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Characterization of ionotropic glutamate receptors in insect neuro-muscular junction
Comparative Biochemistry and Physiology, Part C 149 (2009) 275–280
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Comparative Biochemistry and Physiology, Part C j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c b p c
Characterization of ionotropic glutamate receptors in insect neuro-muscular junction I.M. Fedorova, L.G. Magazanik, D.B. Tikhonov ⁎ I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry RAS, St-Petersburg, Russia
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Article history: Received 4 June 2008 Received in revised form 28 July 2008 Accepted 29 July 2008 Available online 31 July 2008 Keywords: Calliphora vicina Ionotropic glutamate receptors AMPA receptors
a b s t r a c t Pharmacological properties of ionotropic glutamate receptors from Calliphora vicina larvae neuro-muscular junction (C. vicina iGlurs) were studied by two-electrode voltage-clamp technique. Characteristics of the ion channel pore were analyzed using a 26-member series of channel blockers, which includes mono- and dicationic derivatives of adamantane and phenylcyclohexyl. Structure–activity relationships were found to be markedly similar to the Ca2+-permeable AMPA receptors (AMPAR) but not NMDA receptors (NMDAR) channel subtype seen in vertebrates. Like AMPARs the channels of C. vicina iGlurs are sensitive mainly to dicationic compounds with 6–7 spacers between hydrophobic headgroup and terminal aminogroup. Study of the voltage dependence of block demonstrated that, like AMPARs, the C. vicina iGlur channels, are permeable to organic cations with dimensions exceeding 10 Å. Concentration dependence of block suggests the presence of two distinct channel populations with approximately 20-fold different sensitivity to cationic blockers. The recognition domain properties are more complex. Besides glutamate, the channels can be activated by kainate, quisqualate and domoate. Competitive antagonists of AMPAR and NMDAR are virtually inactive against the C. vicina iGlurs as well as allosteric modulators GYKI 52466 and PEPA. Surprisingly, the responses were potentiated 3 times by 100 mkM of cyclothiazide. We conclude that the channel-forming domain of C. vicina iGlurs is AMPAR-like, whereas the recognition domain is specific. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Ionotropic glutamate receptors (iGlurs) play a key role in excitation processes in the CNS of vertebrates. It is well known that different molecular forms of glutamate receptors possess markedly different physiological and pharmacological properties (see Dingledine et al., 1999 for review) that in their turn, significantly affect synaptic characteristics. For example, voltage-dependent block of NMDA receptor (NMDAR) channels by Mg2+ (Nowak et al., 1984; Mayer et al., 1984) is important for triggering long-term potentiation (Herron et al., 1986). Differences in Ca2+ permeability between GluR2-lacking and GluR2containing AMPA receptors (AMPAR) are also important for synaptic plasticity (Jia et al., 1996; Liu and Cull-Candy, 2000). EPSC decay and thus, the carried charge (Lomeli et al., 1994; Mosbacher et al., 1994), depend on whether the AMPAR contains the flip or flop versions of subunit (Sommer et al., 1990). Characteristics of glutamate receptors of vertebrates were studied intensively and large progress was achieved due to combined efforts of many groups employing methods of electrophysiology, molecular pharmacology, molecular and structural biology, and computer modeling. In contrast, much less is known about properties of glutamate receptors of invertebrates. The genome of the fruit fly contains 30 putative glutamate receptors (Littleton and Ganetzky, 2000). According to the modern view, receptors of neuro-muscular junction are ⁎ Corresponding author. Tel.: +7 812 552 3138; fax: +7 812 552 3012. E-mail address: [email protected] (D.B. Tikhonov). 1532-0456/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2008.07.010
composed of the DGluR3, DGluR2D, DGluR2E subunits and either DGluR2A or DGluR2B subunit (review by DiAntonio, 2006). DGluR2Acontaining and DGluR2B-containing iGluRs differ in desensitization and sensitivity to polyamine toxin, but molecular determinants of this difference remain unknown (DiAntonio et al., 1999). Heteromeric subunit composition of Drosophila iGlurs differs markedly from vertebrate iGlurs which are homomers or “dimers of dimers” (Safferling et al., 2001). This could determine some of the difference in properties between invertebrate and vertebrate iGlurs. Comparison of vertebrate and invertebrate receptors is a promising approach for understanding molecular evolution of the receptor protein and evolution of synaptic transmission mechanisms. Such comparison may also help to obtain more detailed insights into functioning of ligand-gated channels. There are some intriguing differences between amino-acid sequences of Drosophila and vertebrate iGlurs, in the ion channel as well as in the recognition domain. For instance, DGluR3 subunit contains the TA motif instead of the QQ motif typical for the AMPARs selectivity filter. DGluR2B subunit contains a Lys residue in the M3 segment, which is atypical for pore-lining segments of cation channels. In DGluR2A subunit the M3 segment contains a unique deletion. The main aim of present study was to characterize post-synaptic glutamate receptors in the neuro-muscular junction of Calliphora vicina larvae and to compare them with the vertebrate AMPAR and NMDAR. Our laboratory developed a series of organic channel blockers that discriminate NMDAR and AMPAR channels and allow us to characterize topography of the binding sites in these channels (Bolshakov
