Investigation of Agonist Recognition and Channel Properties in a

Feb 7, 2018 - Loop C, which differs in length compared to other pentameric ligand-gated ion channels (pLGICs), contributes to ligand recognition throu...
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Article Cite This: Biochemistry XXXX, XXX, XXX−XXX

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Investigation of Agonist Recognition and Channel Properties in a Flatworm Glutamate-Gated Chloride Channel Daniel Callau-Vázquez, Stephan A. Pless, and Timothy Lynagh* Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 160, 2100 Copenhagen, Denmark S Supporting Information *

ABSTRACT: Glutamate-gated chloride channels (GluCls) are neurotransmitter receptors that mediate crucial inhibitory signaling in invertebrate neuromuscular systems. Their role in invertebrate physiology and their absence from vertebrates make GluCls a prime target for antiparasitic drugs. GluCls from flatworm parasites are substantially different from and are much less understood than those from roundworm and insect parasites, hindering the development of potential therapeutics targeting GluCls in flatworm-related diseases such as schistosomiasis. Here, we sought to dissect the molecular and chemical basis for ligand recognition in the extracellular glutamate binding site of SmGluCl-2 from Schistosoma mansoni, using site-directed mutagenesis, noncanonical amino acid incorporation, and electrophysiological recordings. Our results indicate that aromatic residues in ligand binding loops A, B, and C are important for SmGluCl-2 function. Loop C, which differs in length compared to other pentameric ligand-gated ion channels (pLGICs), contributes to ligand recognition through both an aromatic residue and two vicinal threonine residues. We also show that, in contrast to other pLGICs, the hydrophobic channel gate in SmGluCl-2 extends from the 9′ position to the 6′ position in the channel-forming M2 helix. The 6′ and 9′ positions also seem to control sensitivity to the pore blocker picrotoxin.

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structures of GLC-1 from Caenorhabditis elegans, alone or in the presence of agonists, glutamate and ivermectin, and the pore blocker picrotoxin.13,14 This has led to preliminary characterization of the pharmacological profile of ECD and TMD sites for pharmacological modulators of roundworm GluCls.14,15 Agonist−ECD interactions loosely reflect those in the other major inhibitory pLGICs, GABAARs and glycine receptors (GlyRs). These include a cation−π interaction between the agonist amine and one of three highly conserved aromatic residues14,16,17 and a polar interaction between an agonist carboxylate and a highly conserved threonine residue in loop C, a crucial ∼10-amino acid segment of the ECD.14 Flatworm GluCls, however, differ significantly from roundworm GluCls in the amino acids comprising the glutamate binding ECD,18,19 including a loop C that is two or three amino acids longer than in roundworm GluCls and other inhibitory pLGICs (Figure S1). These differences hinder attempts at homology modeling,15 and in fact, our understanding of flatworm GluCls lags well behind that of most other members of the overarching pLGIC family. SmGluCl-2 from S. mansoni possesses the signature amino acid sequence of flatworm (and broader Lophotrochozoan) GluCls, could constitute a drug target in an important parasite, and is thus a useful starting point for investigating flatworm GluCl function.8,20 In the study presented here, we sought to elucidate the interactions between SmGluCl-2 and the agonist

entameric ligand-gated ion channels (pLGICs) are neurotransmitter receptors that play vital signaling roles in animals with complex nervous systems.1,2 Binding of a neurotransmitter (agonist) to an extracellular domain (ECD) activates ionic current through a transmembrane channel domain (TMD). This chemo-electric signal is excitatory, in the case of cation-selective channels, such as muscle acetylcholine receptors, or inhibtory, in the case of anion-selective channels, such as type A γ-aminobutyric acid receptors (GABAARs).2 The pharmacological modulation of pLGICs constitutes a potent means of altering the activity of neurons or muscles in which pLGICs are expressed. Glutamate-gated chloride channels (GluCls) are inhibitory pLGICs restricted to invertebrate lineages, and their absence from vertebrates makes GluCls a prime target for antiparasitic drugs.3 This is demonstrated by ivermectin, an anthelmintic used on billions of humans, livestock, and pets that paralyzes or kills roundworms by activating GluCls, leading to flaccid paralysis.4,5 Flatworm parasites, such as Schistosoma mansoni, which inflicts a huge burden on many of the world’s poorest and urgently needs new treatments,6,7 are not susceptible to ivermectin, as flatworm GluCls differ in their TMD ivermectin binding site.8,9 Consequently, flatworm GluCls have remained relatively unexplored as potential anthelmintic drug targets, despite evidence that glutamatergic signaling is important to flatworm physiology.10−12 A detailed understanding of the pharmacological properties of flatworm GluCls could thus facilitate the discovery of potent modulators of flatworm physiology. Our understanding of GluCl structure and function has been transformed in the past six years, thanks largely to crystal © XXXX American Chemical Society

Received: December 11, 2017 Revised: January 26, 2018

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DOI: 10.1021/acs.biochem.7b01245 Biochemistry XXXX, XXX, XXX−XXX

