14 research outputs found
Data from: Molecular determinants of agonist selectivity in glutamate-gated chloride channels which likely explain the agonist selectivity of the vertebrate glycine and GABAA-ρ receptors
Orthologous Cys-loop glutamate-gated chloride channels (GluClR’s) have been cloned and described electrophysiologically and pharmacologically in arthropods and nematodes (both members of the invertebrate ecdysozoan superphylum). Recently, GluClR’s from Aplysia californica (a mollusc from the lophotrochozoan superphylum) have been cloned and similarly studied. In spite of sharing a common function, the ecdysozoan and lophotrochozoan receptors have been shown by phylogenetic analyses to have evolved independently. The recent crystallization of the GluClR from C. elegans revealed the binding pocket of the nematode receptor. An alignment of the protein sequences of the nematode and molluscan GluClRs showed that the Aplysia receptor does not contain all of the residues defining the binding mode of the ecdysozoan receptor. That the two receptors have slightly different binding modes is not surprising since earlier electrophysiological and pharmacological experiments had suggested that they were differentially responsive to certain agonists. Knowledge of the structure of the C. elegans GluClR has permitted us to generate a homology model of the binding pocket of the Aplysia receptor. We have analyzed the differences between the two binding modes and evaluated the relative significance of their non-common residues. We have compared the GluClRs electrophysiologically and pharmacologically and we have used site-directed mutagenesis on both receptor types to test predictions made from the model. Finally, we propose an explanation derived from the model for why the nematode receptors are gated only by glutamate, whereas the molluscan receptors can also be activated by β-alanine, GABA and taurine. Like the Aplysia receptor, the vertebrate glycine and GABAA-ρ receptors also respond to these other agonists. An alignment of the sequences of the molluscan and vertebrate receptors shows that the reasons we have given for the ability of the other agonists to activate the Aplysia receptor also explain the agonist profile seen in the glycine and GABAA-ρ receptors
Data from: Molecular determinants of agonist selectivity in glutamate-gated chloride channels which likely explain the agonist selectivity of the vertebrate glycine and GABAA-ρ receptors
Orthologous Cys-loop glutamate-gated chloride channels (GluClR’s) have been cloned and described electrophysiologically and pharmacologically in arthropods and nematodes (both members of the invertebrate ecdysozoan superphylum). Recently, GluClR’s from Aplysia californica (a mollusc from the lophotrochozoan superphylum) have been cloned and similarly studied. In spite of sharing a common function, the ecdysozoan and lophotrochozoan receptors have been shown by phylogenetic analyses to have evolved independently. The recent crystallization of the GluClR from C. elegans revealed the binding pocket of the nematode receptor. An alignment of the protein sequences of the nematode and molluscan GluClRs showed that the Aplysia receptor does not contain all of the residues defining the binding mode of the ecdysozoan receptor. That the two receptors have slightly different binding modes is not surprising since earlier electrophysiological and pharmacological experiments had suggested that they were differentially responsive to certain agonists. Knowledge of the structure of the C. elegans GluClR has permitted us to generate a homology model of the binding pocket of the Aplysia receptor. We have analyzed the differences between the two binding modes and evaluated the relative significance of their non-common residues. We have compared the GluClRs electrophysiologically and pharmacologically and we have used site-directed mutagenesis on both receptor types to test predictions made from the model. Finally, we propose an explanation derived from the model for why the nematode receptors are gated only by glutamate, whereas the molluscan receptors can also be activated by β-alanine, GABA and taurine. Like the Aplysia receptor, the vertebrate glycine and GABAA-ρ receptors also respond to these other agonists. An alignment of the sequences of the molluscan and vertebrate receptors shows that the reasons we have given for the ability of the other agonists to activate the Aplysia receptor also explain the agonist profile seen in the glycine and GABAA-ρ receptors
Glutamate docked in the homology model of GluClAc2 (Aplysia californica)
Coordinates of the homology model of GluClAc2, the glutamate-gated chloride channel receptor of Aplysia californica with docked glutamate
Alignment used for homology model building.
<p>Sequence alignment of residues of the two GluCl receptors: the first, a receptor from <i>C. elegans</i>, GluCl<sub>Cryst</sub>, which has been crystalized and which will serve as the template for the homology model of the second, GluCl<i>Ac</i>2, from the <i>Aplysia californica</i>. The numbers above the sequence represent the positions in the alignment based on the truncated receptor from GluCl<sub>Cryst</sub>. The last line describes the secondary structure of GluCl<sub>Cryst</sub> with the blue arrows representing β-sheets and the orange tubes, the α-helices. Throughout the length of the sequences, a light blue highlighting indicates identical or similar residues. Residues binding glutamate in GluCl<sub>Cryst</sub> are surrounded by a black rectangle. These positions are highlighted in green, yellow and red when the aligned residue in GluCl<i>Ac</i>2 is respectively identical, similar or different. Three additional positions are surrounded by a red rectangle and correspond to positions of residues binding glutamate only in the homology model of GluCl<i>Ac</i>2.</p
Responses of mutated GluCl<i>Ac</i>2 and GluClα2b receptors to increasing concentrations of glutamate.