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et al., 2005). With the help of these tools we demonstrated that the ion channel structure of Calliphora iGlurs is remarkably similar to that of Ca2+-permeable AMPAR channels. In contrast to this, recognition domain of the receptor demonstrates specific properties with no direct analogs in vertebrate receptors. 2. Materials and methods 2.1. Electrophysiology Late third stage larvae of C. vicina (Diptera: Calliphoridae) were used. After dissection, the internal organs were removed, so that the preparation consisted only of muscles attached to the cuticle. The ventral ganglion was excised and the segmental nerves were stimulated through the suction electrode. Recordings were made from ventral longitudinal fibers. To eliminate electrical contacts of the recorded fiber with its neighbors, the latter were dissected. This resulted in significant increase of input resistance in the recorded cell. The preparation was perfused with a saline solution containing (in mM): 172 NaCl, 2.5 KCl, 0.5 CaCl2, 8.0 MgCl2, 2.4 NaHCO3, 0.3 H2PO4, and 52 sucrose. pH was adjusted to 7.2 with NaOH or HCl. Experiments were performed at room temperature (20–24 °C). The excitation postsynaptic currents (EPSC) were evoked by nerve stimulation and recorded by a conventional two-electrode voltage clamp using Axoclamp 2B (Axon Instr.) amplifier. The data were filtered at 2 kHz and stored on the computer. Drugs were purchased from Sigma and Tocris. The IEM compounds were synthesized by Dr. V. Gmiro at the Institute of experimental medicine RAMS, St. Petersburg. 2.2. Data analysis and statistics
Mayer et al., 1984). However, in our experiments with C. vicina iGlurs, this effect was not observed. I–V curve deviated from linearity only at holding potentials more negative than −100 mV but still remained monotonous. Organic open-channel blockers serve as powerful tools for analysis of spatial structure of ion channels. Our studies of the structure– activity relationships (Bolshakov et al., 2000, 2003, 2005) in series of channel blockers made possible to develop topographical and molecular models of AMPAR and NMDAR channels (Tikhonov et al., 2002; Tikhonov, 2007). We selected 26 representative mono- and dicationic derivatives of adamantane and phenylcyclohexyl for the present comparative study. Blocking activities of these compounds were estimated by constructing concentration dependencies of their action as described in the Materials and methods section. The results are summarized in Fig. 1. Quantitatively, C. vicina iGlurs sensitivity to blockers differs significantly from both NMDAR and Ca2+-permeable AMPAR. However, there is a strong correlation between C. vicina iGlurs and AMPAR sensitivity to the compounds used. Moreover, all key fingerprint features of AMPAR (see Bolshakov et al., 2005) are well reproduced. First, dicationic compounds are much more effective than their monocationic analogs. Second, the most active dicationic compounds have 6–7 spacers between hydrophobic head and terminal ammonium group. Third, trimethylammonium terminal group provides better activity than aminogroup. These results show strong similarity in molecular organization of the channel binding site in vertebrate AMPARs and C. vicina iGlur. It is known that pentobarbital effectively inhibits Ca2+-impermeable (GluR2-containing) AMPAR of vertebrates whereas GluR2-lacking receptors are much less sensitive to it (Taverna et al., 1994; Yamakura et al., 1995). However, it is still unclear if pentobarbital acts as a
To avoid signal-to-signal fluctuations, signals were digitally averaged from 10 consecutive responses before analysis. To minimize the influence of fluctuations in synaptic delay, the signals were adjusted by the midpoint of their rise. This provided good agreement between the shape (rise and decay) of individual and averaged signals. The concentration dependence of block was analyzed by the classical Hill equation BlockðkÞ ¼ 100= 1 þ ðC=IC50 Þn
ð1Þ
or by the model of binding of drug molecules to two distinct populations of receptor BlockðkÞ ¼ A=ð1 þ C=IC50A Þ þ ð100−AÞ=ð1 þ C=IC50B Þ:
ð2Þ
The concentration dependencies were analyzed separately for each cell, and fitting data were then averaged. This was necessary because of cell to cell variations of relative abundance of the receptor populations. Pooling of experimental data significantly distorted the results. All data are presented as means ± SD from at least five experiments. Significance of the effects was assessed by one-way ANOVA test and considered significant when the p value was less than 0.05. 3. Results Ionotropic receptors are composed of two principal domains, an intramembrane pore-forming domain and an extracellular recognition domain that controls the opening of the pore. Properties of these domains are largely independent and can be studied by use of specific series of ligands. 3.1. Characterization of the ion channel An important feature of the NMDAR ion channel is the voltagedependent block by Mg2+. This effect causes non-monotonous behavior of I–V curve at negative voltages (Nowak et al., 1984;
Fig. 1. Correlations between sensitivity of C. vicina iGlurs and vertebrate NMDA (A) and Ca2+-permeable AMPA (B) receptors to mono- and dicationic blockers. Significant correlation (0.78) is observed only with AMPA receptors.
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shown to be non-monotonous. At low holding potentials blocking action increases with hyperpolarization, but further hyperpolarization causes saturation or even decrease of the blocking action (Fig. 2). This result suggests permeation of large organic cations through the C. vicina iGlur channels, which makes them similar to the Ca2+-permeable AMPARs. On the contrary, vertebrate NMDARs are not permeable to these drugs (Tikhonova et al., 2008). 3.2. Distinct populations of receptors
Fig. 2. Biphasic voltage dependence of channel block suggests permeation of C. vicina iGlurs by organic blockers at large negative voltages. The values are normalized to the IC50 measured at −80 mV (taken as 1). Activities increase between −40 and −80 mV but saturate or even decrease at more negative voltages.
channel blocker (Jackson et al., 2003). In our experiments pentobarbital demonstrated weak inhibitory activity (IC50 = 389 ± 62 μM). Vertebrate Ca2+-permeable AMPARs have rather large channel dimensions. This was revealed by non-monotonous voltage dependence of the channel block (Bahring and Mayer, 1998; Tikhonova et al., 2008). Under high negative voltages blocking molecules permeate through the channel, thus causing a relief from block. In the present study we analyzed voltage dependence of action of IEM-1925 (PhChNH-(CH2)2-NH2), IEM-1676 (Ad-NH-(CH2)5-NMe3), IEM-1755 (Ad-NH(CH2)5-NH2) and IEM-2121 (2MeAd-NH2-(CH2)6-NMe3) on C. vicina iGlurs. As in the case of AMPARs, voltage dependence of block was
Fig. 3. High- and low-sensitive populations of C. vicina iGluRs. (A) Concentration dependence of channel block suggests presence of two populations with different sensitivities. (B) Correlation between sensitivity of Ca2+-permeable AMPA receptors and high-sensitive population of C. vicina iGlurs.