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Figure 1. (A) Top-down and side views of roundworm GLC-1 GluCl (Protein Data Bank entry 3RIF) and amino acid sequence alignment of inhibitory pLGIC subunits, highlighting loop A, B, C, D, and E segments. Circles denote SmGluCl-2 residues investigated in this study. The side chains of the likely equivalent residues in GLC-1 are shown as sticks in the magnified views. (B) Electrophysiological recordings of current responses to glutamate (in millimolar) of oocytes expressing wild-type or mutant SmGluCl-2 channels. (C) Concentration−response curves for functional SmGluCl-2 mutants [mean ± standard error of the mean (n = 5−7)].

incorporation), oocytes were incubated in Leibovitz’s L-15 medium (Life Technologies) with 3 mM L-glutamine, 2.5 mg/ mL gentamycin, and 15 mM HEPES (pH 7.4 with NaOH) until experiments were performed. Electrophysiological Recordings and Data Analysis. One to 5 days after mRNA injection, oocytes were placed one at a time in a small plastic chamber perfused with the bath solution [96 mM NaCl, 2 mM KCl, 1.8 mM BaCl2, and 5 mM HEPES (pH 7.4)] for two electrode voltage clamp experiments. L-Glutamate (hereafter “glutamate”), dissolved in the bath solution, was applied for approximately 10 s with approximately 1 min between subsequent applications using a ValveBank 8 perfusion system (AutoMate Scientific). Picrotoxin was applied or co-applied similarly, as indicated in the figures. For coapplication, the different drugs were mixed immediately prior to experiments and perfused through the same line. Oocytes were clamped at −60 mV, and currents were recorded with microelectrodes (filled with 3 M KCl), an OC-725C amplifier (Warner Instruments), and a Digidata 1550 digitizer (Molecular Devices) at 1 kHz with 200 Hz filtering. Current responses were later analyzed in Clampfit 10 (Molecular Devices) with additional filtering for illustration. To establish glutamate EC50 values and picrotoxin IC50 values, peak current amplitudes and decreases in current amplitude, respectively, were plotted against drug concentration using the four-parameter Hill equation in Prism 6 (GraphPad). Parameters were compared statistically (tests described in the relevant tables) using Prism 6. Amino Acid Sequence Alignments and Molecular Structures. Amino acid sequences were retrieved from UniProt. Brief names are shown in Figure 1A, and extended names and UniProt entries are listed in Figure S1. Sequences were aligned in MUSCLE24 via the European Bioinformatics Institute portal (www.ebi.ac.uk).

glutamate, with the goal of understanding how glutamate interacts with, and thus how potential drugs could modulate, flatworm GluCls.

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MATERIALS AND METHODS Expression of SmGluCl-2 in Xenopus laevis Oocytes. The cDNA of the Sm-GluCl-2.1 (hereafter SmGluCl-2) isoform from S. mansoni in the pT7TS vector8 was used for site-directed mutagenesis with PfuUltra II Fusion HS DNA Polymerase (Agilent Technologies) following the supplier’s instructions using custom-designed primers (Eurofins Genomics). cDNAs were linearized with XbaI (New England Biolabs), and mRNAs were synthesized with the Ambion mMESSAGE mMACHINE T7 transcription kit (ThermoFisher Scientific) and purified in RNeasy columns (Qiagen). Incorporation of noncanonical amino acids (ncAAs) utilized the nonsense suppression method, in which oocytes are coinjected with SmGluCl-2 mRNAs containing a UAG stop codon at the site of interest and aminoacylated tRNA.21 Modified Tetrahymena thermophila tRNA22 DNA oligomers were synthesized commercially (Integrated DNA Technologies), and tRNAs were transcribed with the T7-Scribe transcription kit (Cellscript) and purified with Chroma Spin DEPC-H20 columns (Clontech). Aminoacylation of tRNA with Nvoc-Phe-OPdCpA was performed in vitro using T4 DNA ligase (New England Biolabs), and aminoacyl-tRNA was purified via phenol/chloroform extraction and ethanol precipitation, air-dried, and stored at −80 °C until it was used. Immediately before being injected into oocytes, aminoacyl-tRNA was resuspended in 1 μL of water, and NVOC was removed by a 50 s exposure to ultraviolet light (400 W Xe lamp, Newport). Oocytes were prepared and injected as described previously,23 under license 2014-15-0201-00031 from the Danish Veterinary and Food Administration. After injection of 10 ng (regular mutants) or 40 ng of mRNA (for ncAA B