<p>A: Responses (in pA) of seven mutated GluCl<i>Ac</i>2 receptors to 1, 10, 50 and 100 mM glutamate. B: Responses of five mutated GluClα2B receptors to 1, 10, 50 and 100 mM glutamate.</p
Similar binding modes for the three additional ligands in GluCl<i>Ac</i>2.
<p>2D diagram representing the interactions between the binding pocket residues of the homology model for GluCl<i>Ac</i>2 and (A) β-alanine, (B) GABA and (C) taurine. Ligands are represented in lines. Only the polar hydrogens that are involved in interactions with the receptor are explicitly represented. Residues are depicted as circles in which the residue type, number and position (the latter in parentheses) are written on a colored background which indicates the subunit to which the residue belongs (see Fig. 3 and Fig. 4). Backbone and side chain hydrogen bonds are represented by green and blue arrows, respectively. Salt bridges are represented by purple arrows, π interactions are represented by orange lines. Atom colors as in Fig. 3B.</p
3D representation of the whole ion channel [49] of GluCl<sub>Cryst</sub> (PDB code: 3RIF).
<p>Only two adjacent subunits are explicitly displayed, the three others are represented by the transparent surface. Alpha-helices are represented by tubes, beta-sheets by arrows (β1–β2 sheet in red, loop C in green, M2 helices and M2–M3 loop in blue). Principal and complementary subunits are colored, respectively, in violet and yellow. Glutamate is represented as CPK volumes, R37, V45, R56, and P268 are displayed in ball and stick. According to Calimet et al. V45 from β1–β2 loop and P268 from M2–M3 loop are involved in the gating mechanism. Interestingly the critical residues at positions 37, 54, 56 belong to β1–β2 sheets.</p
Responses to 1 mM glutamate in both WT and mutated GluCl<i>Ac</i>2 and GluClα2b receptors.
<p>A. Mutations in four binding residues of GluCl<i>Ac</i>2: Y96, R98, R135 and Y161 (positions 54, 56, 93 and 119). B. Mutations in GluCl<i>Ac</i>2 of residues L79 alone and L79+R135. These two residues are found at positions 37 and 93 (see Figs. 3 and 4). Only one of the two (R135) belongs to the GluCl<i>Ac</i>2 binding pocket (see the R135A mutation alone in A). C. Mutations in GluClα2b of residues R111 and Q167 found at positions 37 and 93, respectively: R111 alone, and R111+Q167 in a double mutation. Only one of the residues (R111) belongs to the GluClα2b binding pocket. Calibration: A: 1 sec, 500 pA; B: 1 sec, 500 pA; C: 1 sec, 1000 pA.</p
Alignment of the residues from the binding pocket region through the third transmembrane region of several ligand-gated ion channels.
<p>The alignment contains the sequences from four invertebrate glutamate-gated chloride channels (GluCl<sub>Cryst</sub> and GluClα2b from <i>C. Elegans</i> and GluCl<i>Ac</i>1 and GluCl<i>Ac</i>2 from <i>Aplysia californica</i>), and those from two vertebrate receptors: the glycine receptor Glyα1 from <i>Rattus norvegicus</i> (accession #CAC35979 ) and the GABA receptor from <i>Homo sapiens</i>, GABA<sub>A</sub>-ρ1 (accession #EAW48558). The second line represents the secondary structure of GluCl<sub>Cryst</sub>: the blue arrows represent β-sheets; the orange tubes, the α-helices. Loop C and helices M1, M2 and M3 are indicated above the alignment. Positions 37, 54 and 93 are indicated in black boxes. Identical, strongly similar and weakly similar residues are highlighted, respectively, in dark blue, medium blue and light blue. Residues of interest for the binding of glutamate that were unveiled in this article are surrounded by violet rectangles when the residues are on the Principal face, and are surrounded by yellow rectangles when on the Complementary face. Residues surrounded by a red rectangle are involved in the opening/gating mechanism of the ion channel. The importance of a conserved proline in the M2–M3 extracellular loop will be discussed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108458#pone-0108458-g010" target="_blank">Figure 10</a>.</p
Glutamate bound to GluCl<sub>Cryst</sub> and to the homology model of GluCl<i>Ac</i>2.
<p>A. 3D representation of the binding pocket of glutamate at the interface between the two subunits of the crystallographic structure of GluCl<sub>Cryst</sub> (Left) and the model of GluCl<i>Ac</i>2 (Right). The Principal and Complementary subunits are displayed in violet and green/yellow, respectively. The bound glutamate is represented by a ball and stick display. The residues for four important positions in the alignment are represented as sticks, and their names and residue numbers are written in green. B. 2D diagram representing the interactions between glutamate and each of the two receptors: GluCl<sub>Cryst</sub> (Left) and GluCl<i>Ac</i>2 (Right) as displayed in 3A. Glutamate is represented in lines, and adopts two different conformations reflecting the respective bindings at the two receptors. Only the polar hydrogens that are involved in interactions with the receptor are explicitly represented. Residues are depicted as circles in which the residue type, number and position (the latter in parentheses) are written on a colored background which indicates the subunit to which the residue belongs (see Fig. 3A). Backbone and side chain hydrogen bonds are represented by green and blue arrows, respectively. Salt bridges are represented by violet arrows and π interactions are represented by orange lines. Atom color code: carbon gray, oxygen red, nitrogen blue, hydogens white in A, black in B.</p