Analysis of the concentration dependence of block shows that the apparent Hill coefficient is unusually small. For all compounds this value was less than 1 (0.5–0.8). The simple reaction scheme cannot explain this effect. However, if we assume that there are two populations of channels with different sensitivities, the small slope is readily explained. Fig. 3A shows fitting of the concentration dependence of block by IEM-1925. Fitting by the Hill equation gives IC50 = 34 μM and a Hill coefficient of 0.7. The two population models (Eq. 2) give IC50(A) = 10 μM and IC50(B) = 170 μM; the relative abundance of population A is 51%. The same analysis was performed for several other compounds. For all compounds tested the relative abundance of populations was estimated as 40–60%. The obtained IC50 values for the high-sensitive population (A) were only 2–4 times higher than for vertebrate AMPARs and show good structure–activity correlation (Fig. 3B).Thus, the high-sensitive population of C. vicina iGlurs demonstrates not only qualitative agreement with AMPARs, but also a similarity of quantitative values of IC50. It is more difficult to draw conclusions about the low-sensitive population. The obtained IC50 values for low-sensitive population are 10–30 times larger than for high-sensitive population. The values are scattered and specific structure–activity relationships were not observed. This could be because fitting of the experimental curve for three parameters is not quite stable: small fluctuations of individual data lead to significant change of values. In some experiments the deep block (N70%) was not
Fig. 4. Effect of bath application of glutamate agonists on EPSC. (A) Representative recording in control and after bath application of glutamate. Decrease of EPSC amplitude is accompanied by slowing-down of decay. (B) Concentration dependence of the effect of bath application of drugs.
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achieved, and correct estimation of IC50 values for low-sensitive population was difficult. 3.3. Characterization of the recognition domain Many agonists of glutamate receptors induce only transient response followed by deep desensitization. Therefore, slow bath application of agonists is not a suitable approach. To examine effects of putative agonists we employed the effect of “cross-desensitization” (James et al., 1980). If agonist applied to the bath solution desensitizes some receptors, the number of receptors available for activation by synaptic glutamate is reduced. Thus, the effect of agonist applied to the bath solution should induce a reduction of EPSC amplitude. Control experiments were performed with bath application of glutamate. As expected, glutamate caused concentration-dependent decrease of EPSC with IC50 value of 180 ± 20 μM (Fig. 4). Decrease of the EPSC amplitude was accompanied by significant slowing of the EPSC decay and rise of holding current (36 ± 13 nA). This current can be due to non-desensitizing fraction of the response caused by bath-applied glutamate. However, glutamate action is non-specific; it can activate some current via metabotropic receptors (see, e.g. Sekizawa and Bonham, 2006). In agreement with the latter possibility, 100 μM IEM1925 induced only about 10% inhibition of the steady-state response to glutamate. After that we tested the effect of NMDA, AMPA, kainate, quisqualate and domoate. Effect of quisqualate was similar to the effect of glutamate. The drug caused reduction of EPSC amplitude, slowing-down of the decay and induced non-desensitizing current. 1 mM of NMDA was ineffective. We observed neither increase of holding current, nor changes of EPSCs. Application of NMDA together with 100 μM glycine produced only a minor (10%) reduction of EPSC. No cross-desensitization effect was seen after application of AMPA in concentrations up to 1 mM. On the contrary, 100 μM domoate and 1 mM kainate caused about 50% decrease of the EPSC amplitude. We observed no increase of holding current after application of domoate. Kainate and quisqualate induced non-desensitizing currents, which were effectively inhibited by active channel blockers (Fig. 5). Competitive antagonists in experiments with bath application should also decrease EPSC amplitude (of course, without producing current themselves). We tested action of APV and CNQX as NMDAR and non-NMDAR antagonists, respectively. Both in concentration of 1 mM did not substantially affect EPSC amplitude. Several allosterically acting agents such as GYKI 52466, aniracetam, PEPA, concanavalin A, cyclothiazide (CTZ) modulate GluRs of vertebrates (Donevan and Rogawski, 1993; Isaacson and Nicoll, 1991; Partin et al., 1993; Yamada and Tang 1993; Mathers and Usherwood, 1976). It was demonstrated previously that concanavalin A is unable to potentiate EPSCs in the Calliphora neuro-muscular junction (Magazanik et al., 1992). GYKI 52466, aniracetam and PEPA in our experiments were inactive. CTZ, surprisingly, produced a strong effect (Fig. 6).
Fig. 5. Steady-state current induced by bath application of kainate and its blockade by IEM-1676.
Fig. 6. Effect of CTZ on EPSC. (A) Representative recordings in control, after bath application of 100 μM CTZ and after CTZ wash-out. Significant and reversible increase of the EPSC amplitude is not accompanied by change of shape of the response. (B) Crossdesensitization by glutamate and kainate in control and in the presence of CTZ. The values do not differ significantly, indicating that effects are independent.