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RESULTS Identification of Determinants of Glutamate Potency in SmGluCl-2. A conserved structural feature of pLGICs is the “aromatic box”, three to five aromatic side chains that surround the agonist.25 In inhibitory pLGICs that bind GABA, glycine, or glutamate, this is primarily comprised of tyrosine and/or phenylalanine residues from loops A, B, and C (Figure 1A). To test possible contributions of these residues to glutamate sensitivity in SmGluCl-2, we mutated F92 (loop A), Y152 (loop B), and Y202 (loop C) individually to leucine. This essentially retained the hydrophobicity but removed the aromaticity of these side chains. Each of these mutations resulted in the loss of glutamate sensitivity (Figure 1B), suggesting that each aromatic side chain is important for glutamate recognition or receptor expression, assembly, or trafficking. Consistent with the important role of aromaticity at these positions, substitution of Y152 and Y202 with phenylalanine had much milder effects on function (Figure 1B). Both Y152F and Y202F mutants showed decreased glutamate potency compared to that of the wild type (WT), however [Figure 1C, EC50 values [95% confidence interval (CI)] of 21 (19−23) μM for the WT, 152 (139−165) μM for Y152F, and 91 (71−112) μM for Y202F (n = 6 or 7)], indicating that the hydroxyl groups of these side chains may contribute to glutamate potency. Another conserved feature of inhibitory pLGICs is a threonine (or serine) residue three positions upstream of the loop C aromatic (Figure 1A), whose mutation decreases agonist potency in GlyRs, GABAARs, and GluCls.26−28 Homology modeling of SmGluCl-2 is hampered by the fact that loop C is longer than in other channels, and one of two threonines in this part of loop C could foreseeably interact with the agonist (Figure 1A and Figure S1). To test the potential roles of both of these SmGluCl-2 threonine residues, they were individually substituted for alanine, removing hydroxyl and methyl groups, or for serine, retaining the hydroxyl moiety but removing a methyl group. Whereas T197A and T199A mutations both abolished responses to glutamate, T197S and T199S mutations preserved responses to glutamate (Figure 1B), indicating a role for both hydroxyl groups in channel expression or function. EC50 values for T197S [2.0 (1.3−2.7) mM (n = 5)] and T199S [2.7 (1.8−3.6) mM (n = 6)] were substantially increased compared to that of the WT, suggesting that the hydroxyl group alone at these positions is not sufficient for high glutamate potency. Thus, we cannot conclude which residue is more likely to interact with the agonist. To rule out the possibility that threonine mutations have some nonspecific effect on channel function, we also mutated threonine 203, one position downstream of Y202 (Figure 1A), to alanine. T203A channel function was very similar to that of the WT (Figure 1B,C), ruling out the possibility that loop C mutations randomly affect channel function. Finally, we sought to establish if conserved arginine and serine residues from the complementary face of the ligand binding site contribute to glutamate potency in SmGluCl-2. Alanine and lysine substitution of R54 abolished responses to glutamate (Figure 1B), indicating that positive charge alone at this loop D side chain is insufficient for high glutamate potency. Alanine substitution of S122 abolished responses to glutamate (Figure 1B), and although threonine substitution preserved responses to glutamate, S122T channels showed markedly reduced sensitivity [Figure 1C; EC50 = 668 (455−880) μM (n

= 7)]. Thus, the carboxylate terminus of glutamate is likely to interact with complemetnary face arginine and serine residues, as in other pLGICs. SmGluCl-2 Loop A, B, and C Aromatic Residues. These results hint at an important role of aromaticity of loop A, B, and C residues in the structure or function of SmGluCl-2, but they are not really informative with respect to the chemical nature of potential interactions with the agonist. We therefore turned to noncanonical amino acid (ncAA) incorporation, enabling the substitution of electron-withdrawing groups that incrementally decrease the cation−π binding ability of aromatic side chains.29 A correlation between increases in agonist EC50 and decreased cation−π binding ability (increasing level of fluorination) at a particular position would be indicative of a cation−π interaction. To this end, phenylalanine (Phe) or derivatives F1-Phe, F2-Phe, and F3-Phe (Figure 2A) were incorporated into positions 92, 152 or 202 by co-injecting F92TAG, Y152TAG, or Y202TAG mutant cRNA with ncAAcylated tRNA. As the incorporation of Phe into positions 152 and 202 essentially removes a hydroxyl moiety from these side chains, we also generated the conventional mutant constructs Y152F and Y202F and compared them to the WT. Both substitutions caused a significant increase in glutamate EC50 values (Table 1), which reflects the increase in glutamate EC50 observed upon mutation of the equivalent loop B tyrosine in roundworm GluCls but contrasts with the minimal effects upon mutating the equivalent loop C tyrosine in roundworm GluCls.20,27 Despite the decreased maximum current amplitude compared to those of the WT and conventional mutant constructs, concentration−response data for the 92Phe and 152Phe ncAA constructs overlapped closely with those of the WT and Y152F conventional constructs (Figure 2B−E), suggesting that ncAAcontaining channels recapitulated the function of conventional channels. As a control experiment for specific incorporation of ncAAs, we also measured the glutamate-gated current amplitude at oocytes injected with TAG mutant cRNA and non-aminoacylated tRNA. In each case, the current was negligible (Figure 2B), indicating little, if any, unspecific incorporation of endogenous amino acids that could confound the interpretation of results. Although 92Phe- and 152Pheexpressing oocytes yielded glutamate-gated currents as great as 500 nA, the current amplitude decreased substantially with fluorinated derivatives and was small for all 202-ncAA constructs (Figure 2B). This meant that only Phe-, F1-Phe-, and F2-Phe-incorporating mutants could be compared for positions 92 and 152, and only Phe- and F1-Phe-incorporating receptors could be compared for the 202 position. With regard to F92 in loop A, F1-Phe ncAA substitution caused no significant decrease in glutamate sensitivity (Figure 2D), suggesting that the electron-rich face of this phenylalanine residue plays no major role in glutamate recognition. In contrast, at Y152 and Y202, F1-Phe substitution caused substantial decreases in glutamate sensitivity relative to Y152F or Y202F (Figure 2C,E,F), raising the possibility that π electrons at these positions may contribute to glutamate recognition. However, this trend did not continue for an increasing level of fluorination at position Y152 (Figure 2G), which is inconsistent with a cation−π interaction with the agonist. For position 202, we could not assess glutamate sensitivity with an increasing level of fluorination, as glutamategate currents were negligible (Figure 2B). Thus, loop A and B aromatic side chains, although important for glutamate sensitivity, seem not to form cation−π interactions with the C

