oligomers cannot be ignored when analyzing the properties of ligand (pMHC) recognition and signal transduction. As with other membrane receptors, the existence of TCR oligomers points out to cooperativity phenomena. We review the data in support of conformational changes in the TCR as the basic principle to transduce the activation signal to the cytoplasm and the incipient data suggesting cooperativity within nanoclusters.

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Conformational changes in extracellular loop 2 associated with signal transduction in the glycine receptor

Cited by 3 publications

References 34 publications

Charge and Geometry of Residues in the Loop 2 β Hairpin Differentially Affect Agonist and Ethanol Sensitivity in Glycine Receptors

Charge and Geometry of Residues in the Loop 2 β Hairpin Differentially Affect Agonist and Ethanol Sensitivity in Glycine Receptors

Contributions of Conserved Residues at the Gating Interface of Glycine Receptors

TCR Nanoclusters as the Framework for Transmission of Conformational Changes and Cooperativity

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