100 μM of CTZ potentiated the EPSC response by 3 times. The effect was fully reversible. It should be noted that the response shape was not affected by CTZ. Finally, we tested the effect of co-application of CTZ with glutamate and kainate. In both cases cross-desensitization effect caused by agonists was not changed in the presence of CTZ. 4. Discussion In the present study we demonstrated that pharmacological characteristics of the ion channel of C. vicina iGlurs are remarkably similar to those of Ca2+-permeable AMPARs. Because the blocking drugs were selected from our previous studies to characterize organization of the binding site in glutamate receptor channels, we can conclude that topography of the binding sites for open-channel blockers in C. vicina iGlurs and Ca2+-permeable AMPARs is the same. Certainly, it does not mean that the structures are identical. The pore diameters of these channels allow permeations of organic compounds with dimensions exceeding 10 Å. The spatial localization of hydrophobic and nucleophilic groups is similar, resulting in strong correlation of the structure–activity relationships. Besides these similarities, we cannot rule out some differences in the channel structure that do not significantly affect the action of the channel blockers used. It is interesting to compare the properties of C. vicina iGlur channels with the available data on iGlurs of other invertebrates. iGlurs of locust muscle were intensively studied by P. Usherwood and his colleagues. These channels are sensitive to polyamine-amides like philanthotoxin (PhTX). Study of series of PhTX analogs revealed complex structure– activity relationships (Bruce et al., 1990; Sudan et al., 1995; Strømgaard et al., 2000). This complexity can be explained (at least partially) by conformation flexibility of philanthotoxins, which can form intramolecular hydrogen bonds (Tikhonov et al., 2000). However, the general
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conclusion about determinants of philanthotoxin action on GluRs in locust muscle is that charged polyamine “tail” is critically important (Mellor and Usherwood, 2004). This pattern is typical for vertebrate Ca2+-permeable AMPARs and C. vicina iGlurs but not for vertebrate NMDARs. Previously, we studied iGluRs of mollusk neurons (Samoilova et al., 1997; Magazanik et al., 2002). Although the series of blocking compounds we used was less representative, the results strongly demonstrated low activity of monocationic compounds; dependence of activity of dicationic compounds on the chain length was the same as in AMPARs and C. vicina iGlurs. Moreover, blocking activities (IC50 values) were quantitatively similar on mollusk glutamate receptors and vertebrate AMPARs. Analysis of the concentration dependence of block revealed two populations of iGlurs with 10–20-fold difference in sensitivity to channel blockers. However, even for the highly-sensitive population the estimated IC50 values are 2–5 times larger than the values measured on vertebrate AMPARs. This discrepancy might be due to the different experimental protocols used. Activities against AMPARs were measured as inhibition of stationary current evoked by kainate application on isolated neurons. Activities against C. vicina iGlurs were estimated from the decrease of EPSCs. In the latter case, non-stationary conditions can affect results because of the use-dependence of channel block. It is difficult to characterize the population of low-sensitive iGluRs in Calliphora neuro-muscular junction. For all compounds tested the IC50 value for block of low-sensitive receptors exceeded 100 μM. Therefore, we cannot say if low sensitivity is due to the difference in channel topography. It is possible that some additional factor decreases activity of cationic blockers. For instance, the Lys residue in M3 segment of DGluR2A subunit can play such a role. In agreement with this possibility, Drosophila iGlurs containing DGluR2A are less sensitive to argiotoxin than the DGluR2A-lacking receptors (DiAntonio et al., 1999). It is tempting to speculate that the high- and lowsensitive C. vicina iGlurs resemble Ca2+-permeable and Ca2+-impermeable AMPARs, since the latter are low-sensitive to cationic blockers too (Magazanik et al., 1997; Bolshakov et al., 2005). However, weak sensitivity of C. vicina iGlurs to pentobarbital evidences against this idea. Pentobarbital activity in our experiments was low and pentobarbital action did not reveal subpopulations of receptors. It is more likely that the aforementioned local differences in the binding site sequence are responsible for high or low sensitivity of C. vicina iGlurs to channel blockers. Properties of the recognition domain are much more complex and no direct analogy with vertebrate receptors can be drawn. Besides glutamate, C. vicina iGlurs can be activated only by domoate, quisqualate and kainate. Competitive antagonists of both AMPAR and NMDAR were inactive. It should be noted that non-NMDA invertebrate receptors demonstrate a diversity of pharmacological characteristics. Thus, quisqualate is active in many preparations (see e.g. Dudel et al., 1988; Dierkes et al., 1996; Brierley et al., 1997; Kimura et al., 2001; Muller et al., 2003; Holden-Dye and Walker, 2006; Scannell et al., 2008). Kainate produces the same effect as quisqualate and glutamate on leech neurons (Dierkes et al., 1996), Ascaridia galli and Caenorhabditis elegans (Holden-Dye and Walker, 2006) and in the pond snail, Helisoma trivolvis (Scannell et al., 2008) but is ineffective on the locust muscle (Dudel et al., 1988), ganglion cells of Aplysia (Kimura et al., 2001) and produces only a small effect on Lymnaea neurons (Brierley et al., 1997). DNQX (or CNQX) often block the invertebrate non-NMDA receptors (Dierkes et al., 1996; Kimura et al., 2001; Muller et al., 2003; Lima et al., 2003). Our results demonstrate closest similarity with data reported for motoneurons of the lobster cardiac ganglion (Hashemzadeh-Gargari and Freschi, 1992). In that work the rank order of the agonists potencies was quisqualate greater than glutamate, glutamate greater than kainate. Kainate, unlike the other agonists, showed no desensitization. CNQX and DNQX only partially blocked the responses