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Biochemistry Table 1. Parameters for Glutamate Concentration− Response Dataa EC50 (μM) (95% CI) WT 92-Phe 92-F1-Phe 92-F2-Phe 152-Phe 152-F1-Phe 152-F2-Phe 202-Phe 202-F1-Phe

21 38 40 161 207 1284 1587 76 1511

(18−23) (26−51) (33−48) (76−245) (106−308) (468−2099) (830−2343) (32−120) (985−2038)

nH (95% CI)

n

1.6 1.5 1.5 1.4 1.5 1.4 1.4 1.4 1.8

4 4 4 4 4 9 6 4 4

(1.2−1.9) (0.9−2.0) (0.8−2.2) (0.5−2.3) (0.8−2.3) (1.0−1.7) (0.7−2.0) (1.0−1.7) (0.9−2.7)

a

Peak current responses to increasing concentrations of glutamate were fit with the Hill equation at n cells. EC50 and nH were then averaged.

in glutamate sensitivity that we could not measure currents in a reasonable concentration range. Channel Pore Mutations Increase the Glutamate Sensitivity of Loop B and C Mutants. Due to the absence of measurable activity with the 202F2-Phe substitution, we could not assess the role of π electron density at this position. Similarly, T197A and T199A channels were completely insensitive to glutamate, preventing us from ascribing the suspected role in glutamate recognition to one of these hydroxyl side chains. We therefore sought to generate a new SmGluCl-2 “template” with glutamate sensitivity that is higher than that of the WT template, on which these mutations could be assessed within a practical concentration range. As mutations in the first membrane-spanning (M1) helix and the pore-lining M2 helix have been shown to enhance glutamate sensitivity in other GluCls,27,30 we generated A220L, A220F, and A220W (M1) and L6′S, L6′T, L9′S, and L9′T (M2) SmGluCl-2 mutants (Figure 3A). M1 mutations A220F and A220W caused an increase in glutamate sensitivity, which is evident in the leftward shift of the concentration−response relationships (Figure 3B). The A220L mutation, involving a smaller but still substantial increase in side chain size, did not cause a change in glutamate potency (Figure 3B), perhaps reflecting the fact that roundworm and insect GluCls possessing leucine, isoleucine, and valine at this position show glutamate sensitivity similar to that of WT SmGluCl-2.31−33 A220F and A220W mutations also caused significant decreases in the cooperativity coefficient for activation by glutamate (Table 2). This differs from the effects of analogous mutations in heteromeric GLC1/ GLC2 roundworm GluCls, perhaps because there are different interactions between adjacent subunits in this intersubunit pocket in homomeric and heteromeric receptors.30 In other inhibitory pLGICs, the substitution of the porelining M2 L9′ residue for smaller, polar serine, and threonine residues has been shown to cause increases in agonist sensitivity by essentially removing a hydrophobic gate in the midchannel.27,34,35 We considered that similar substitutions at SmGluCl-2 L6′, a threonine in most inhibitory pLGICs (Figure S1), might have a similar effect. In SmGluCl-2, although the L6′S glutamate EC50 was lower than that of the WT (Table 2), the other L6′ and L9′ substitutions caused no change or even a decrease in glutamate potency (Figure 3C−F and Table 2). As is often the case with M2 mutations, each mutation caused some constitutive current, which was blocked by the inhibitory pLGIC blocker picrotoxin (Figure 3C; but see Effects of M2 Mutations on Channel Block by Picrotoxin). Curiously,

Figure 2. (A) Noncanonical amino acid (ncAA) structures (L-isomers were used). (B) Example responses to indicated glutamate concentrations (in millimolar) at oocytes expressing indicated conventional (Y152F) or ncAA-incorporating (others) constructs. Grayscale bars: x, 30 s; y as indicated (microamperes). A dash indicates control oocytes where TAG mutant cRNA was injected with non-aminoacylated tRNA. (C) Mean ± standard error of the mean (SEM) (n = 4−9) of maximum glutamate-gated current amplitude. (D−F) Average glutamate concentration−response data [mean ± SEM (n = 3−7)] at oocytes injected with Phe92TAG, Tyr152TAG, or Tyr202TAG cRNAs together with indicated ncAA. Black symbols and curves show data for the WT or conventional mutants. (G) Relationship between the increase in glutamate EC50 and theoretical cation−π binding ability.

agonist. We cannot conclude if the contribution of Y202 to glutamate sensitivity occurs via a cation−π interaction or otherwise, due either to poor expression or to such a decrease D

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dose−response relationships that were similar to those for activation of WT channels (Figure 3F). These phenotypes were not promising with regard to a high-sensitivity template for mutagenesis, but we chose to go ahead and test them in combination with ECD mutations, as the combined effects of agonist binding site mutations and channel mutations are not easy to predict.27,36 From the M1 mutants, we selected A220W for further testing as a high-sensitivity template. To test for restoration of glutamate sensitivity by M1 and M2 mutations, we generated several double-mutant channels combining the T197A or T199A mutation with the A220W mutation or with one of the M2 mutations. T197A/A220W and T199A/A220W channels showed no response to 30 mM glutamate (Figure 4A), suggesting that the M1 mutation cannot

Figure 3. (A) Top-down view of all five M2 helices (gray circle) and helices M1−M4 from one subunit (cyan) in the roundworm GLC-1 structure (Protein Data Bank entry 3RIF; divergent SmGluCl-2 equivalent in parentheses). (B, D, and F) Normalized glutamate (Glu)-induced current for WT, A220, and L6′S mutants (I/Imax) or glutamate-induced decrease in constitutive current for L9′ mutants (Decr/Decrmax) for WT SmGluCl-2 and the indicated mutants [mean ± SEM (n = 5−7)]. (C and E) Typical recording at oocytes expressing L6′S and L9′S SmGluCl-2 mutants, respectively. PTX, picrotoxin. Scale bars: x, 20 s; y, 0.2 μA.

