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_Cryst, 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_Cryst. The last line describes the secondary structure of GluCl_Cryst 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_Cryst 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

3D representation of the whole ion channel [49] of GluCl_Cryst (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

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

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

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_Cryst 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_A-ρ1 (accession #EAW48558). The second line represents the secondary structure of GluCl_Cryst: 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

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

Summary of the interactions of glutamate in GluCl_Cryst and GluCl<i>Ac</i>2.

<p>List of residues (as identified from the crystallographic structure of GluCl_Cryst and predicted by the GluCl<i>Ac</i>2 homology model) accompanied by their position number in the alignment of Fig. 2 and a description of the type of interactions in which they are involved. Residue numbers are given for the crystalized receptor (GluCl_Cryst), the receptor used in the electrophysiological experiments (GluClα2b), and the receptor that figures in the homology model (GluCl<i>Ac</i>2) from left to right, respectively. Rows in green, yellow and red indicate identical, similar and different aligned residues, respectively.</p

Orthologs of GluCl<i>Ac</i>2 (A) and GluCl<i>Ac</i>1 (B) obtained from three other lophotrochozoan species.

<p>A: Sequence alignment of predicted proteins from <i>Capitella teleta</i> (Ct), <i>Helobdella robusta</i> (<i>Hr</i>), and <i>Lottia gigantea</i> (<i>Lg</i>) with residues of the GluCl<i>Ac</i>2 receptor from <i>Aplysia californica</i> (<i>Ac</i>). The binding residues predicted by the homology model of GluCl<i>Ac</i>2 are bold and surrounded by either violet (P subunit) or yellow (C subunit) rectangles. Residue numbers from GluCl<i>Ac</i>2 are indicated above the alignment. B: Alignment of GluCl<i>Ac</i>1 and the second <i>Lottia gigantea</i> sequence (Lg_125242) which, like GluCl<i>Ac</i>1, fails to have an arginine at the position corresponding to the R98 of GluCl<i>Ac</i>2 (position 95 in GluCl<i>Ac</i>1). Furthermore neither Lg_125242 nor GluCl<i>Ac</i>1 have a tyrosine aligned with the position corresponding to Y161 of GluCl<i>Ac</i>2 (position 158 in GluCl<i>Ac</i>1).</p

Differential sensitivity of GluCl<i>Ac</i>2 and GluClα2b receptors to glutamate, GABA, β-alanine and taurine.

<p><b>Column labeled I:</b><u>Traces preceded by Ac</u>: Responses of GluCl<i>Ac</i>2 to a 1-sec application of (A) 1 mM glutamate, 1 mM GABA and 10 mM GABA; (B) 1 mM glutamate, 1 mM β-alanine and 10 mM β-alanine; and (C) to 1 mM glutamate, 1 mM taurine and 10 mM taurine. <u>Traces preceded by α2b</u>: Responses of GluClα2b to a 1-sec application of (A) 1 mM glutamate, 10 mM GABA and 50 mM GABA; (B) 1 mM glutamate, 10 mM β-alanine and 50 mM β-alanine; and (C) 1 mM glutamate, 10 mM taurine and 50 mM taurine. <b>Column labeled II:</b><u>Traces preceded by Ac′</u>: Responses of GluCl<i>Ac</i>2 to a 1-sec application of glutamate before and after a 3-min application of (A) 1 mM GABA; (B) 1 mM β-alanine, and (C) 1 mM taurine. <u>Traces preceded by α2b</u>: Responses of GluClα2b to a 1-sec application of glutamate before and after a 3-min application of (A) 10 mM GABA; (B) 10 mM β-alanine, and (C) 10 mM taurine. All applications of glutamate or other agonists were separated by a 3-min interval (during which the control solution bathed the cell), except in column II where a second agonist (GABA, β-alanine, or taurine) was applied during the 3-min interval separating glutamate applications.</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