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even in concentration of hundreds μM, and concanavalin A was ineffective. However, it would be premature to speculate about structural similarities (differences) between recognition domain of C. vicina iGluRs and other non-NMDA receptors, because such conclusions require systematic study with use of homologous series of drugs, as we did in this work with the ion channel. CTZ, but not concanavalin A, significantly potentiated the EPSC in our experiments. Potentiation by cyclothiazide of recombinant veretebrate receptor responses in Xenopus oocytes showed selectivity for AMPA versus kainate receptors. In contrast, concanavalin A strongly potentiated responses at kainate but not AMPA receptors (Partin et al., 1993). It should be noted that the mechanism of action of CTZ on C. vicina iGlurs and AMPARs is not identical. If CTZ reduces desensitization, the EPSC should become longer. But it is not the case. Careful kinetic analysis of CTZ action on vertebrate AMPARs was performed by Partin et al. (1996). According to their results, CTZ induces dual effects: increase of agonist affinity and reduction of the onset rate of desensitization. We hypothesize that on C. vicina iGlurs CTZ only increases agonist affinity without affecting desensitization rate. In the present work we studied pharmacological characteristics of ionotropic glutamate receptors from C. vicina neuro-muscular junction and compared receptor properties with those of vertebrate receptors. The channel pore was well-characterized using a homologous series of channel blockers as molecular probes. We found close similarity between the ion channels of C. vicina iGlurs and vertebrate AMPARs. However, glutamate receptors in Calliphora neuro-muscular junction are not homogeneous, and according to their sensitivity to channel blockers they can be subdivided into high- and low-sensitive populations. Molecular origin of low sensitivity remains unclear. Properties of the recognition domain of C. vicina iGlurs make them markedly different from all known ionotropic glutamate receptors. In the present work we just outlined the problem; solution will require serious additional efforts. Further analysis is expected to reveal molecular determinants of drug action on the recognition domain. This will contribute to our understanding of functioning of the ionotropic glutamate receptors and of molecular evolution in these proteins. Acknowledgements This work is supported by RFBR grants 08-04-00326 and 04-00617, grant for Russian scientific schools 4821.2008.4 and by a grant from program “Molecular and Cellular Biology”, RAS. References Bahring, R., Mayer, M.L., 1998. An analysis of philanthotoxin block for recombinant rat GluR6(Q) glutamate receptor channels. J. Physiol. 509, 635–650. Bolshakov, K.V., Tikhonov, D.B., Gmiro, V.E., Magazanik, L.G., 2000. Different arrangement of hydrophobic and nucleophilic components of channel binding sites in Nmethyl-D-aspartate and AMPA receptors of rat brain is revealed by channel blockade. Neurosci. Lett. 291, 101–104. Bolshakov, K.V., Gmiro, V.E., Tikhonov, D.B., Magazanik, L.G., 2003. Determinants of trapping block of N-methyl-D-aspartate receptor channels. J. Neurochem. 87, 56–65. Bolshakov, K.V., Kim, K.H., Potapjeva, N.N., Gmiro, V.E., Tikhonov, D.B., Usherwood, P.N., Mellor, I.R., Magazanik, L.G., 2005. Design of antagonists for NMDA and AMPA receptors. Neuropharmacology 49, 144–155. Brierley, M.J., Yeoman, M.S., Benjamin, P.R., 1997. Glutamate is the transmitter for N2v retraction phase interneurons of the Lymnaea feeding system. J. Neurophysiol. 78, 3408–3414. Bruce, M., Bukownik, R., Eldefrawi, A.T., Eldefrawi, M.E., Goodnow Jr., R., Kallimopoulos, T., Konno, K., Nakanishi, K., Niwa, M., Usherwood, P.N., 1990. Structure–activity relationships of analogues of the wasp toxin philanthotoxin: non-competitive antagonists of quisqualate receptors. Toxicon 28, 1333–1346. DiAntonio, A., 2006. Glutamate receptors at the Drosophila neuromuscular junction. Int. Rev. Neurobiol 75, 165–179. DiAntonio, A., Petersen, S.A., Heckmann, M., Goodman, C.S., 1999. Glutamate receptor expression regulates quantal size and quantal content at the Drosophila neuromuscular junction. J. Neurosci. 19, 3023–3032. Dierkes, P.W., Hochstrate, P., Schlue, W.R., 1996. Distribution and functional properties of glutamate receptors in the leech central nervous system. J. Neurophysiol. 75, 2312–2321.
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Comparative Biochemistry and Physiology, Part C j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c b p c
Characterization of ionotropic glutamate receptors in insect neuro-muscular junction I.M. Fedorova, L.G. Magazanik, D.B. Tikhonov ⁎ I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry RAS, St-Petersburg, Russia
a r t i c l e
i n f o
Article history: Received 4 June 2008 Received in revised form 28 July 2008 Accepted 29 July 2008 Available online 31 July 2008 Keywords: Calliphora vicina Ionotropic glutamate receptors AMPA receptors
a b s t r a c t Pharmacological properties of ionotropic glutamate receptors from Calliphora vicina larvae neuro-muscular junction (C. vicina iGlurs) were studied by two-electrode voltage-clamp technique. Characteristics of the ion channel pore were analyzed using a 26-member series of channel blockers, which includes mono- and dicationic derivatives of adamantane and phenylcyclohexyl. Structure–activity relationships were found to be markedly similar to the Ca2+-permeable AMPA receptors (AMPAR) but not NMDA receptors (NMDAR) channel subtype seen in vertebrates. Like AMPARs the channels of C. vicina iGlurs are sensitive mainly to dicationic compounds with 6–7 spacers between hydrophobic headgroup and terminal aminogroup. Study of the voltage dependence of block demonstrated that, like AMPARs, the C. vicina iGlur channels, are permeable to organic cations with dimensions exceeding 10 Å. Concentration dependence of block suggests the presence of two distinct channel populations with approximately 20-fold different sensitivity to cationic blockers. The recognition domain properties are more complex. Besides glutamate, the channels can be activated by kainate, quisqualate and domoate. Competitive antagonists of AMPAR and NMDAR are virtually inactive against the C. vicina iGlurs as well as allosteric modulators GYKI 52466 and PEPA. Surprisingly, the responses were potentiated 3 times by 100 mkM of cyclothiazide. We conclude that the channel-forming domain of C. vicina iGlurs is AMPAR-like, whereas the recognition domain is specific. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Ionotropic glutamate receptors (iGlurs) play a key role in excitation processes in the CNS of vertebrates. It is well known that different molecular forms of glutamate receptors possess markedly different physiological and pharmacological properties (see Dingledine et al., 1999 for review) that in their turn, significantly affect synaptic characteristics. For example, voltage-dependent block of NMDA receptor (NMDAR) channels by Mg2+ (Nowak et al., 1984; Mayer et al., 1984) is important for triggering long-term potentiation (Herron et al., 1986). Differences in Ca2+ permeability between GluR2-lacking and GluR2containing AMPA receptors (AMPAR) are also important for synaptic plasticity (Jia et al., 1996; Liu and Cull-Candy, 2000). EPSC decay and thus, the carried charge (Lomeli et al., 1994; Mosbacher et al., 1994), depend on whether the AMPAR contains the flip or flop versions of subunit (Sommer et al., 1990). Characteristics of glutamate receptors of vertebrates were studied intensively and large progress was achieved due to combined efforts of many groups employing methods of electrophysiology, molecular pharmacology, molecular and structural biology, and computer modeling. In contrast, much less is known about properties of glutamate receptors of invertebrates. The genome of the fruit fly contains 30 putative glutamate receptors (Littleton and Ganetzky, 2000). According to the modern view, receptors of neuro-muscular junction are ⁎ Corresponding author. Tel.: +7 812 552 3138; fax: +7 812 552 3012. E-mail address: [email protected] (D.B. Tikhonov). 1532-0456/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2008.07.010