Figure 4. (A) Example responses to 30 mM glutamate (black bars) at oocytes expressing double mutants. (B and C) Normalized glutamategated current (I/Imax) at the indicated mutants [mean ± SEM (n = 5− 7)].

restore glutamate sensitivity on loop C mutants, despite the increased glutamate sensitivity of A220W alone compared to that of the WT. Similarly, the combination of an L6′T, L9′S, or L9′T mutation with a T197A or T199A mutation resulted in no measurable activity (Figure 4A). In contrast, the L6′S mutation conferred glutamate sensitivity on both T197A and T199A channels (Figure 4A,B). The constitutive activity seen with the L6′S single mutant was still present but smaller in the L6′S/ T197A and L6′S/T199A double mutants. Thus, the L6′S mutation allows us to compare the effects of loop C threonine mutations. Relative to the L6′S mutation (EC50 = 11 μM), the T197A and T199A mutations caused 30- and 1000-fold reductions in glutamate sensitivity, respectively (Figure 4B). The L6′S mutation also increased the glutamate sensitivity of Y152F and Y202F mutants (Figure 4C), further indicating that it provides a high-sensitivity template on which binding site mutations, including ncAAs, might be tested. However, when the L6′S mutation was combined with the Y152TAG and Y202TAG constructs, the expression of these constructs was even poorer than with the single Y152TAG and Y202TAG constructs, as inferred from the current amplitude upon application of 30 mM glutamate (not shown). We are left to speculate that the Y202 side chain could contribute to a cation−π interaction, as the loop A and loop B positions that were assessed seem not to (Figure 2G).

Table 2. Parameters for Glutamate Concentration− Response Data at M1 and M2 Single Mutantsa EC50 (μM) (95% CI) WT A220L A220F A220W L6′S L6′T

22 (17−27) 15 (14−17) 4.6 (2.0−7.3)b 4.0 (1.6−6.3)b 11.3 (6.6−16) 280(260−300)c

nH (±95% CI)

n

2.4 2.0 1.0 1.0 2.4 1.3

6 7 7 8 7 7

(1.8−2.9) (1.8−2.1) (0.6−1.3)c (0.7−1.2)c (1.8−3.0) (1−2−1.4)c

a

Peak current responses to increasing concentrations of glutamate were fit with the Hill equation at n cells. EC50 and nH were then averaged. bP < 0.05 compared to the WT value in one-way analysis of variance with Dunnett’s multiple-comparison test. cP < 0.001 compared to the WT value in one-way analysis of variance with Dunnett’s multiple-comparison test.

glutamate was essentially converted to an inhibitor in L9′S and L9′T mutants, as increasing concentrations of glutamate caused a dose-dependent decrease in the constitutive activity of these channels (Figure 3E), although small increases in current were observed with low glutamate concentrations or at the very start of glutamate application (Figure 3E, inset). Plotting the glutamate-induced decrease in current against glutamate concentration for L9′S and L9′T mutants yielded sigmoidal E

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Biochemistry Effects of M2 Mutations on Channel Block by Picrotoxin. Finally, we performed additional experiments with picrotoxin, as 1 mM picrotoxin seemed to block constitutive current at L6′ mutants completely, but that at L9′ mutants incompletely. This seemed unusual, as 1 mM picrotoxin is sufficient to block glutamate-gated current at several GluCls.32,33 Furthermore, agonists tend to decrease the potency of picrotoxin,37 and we expected that constitutive current in the absence of agonist would be more readily blocked than agonist-gated current. Testing the effects of picrotoxin at various concentrations showed that at L6′S and L6′T mutants, the constitutive current was essentially completely blocked at picrotoxin concentrations between 10 and 100 μM (Figure 5A). In contrast, inhibition of the

interactions, the low potency at L9′ channels cannot be explained by the absence of polar 6′ side chains. Perhaps the open channel conformation of L6′S/T channels differs from that of L9′S/T channels. We also noted that the picrotoxin-induced block of L6′S channels was slow to wash out. Comparing panels A and C of Figure 5 shows that upon the return to the control bath solution, 30 μM picrotoxin-induced block of L6′T channels rapidly diminishes. In contrast, upon the return to the control bath solution, 1 μM picrotoxin-induced block of L6′S channels remains for some 50 s, and only after glutamate application and removal is the original constitutive current retrieved. Also, we observed that in the continued presence of subsaturating picrotoxin concentrations, block increased slowly with time. Comparing the first, brief application and the later, longer application of 0.3 μM picrotoxin in Figure 5C illustrates this. The concentration−response data in Figure 5E refer to the block after 10 s applications of each picrotoxin concentration. The fact that picrotoxin becomes trapped in L6′S but not in L6′T channels provides further evidence that the open channel conformation differs in the various constitutively active mutant channels.

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DISCUSSION GluCls constitute important antiparasitic drug targets, because of their physiological role in invertebrates and their absence from vertebrates. In this study, we sought to establish chemical interactions contributing to glutamate recognition by SmGluCl2, as the structure and function of flatworm GluCls are poorly understood compared to those of roundworm GluCls. In particular, we sought to establish if a particular aromatic residue in SmGluCl-2 contributes to glutamate recognition via a cation−π interaction with the agonist amine and if two threonine residues in the long loop C of SmGluCl-2 contribute to glutamate sensitivity. Although we were unable to dissect these interactions precisely, we found that both threonine residues are important for glutamate sensitivity and the aromatic side chains from each ligand binding loop are important for expression and/or glutamate sensitivity. Furthermore, we show that SmGluCl-2 differs significantly from other pLGICs in the role of crucial channel-forming M2 residues. Residues Contributing to Glutamate Recognition. Leucine substitution of the loop A phenylalanine and loop B and C tyrosine residues in SmGluCl-2 abolished glutamategated currents, suggesting that these aromatic residues are important to expression or function. Removal of the hydroxyl moieties in loop B and C tyrosine residues resulted in functional channels, but with moderately decreased glutamate sensitivity. This is consistent with the loop B tyrosine forming functionally significant polar interactions with a conserved loop E serine residue on the opposite face of the binding site (Figure 1). In SmGluCl-2, alanine and threonine substitutions at this position abolished and decreased 30-fold, respectively, glutamate sensitivity. This seems to reflect the ∼500-fold decrease in glutamate sensitivity of the S to A mutation at this position in roundworm GluCls20,27 and is far greater than the decrease in agonist sensitivity of the equivalent S to A mutation in other receptors (below). Perhaps the greater effect of this mutation in GluCls reflects simultaneous interactions with the loop B tyrosine and with the agonist carboxylate, whereas in GlyRs, it cannot interact with the nonpolar phenylalanine in loop B.

Figure 5. Effects of picrotoxin on SmGluCl-2. (A−D) Example recordings of current during various applications of glutamate (black bars, concentrations in micromolar) or picrotoxin (red bars, concentrations in micromolar). (E) Current amplitude during application of increasing picrotoxin concentrations, normalized to that at rest (mutants) or during application of glutamate alone (WT) [mean ± SEM (n = 5−8)].

constitutive current at L9′S and L9′T mutants appeared only at concentrations between 100 μM and 1 mM (Figure 5B). We considered that in L9′S and L9′T mutants, the leucine side chain at the 6′ position might preclude polar picrotoxin−T6′ interactions that are suggested to contribute to picrotoxin affinity at its site deep in the pore.14,38 However, when we tested picrotoxin inhibition of glutamate-gated currents at WT SmGluCl-2, which also contains the 6′-leucine side chain, the inhibitory potency of picrotoxin was still noticeably greater than at L9′S and L9′T channels (Figure 5D,E). Thus, although the introduction of the hydroxyl side chain to the 6′ position could enhance picrotoxin block by allowing favorable polar F

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Biochemistry

loop B tyrosine closer to the glutamate amine.15 This discrepancy is perhaps not surprising, given the differences described above (introductory section and Figure 1) for roundworm and flatworm GluCls. Nonetheless, the importance of the aromatic box and the importance of conserved complementary face arginine and serine residues that we have identified together suggest that the orientation of glutamate in the SmGluCl-2 binding site is similar to that of glutamate, glycine, and GABA in other inhibitory pLGICs. Pore-Lining M2 Residues. A fundamental feature of pLGICs is the presence of a hydrophobic leucine residue at the 9′ position in the middle of the channel pore, which is swung or tilted away from the pore axis during channel activation to allow ion permeation.13,48,49 Toward the intracellular end of the channel, highly conserved polar side chains, including serine or threonine residues at the 6′ and 2′ positions, contribute to ion conduction and channel block by compounds such as picrotoxin.14,38,50 In trying to alter channel activation by glutamate, we investigated the effects of mutations at the 6′ and 9′ positions of SmGluCl-2. In stark contrast to most other pLGICs, both inhibitory and excitatory, wild-type SmGluCl-2 contains a leucine residue at the 6′ position (see Figure S1). We considered that the L6′S and L6′T SmGluCl-2 mutants might differ little from WT and simply reflect other pLGICs with endogenous hydrophilic side chains at this position. However, L6′S and L6′T channels were constitutively active, and the L6′S mutation tended to increase glutamate potency alone and in combination with other mutations. L9′S and L9′T mutations also resulted in constitutively active channels. The level of constitutive activation may have been greater in L9′S and L9′T channels, as glutamate could scarcely activate additional current through these mutants, instead causing only a substantial decrease in constitutive current. We interpret these results as evidence that the hydrophobic gate extends from L9′ to L6′ in SmGluCl-2. This extended hydrophobic gate may be conserved in several other flatworm GluCls, including those from S. mansoni, based on the presence of a 6′ leucine residue.8 The constitutive activity of L6′S/T and L9′S/T mutants was blocked by picrotoxin, although the potency was some 1000fold greater at L6′S/T mutants. This greater potency could originate from favorable polar interactions between picrotoxin oxygen atoms and the hydroxyl side chains introduced by L6′S/ T mutations, which would reflect the results at GlyRs, where T6′V (but also T6′S) mutations drastically decrease picrotoxin potency.38 The roundworm GLC-1/picrotoxin X-ray crystal structure shows picrotoxin oxygen atoms in reasonable proximity of T6′ side chains, although substantially closer to T2′ side chains, one helical turn below.14 Alternatively, the different potency of picrotoxin at L6′S/T and L9′S/T mutations could be due to effects of the mutations on the overall pore conformation or on the transitions between different conformational states. If so, it would seem surprising that the more “completely” active L9′S/T mutants (which are not additionally activated by glutamate) are not more readily blocked by the drug. We therefore tentatively conclude that picrotoxin has difficulty penetrating the bulky L6′ residues in the L9′S/T mutants.