composed of the DGluR3, DGluR2D, DGluR2E subunits and either DGluR2A or DGluR2B subunit (review by DiAntonio, 2006). DGluR2Acontaining and DGluR2B-containing iGluRs differ in desensitization and sensitivity to polyamine toxin, but molecular determinants of this difference remain unknown (DiAntonio et al., 1999). Heteromeric subunit composition of Drosophila iGlurs differs markedly from vertebrate iGlurs which are homomers or “dimers of dimers” (Safferling et al., 2001). This could determine some of the difference in properties between invertebrate and vertebrate iGlurs. Comparison of vertebrate and invertebrate receptors is a promising approach for understanding molecular evolution of the receptor protein and evolution of synaptic transmission mechanisms. Such comparison may also help to obtain more detailed insights into functioning of ligand-gated channels. There are some intriguing differences between amino-acid sequences of Drosophila and vertebrate iGlurs, in the ion channel as well as in the recognition domain. For instance, DGluR3 subunit contains the TA motif instead of the QQ motif typical for the AMPARs selectivity filter. DGluR2B subunit contains a Lys residue in the M3 segment, which is atypical for pore-lining segments of cation channels. In DGluR2A subunit the M3 segment contains a unique deletion. The main aim of present study was to characterize post-synaptic glutamate receptors in the neuro-muscular junction of Calliphora vicina larvae and to compare them with the vertebrate AMPAR and NMDAR. Our laboratory developed a series of organic channel blockers that discriminate NMDAR and AMPAR channels and allow us to characterize topography of the binding sites in these channels (Bolshakov
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et al., 2005). With the help of these tools we demonstrated that the ion channel structure of Calliphora iGlurs is remarkably similar to that of Ca2+-permeable AMPAR channels. In contrast to this, recognition domain of the receptor demonstrates specific properties with no direct analogs in vertebrate receptors. 2. Materials and methods 2.1. Electrophysiology Late third stage larvae of C. vicina (Diptera: Calliphoridae) were used. After dissection, the internal organs were removed, so that the preparation consisted only of muscles attached to the cuticle. The ventral ganglion was excised and the segmental nerves were stimulated through the suction electrode. Recordings were made from ventral longitudinal fibers. To eliminate electrical contacts of the recorded fiber with its neighbors, the latter were dissected. This resulted in significant increase of input resistance in the recorded cell. The preparation was perfused with a saline solution containing (in mM): 172 NaCl, 2.5 KCl, 0.5 CaCl2, 8.0 MgCl2, 2.4 NaHCO3, 0.3 H2PO4, and 52 sucrose. pH was adjusted to 7.2 with NaOH or HCl. Experiments were performed at room temperature (20–24 °C). The excitation postsynaptic currents (EPSC) were evoked by nerve stimulation and recorded by a conventional two-electrode voltage clamp using Axoclamp 2B (Axon Instr.) amplifier. The data were filtered at 2 kHz and stored on the computer. Drugs were purchased from Sigma and Tocris. The IEM compounds were synthesized by Dr. V. Gmiro at the Institute of experimental medicine RAMS, St. Petersburg. 2.2. Data analysis and statistics
Mayer et al., 1984). However, in our experiments with C. vicina iGlurs, this effect was not observed. I–V curve deviated from linearity only at holding potentials more negative than −100 mV but still remained monotonous. Organic open-channel blockers serve as powerful tools for analysis of spatial structure of ion channels. Our studies of the structure– activity relationships (Bolshakov et al., 2000, 2003, 2005) in series of channel blockers made possible to develop topographical and molecular models of AMPAR and NMDAR channels (Tikhonov et al., 2002; Tikhonov, 2007). We selected 26 representative mono- and dicationic derivatives of adamantane and phenylcyclohexyl for the present comparative study. Blocking activities of these compounds were estimated by constructing concentration dependencies of their action as described in the Materials and methods section. The results are summarized in Fig. 1. Quantitatively, C. vicina iGlurs sensitivity to blockers differs significantly from both NMDAR and Ca2+-permeable AMPAR. However, there is a strong correlation between C. vicina iGlurs and AMPAR sensitivity to the compounds used. Moreover, all key fingerprint features of AMPAR (see Bolshakov et al., 2005) are well reproduced. First, dicationic compounds are much more effective than their monocationic analogs. Second, the most active dicationic compounds have 6–7 spacers between hydrophobic head and terminal ammonium group. Third, trimethylammonium terminal group provides better activity than aminogroup. These results show strong similarity in molecular organization of the channel binding site in vertebrate AMPARs and C. vicina iGlur. It is known that pentobarbital effectively inhibits Ca2+-impermeable (GluR2-containing) AMPAR of vertebrates whereas GluR2-lacking receptors are much less sensitive to it (Taverna et al., 1994; Yamakura et al., 1995). However, it is still unclear if pentobarbital acts as a
To avoid signal-to-signal fluctuations, signals were digitally averaged from 10 consecutive responses before analysis. To minimize the influence of fluctuations in synaptic delay, the signals were adjusted by the midpoint of their rise. This provided good agreement between the shape (rise and decay) of individual and averaged signals. The concentration dependence of block was analyzed by the classical Hill equation BlockðkÞ ¼ 100= 1 þ ðC=IC50 Þn
ð1Þ
or by the model of binding of drug molecules to two distinct populations of receptor BlockðkÞ ¼ A=ð1 þ C=IC50A Þ þ ð100−AÞ=ð1 þ C=IC50B Þ:
ð2Þ
The concentration dependencies were analyzed separately for each cell, and fitting data were then averaged. This was necessary because of cell to cell variations of relative abundance of the receptor populations. Pooling of experimental data significantly distorted the results. All data are presented as means ± SD from at least five experiments. Significance of the effects was assessed by one-way ANOVA test and considered significant when the p value was less than 0.05. 3. Results Ionotropic receptors are composed of two principal domains, an intramembrane pore-forming domain and an extracellular recognition domain that controls the opening of the pore. Properties of these domains are largely independent and can be studied by use of specific series of ligands. 3.1. Characterization of the ion channel An important feature of the NMDAR ion channel is the voltagedependent block by Mg2+. This effect causes non-monotonous behavior of I–V curve at negative voltages (Nowak et al., 1984;
Fig. 1. Correlations between sensitivity of C. vicina iGlurs and vertebrate NMDA (A) and Ca2+-permeable AMPA (B) receptors to mono- and dicationic blockers. Significant correlation (0.78) is observed only with AMPA receptors.