The large decrease in glutamate sensitivity upon phenylalanine substitution of the loop C tyrosine contrasts the minimal effects of Y to F mutations at the equivalent loop C position in roundworm GluCls.20,27 Similarly, the relatively conservative serine substitution of loop C threonine residues 197 and 199 caused ∼100-fold decreases in glutamate sensitivity in SmGluCl-2 (and alanine substitution abolished responses to glutamate). These effects are also substantially greater than those observed with equivalent substitutions in roundworm GluCls 20,27 and in vertebrate GlyRs and GABAARs.26,28 Loop C of SmGluCl-2 is two or three amino acids longer than in other inhibitory pLGICs (Figure S1), and this greater length seems to be conserved throughout other lophotrochozoan GluCls.8,19 Thus, loop C contributions to binding site structure and function seem to differ significantly in flatworm GluCls and roundworm GluCls and other inhibitory pLGICs. The large decreases in agonist sensitivity with most mutations make it difficult to establish whether threonine 197 or threonine 199 contributes more markedly to glutamate recognition. The comparison of T197A and T199A mutations on the L6′S high-sensitivity background showed 30- and 1000fold reductions in glutamate sensitivity, respectively, but T199S was only marginally less sensitive to glutamate than T197S was (both on the WT background). The question of whether these effects are due to a direct interaction with the agonist is difficult to answer, as the mutation of the threonine known to interact with the agonist in several other inhibitory pLGICs decreases agonist sensitivity as little as 80-fold but as much as 1000fold.26,27,39 Furthermore, the mutation of certain residues that do not bind the agonist directly can also increase EC50 values 100−1000-fold.23,40,41 With regard to conserved arginine and serine residues from loops D and E of the complementary face, again, we observed somewhat unique responses of SmGluCl-2 to mutation. Studies with vertebrate GlyRs and GABAARs and roundworm GABAARs and GluCls show that conservative (e.g., R to K and S to T) substitutions at these positions affect agonist potency to an extent that is smaller than that of nonconservative substitutions (e.g., alanine substitution).20,27,42−46 Unlike results in many of those studies, however, where even R to A and S to A mutations are tolerated to some extent, in SmGluCl-2, R54A, R54K, and S122A substitutions abolished activity, and even the conservative S122T mutation caused a 30-fold decrease in agonist sensitivity. Unfortunately, our attempts to dissect the chemical interactions between the aromatic residues and the agonist were unsuccessful, due to poor incorporation of phenylalanine derivatives of differing cation−π binding ability into the loop A, B, and particularly the loop C sites. Most definitive studies of cation−π interactions in inhibitory pLGICs correlate functional data with four to five derivatives of differing cation−π ability.16,17,47 For SmGluCl-2 loop A and B positions, we could measure functional data with three derivatives, which was not indicative of cation−π interactions. For the loop C position, however, we could measure glutamate sensitivity only at receptors containing two different derivatives, which did show a substantial decrease in glutamate potency with decreasing π electron density on the aromatic face. We therefore suspect that the SmGluCl-2 loop C tyrosine interacts with the agonist amine via a cation−π interaction. This differs from a previous prediction of ours, in which homology modeling based on roundworm GLC-1 placed the aromatic face of the SmGluCl-2

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CONCLUSIONS The flatworm SmGluCl-2 is activated by glutamate with a potency similar to that of roundworm GluCls, despite substantial divergence in loop C and in pore-lining M2 G

DOI: 10.1021/acs.biochem.7b01245 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