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shown to be non-monotonous. At low holding potentials blocking action increases with hyperpolarization, but further hyperpolarization causes saturation or even decrease of the blocking action (Fig. 2). This result suggests permeation of large organic cations through the C. vicina iGlur channels, which makes them similar to the Ca2+-permeable AMPARs. On the contrary, vertebrate NMDARs are not permeable to these drugs (Tikhonova et al., 2008). 3.2. Distinct populations of receptors
Fig. 2. Biphasic voltage dependence of channel block suggests permeation of C. vicina iGlurs by organic blockers at large negative voltages. The values are normalized to the IC50 measured at −80 mV (taken as 1). Activities increase between −40 and −80 mV but saturate or even decrease at more negative voltages.
channel blocker (Jackson et al., 2003). In our experiments pentobarbital demonstrated weak inhibitory activity (IC50 = 389 ± 62 μM). Vertebrate Ca2+-permeable AMPARs have rather large channel dimensions. This was revealed by non-monotonous voltage dependence of the channel block (Bahring and Mayer, 1998; Tikhonova et al., 2008). Under high negative voltages blocking molecules permeate through the channel, thus causing a relief from block. In the present study we analyzed voltage dependence of action of IEM-1925 (PhChNH-(CH2)2-NH2), IEM-1676 (Ad-NH-(CH2)5-NMe3), IEM-1755 (Ad-NH(CH2)5-NH2) and IEM-2121 (2MeAd-NH2-(CH2)6-NMe3) on C. vicina iGlurs. As in the case of AMPARs, voltage dependence of block was
Fig. 3. High- and low-sensitive populations of C. vicina iGluRs. (A) Concentration dependence of channel block suggests presence of two populations with different sensitivities. (B) Correlation between sensitivity of Ca2+-permeable AMPA receptors and high-sensitive population of C. vicina iGlurs.
Analysis of the concentration dependence of block shows that the apparent Hill coefficient is unusually small. For all compounds this value was less than 1 (0.5–0.8). The simple reaction scheme cannot explain this effect. However, if we assume that there are two populations of channels with different sensitivities, the small slope is readily explained. Fig. 3A shows fitting of the concentration dependence of block by IEM-1925. Fitting by the Hill equation gives IC50 = 34 μM and a Hill coefficient of 0.7. The two population models (Eq. 2) give IC50(A) = 10 μM and IC50(B) = 170 μM; the relative abundance of population A is 51%. The same analysis was performed for several other compounds. For all compounds tested the relative abundance of populations was estimated as 40–60%. The obtained IC50 values for the high-sensitive population (A) were only 2–4 times higher than for vertebrate AMPARs and show good structure–activity correlation (Fig. 3B).Thus, the high-sensitive population of C. vicina iGlurs demonstrates not only qualitative agreement with AMPARs, but also a similarity of quantitative values of IC50. It is more difficult to draw conclusions about the low-sensitive population. The obtained IC50 values for low-sensitive population are 10–30 times larger than for high-sensitive population. The values are scattered and specific structure–activity relationships were not observed. This could be because fitting of the experimental curve for three parameters is not quite stable: small fluctuations of individual data lead to significant change of values. In some experiments the deep block (N70%) was not
Fig. 4. Effect of bath application of glutamate agonists on EPSC. (A) Representative recording in control and after bath application of glutamate. Decrease of EPSC amplitude is accompanied by slowing-down of decay. (B) Concentration dependence of the effect of bath application of drugs.
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achieved, and correct estimation of IC50 values for low-sensitive population was difficult. 3.3. Characterization of the recognition domain Many agonists of glutamate receptors induce only transient response followed by deep desensitization. Therefore, slow bath application of agonists is not a suitable approach. To examine effects of putative agonists we employed the effect of “cross-desensitization” (James et al., 1980). If agonist applied to the bath solution desensitizes some receptors, the number of receptors available for activation by synaptic glutamate is reduced. Thus, the effect of agonist applied to the bath solution should induce a reduction of EPSC amplitude. Control experiments were performed with bath application of glutamate. As expected, glutamate caused concentration-dependent decrease of EPSC with IC50 value of 180 ± 20 μM (Fig. 4). Decrease of the EPSC amplitude was accompanied by significant slowing of the EPSC decay and rise of holding current (36 ± 13 nA). This current can be due to non-desensitizing fraction of the response caused by bath-applied glutamate. However, glutamate action is non-specific; it can activate some current via metabotropic receptors (see, e.g. Sekizawa and Bonham, 2006). In agreement with the latter possibility, 100 μM IEM1925 induced only about 10% inhibition of the steady-state response to glutamate. After that we tested the effect of NMDA, AMPA, kainate, quisqualate and domoate. Effect of quisqualate was similar to the effect of glutamate. The drug caused reduction of EPSC amplitude, slowing-down of the decay and induced non-desensitizing current. 1 mM of NMDA was ineffective. We observed neither increase of holding current, nor changes of EPSCs. Application of NMDA together with 100 μM glycine produced only a minor (10%) reduction of EPSC. No cross-desensitization effect was seen after application of AMPA in concentrations up to 1 mM. On the contrary, 100 μM domoate and 1 mM kainate caused about 50% decrease of the EPSC amplitude. We observed no increase of holding current after application of domoate. Kainate and quisqualate induced non-desensitizing currents, which were effectively inhibited by active channel blockers (Fig. 5). Competitive antagonists in experiments with bath application should also decrease EPSC amplitude (of course, without producing current themselves). We tested action of APV and CNQX as NMDAR and non-NMDAR antagonists, respectively. Both in concentration of 1 mM did not substantially affect EPSC amplitude. Several allosterically acting agents such as GYKI 52466, aniracetam, PEPA, concanavalin A, cyclothiazide (CTZ) modulate GluRs of vertebrates (Donevan and Rogawski, 1993; Isaacson and Nicoll, 1991; Partin et al., 1993; Yamada and Tang 1993; Mathers and Usherwood, 1976). It was demonstrated previously that concanavalin A is unable to potentiate EPSCs in the Calliphora neuro-muscular junction (Magazanik et al., 1992). GYKI 52466, aniracetam and PEPA in our experiments were inactive. CTZ, surprisingly, produced a strong effect (Fig. 6).
Fig. 5. Steady-state current induced by bath application of kainate and its blockade by IEM-1676.