(4) Campbell, W. C. (2016) Ivermectin: A Reflection on Simplicity (Nobel Lecture). Angew. Chem., Int. Ed. 55, 10184−10189. (5) Omura, S. (2008) Ivermectin: 25 years and still going strong. Int. J. Antimicrob. Agents 31, 91−98. (6) Crellen, T., Walker, M., Lamberton, P. H., Kabatereine, N. B., Tukahebwa, E. M., Cotton, J. A., and Webster, J. P. (2016) Reduced Efficacy of Praziquantel Against Schistosoma mansoni Is Associated With Multiple Rounds of Mass Drug Administration. Clin. Infect. Dis. 63, 1151−1159. (7) King, C. H. (2010) Parasites and poverty: the case of schistosomiasis. Acta Trop. 113, 95−104. (8) Dufour, V., Beech, R. N., Wever, C., Dent, J. A., and Geary, T. G. (2013) Molecular cloning and characterization of novel glutamategated chloride channel subunits from Schistosoma mansoni. PLoS Pathog. 9, e1003586. (9) Lynagh, T., and Lynch, J. W. (2012) Ivermectin binding sites in human and invertebrate Cys-loop receptors. Trends Pharmacol. Sci. 33, 432−441. (10) Brownlee, D. J., and Fairweather, I. (1996) Immunocytochemical localization of glutamate-like immunoreactivity within the nervous system of the cestode Mesocestoides corti and the trematode Fasciola hepatica. Parasitol. Res. 82, 423−427. (11) Miller, C. L., Day, T. A., Bennett, J. L., and Pax, R. A. (1996) Schistosoma mansoni: L-glutamate-induced contractions in isolated muscle fibers; evidence for a glutamate transporter. Exp. Parasitol. 84, 410−419. (12) Zamanian, M., Kimber, M. J., McVeigh, P., Carlson, S. A., Maule, A. G., and Day, T. A. (2011) The repertoire of G protein-coupled receptors in the human parasite Schistosoma mansoni and the model organism Schmidtea mediterranea. BMC Genomics 12, 596. (13) Althoff, T., Hibbs, R. E., Banerjee, S., and Gouaux, E. (2014) Xray structures of GluCl in apo states reveal a gating mechanism of Cysloop receptors. Nature 512, 333−337. (14) Hibbs, R. E., and Gouaux, E. (2011) Principles of activation and permeation in an anion-selective Cys-loop receptor. Nature 474, 54− 60. (15) Lynagh, T., Cromer, B. A., Dufour, V., and Laube, B. (2014) Comparative pharmacology of flatworm and roundworm glutamategated chloride channels: Implications for potential anthelmintics. Int. J. Parasitol.: Drugs Drug Resist. 4, 244−255. (16) Padgett, C. L., Hanek, A. P., Lester, H. A., Dougherty, D. A., and Lummis, S. C. (2007) Unnatural amino acid mutagenesis of the GABA(A) receptor binding site residues reveals a novel cation-pi interaction between GABA and beta 2Tyr97. J. Neurosci. 27, 886−892. (17) Pless, S. A., Millen, K. S., Hanek, A. P., Lynch, J. W., Lester, H. A., Lummis, S. C., and Dougherty, D. A. (2008) A cation-pi interaction in the binding site of the glycine receptor is mediated by a phenylalanine residue. J. Neurosci. 28, 10937−10942. (18) Blarre, T., Bertrand, H. O., Acher, F. C., and Kehoe, J. (2014) Molecular determinants of agonist selectivity in glutamate-gated chloride channels which likely explain the agonist selectivity of the vertebrate glycine and GABAA-rho receptors. PLoS One 9, e108458. (19) Kehoe, J., Buldakova, S., Acher, F., Dent, J., Bregestovski, P., and Bradley, J. (2009) Aplysia cys-loop glutamate-gated chloride channels reveal convergent evolution of ligand specificity. J. Mol. Evol. 69, 125− 141. (20) Lynagh, T., Beech, R. N., Lalande, M. J., Keller, K., Cromer, B. A., Wolstenholme, A. J., and Laube, B. (2015) Molecular basis for convergent evolution of glutamate recognition by pentameric ligandgated ion channels. Sci. Rep. 5, 8558. (21) Dougherty, D. A., and Van Arnam, E. B. (2014) In vivo incorporation of non-canonical amino acids by using the chemical aminoacylation strategy: a broadly applicable mechanistic tool. ChemBioChem 15, 1710−1720. (22) Nowak, M. W., Gallivan, J. P., Silverman, S. K., Labarca, C. G., Dougherty, D. A., and Lester, H. A. (1998) In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system. Methods Enzymol. 293, 504−529.

residues. Loop C is longer than in other GluCls, and two threonine residues in loop C seem to contribute to glutamate sensitivity. Loop D and E arginine and serine residues are important for ligand recognition, and aromatic residues in loops A, B, and C seem to be important for expression or function of the receptor, although we could not identify the precise chemical basis of their contribution to glutamate recognition. Finally, and in contrast to other pLGICs, the hydrophobic gate in SmGluCl-2 seems to extend from L9′ to L6′ in the channel pore.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b01245. One figure containing an amino acid sequence alignment (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +45 35321535. ORCID

Stephan A. Pless: 0000-0001-6654-114X Timothy Lynagh: 0000-0003-4888-4098 Funding

T.L. was supported by a Sapere Aude Research Talent award (4092-00348) from the Danish Council for Independent Research and a Lundbeck Postdoctoral Fellowship (R1712014-558). S.A.P. was supported by a Lundbeck Foundation Fellowship (R139-2012-12390). The work was also supported by grants from the Carlsberg Foundation (2013 01 0439) and the Novo Nordisk Foundation (11767). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the technical assistance of Janne M. Colding and Britt Klein Tannehill. ABBREVIATIONS ECD, extracellular domain; GABAAR, type A γ-aminobutyric acid receptor; GluCl, glutamate-gated chloride channel; GlyR, glycine receptor; ncAA, noncanonical amino acid; pLGIC, pentameric ligand-gated ion channel; TMD, transmembrane domain.

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REFERENCES

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DOI: 10.1021/acs.biochem.7b01245 Biochemistry XXXX, XXX, XXX−XXX