Fig. 6. Effect of CTZ on EPSC. (A) Representative recordings in control, after bath application of 100 μM CTZ and after CTZ wash-out. Significant and reversible increase of the EPSC amplitude is not accompanied by change of shape of the response. (B) Crossdesensitization by glutamate and kainate in control and in the presence of CTZ. The values do not differ significantly, indicating that effects are independent.
100 μM of CTZ potentiated the EPSC response by 3 times. The effect was fully reversible. It should be noted that the response shape was not affected by CTZ. Finally, we tested the effect of co-application of CTZ with glutamate and kainate. In both cases cross-desensitization effect caused by agonists was not changed in the presence of CTZ. 4. Discussion In the present study we demonstrated that pharmacological characteristics of the ion channel of C. vicina iGlurs are remarkably similar to those of Ca2+-permeable AMPARs. Because the blocking drugs were selected from our previous studies to characterize organization of the binding site in glutamate receptor channels, we can conclude that topography of the binding sites for open-channel blockers in C. vicina iGlurs and Ca2+-permeable AMPARs is the same. Certainly, it does not mean that the structures are identical. The pore diameters of these channels allow permeations of organic compounds with dimensions exceeding 10 Å. The spatial localization of hydrophobic and nucleophilic groups is similar, resulting in strong correlation of the structure–activity relationships. Besides these similarities, we cannot rule out some differences in the channel structure that do not significantly affect the action of the channel blockers used. It is interesting to compare the properties of C. vicina iGlur channels with the available data on iGlurs of other invertebrates. iGlurs of locust muscle were intensively studied by P. Usherwood and his colleagues. These channels are sensitive to polyamine-amides like philanthotoxin (PhTX). Study of series of PhTX analogs revealed complex structure– activity relationships (Bruce et al., 1990; Sudan et al., 1995; Strømgaard et al., 2000). This complexity can be explained (at least partially) by conformation flexibility of philanthotoxins, which can form intramolecular hydrogen bonds (Tikhonov et al., 2000). However, the general
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conclusion about determinants of philanthotoxin action on GluRs in locust muscle is that charged polyamine “tail” is critically important (Mellor and Usherwood, 2004). This pattern is typical for vertebrate Ca2+-permeable AMPARs and C. vicina iGlurs but not for vertebrate NMDARs. Previously, we studied iGluRs of mollusk neurons (Samoilova et al., 1997; Magazanik et al., 2002). Although the series of blocking compounds we used was less representative, the results strongly demonstrated low activity of monocationic compounds; dependence of activity of dicationic compounds on the chain length was the same as in AMPARs and C. vicina iGlurs. Moreover, blocking activities (IC50 values) were quantitatively similar on mollusk glutamate receptors and vertebrate AMPARs. Analysis of the concentration dependence of block revealed two populations of iGlurs with 10–20-fold difference in sensitivity to channel blockers. However, even for the highly-sensitive population the estimated IC50 values are 2–5 times larger than the values measured on vertebrate AMPARs. This discrepancy might be due to the different experimental protocols used. Activities against AMPARs were measured as inhibition of stationary current evoked by kainate application on isolated neurons. Activities against C. vicina iGlurs were estimated from the decrease of EPSCs. In the latter case, non-stationary conditions can affect results because of the use-dependence of channel block. It is difficult to characterize the population of low-sensitive iGluRs in Calliphora neuro-muscular junction. For all compounds tested the IC50 value for block of low-sensitive receptors exceeded 100 μM. Therefore, we cannot say if low sensitivity is due to the difference in channel topography. It is possible that some additional factor decreases activity of cationic blockers. For instance, the Lys residue in M3 segment of DGluR2A subunit can play such a role. In agreement with this possibility, Drosophila iGlurs containing DGluR2A are less sensitive to argiotoxin than the DGluR2A-lacking receptors (DiAntonio et al., 1999). It is tempting to speculate that the high- and lowsensitive C. vicina iGlurs resemble Ca2+-permeable and Ca2+-impermeable AMPARs, since the latter are low-sensitive to cationic blockers too (Magazanik et al., 1997; Bolshakov et al., 2005). However, weak sensitivity of C. vicina iGlurs to pentobarbital evidences against this idea. Pentobarbital activity in our experiments was low and pentobarbital action did not reveal subpopulations of receptors. It is more likely that the aforementioned local differences in the binding site sequence are responsible for high or low sensitivity of C. vicina iGlurs to channel blockers. Properties of the recognition domain are much more complex and no direct analogy with vertebrate receptors can be drawn. Besides glutamate, C. vicina iGlurs can be activated only by domoate, quisqualate and kainate. Competitive antagonists of both AMPAR and NMDAR were inactive. It should be noted that non-NMDA invertebrate receptors demonstrate a diversity of pharmacological characteristics. Thus, quisqualate is active in many preparations (see e.g. Dudel et al., 1988; Dierkes et al., 1996; Brierley et al., 1997; Kimura et al., 2001; Muller et al., 2003; Holden-Dye and Walker, 2006; Scannell et al., 2008). Kainate produces the same effect as quisqualate and glutamate on leech neurons (Dierkes et al., 1996), Ascaridia galli and Caenorhabditis elegans (Holden-Dye and Walker, 2006) and in the pond snail, Helisoma trivolvis (Scannell et al., 2008) but is ineffective on the locust muscle (Dudel et al., 1988), ganglion cells of Aplysia (Kimura et al., 2001) and produces only a small effect on Lymnaea neurons (Brierley et al., 1997). DNQX (or CNQX) often block the invertebrate non-NMDA receptors (Dierkes et al., 1996; Kimura et al., 2001; Muller et al., 2003; Lima et al., 2003). Our results demonstrate closest similarity with data reported for motoneurons of the lobster cardiac ganglion (Hashemzadeh-Gargari and Freschi, 1992). In that work the rank order of the agonists potencies was quisqualate greater than glutamate, glutamate greater than kainate. Kainate, unlike the other agonists, showed no desensitization. CNQX and DNQX only partially blocked the responses
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even in concentration of hundreds μM, and concanavalin A was ineffective. However, it would be premature to speculate about structural similarities (differences) between recognition domain of C. vicina iGluRs and other non-NMDA receptors, because such conclusions require systematic study with use of homologous series of drugs, as we did in this work with the ion channel. CTZ, but not concanavalin A, significantly potentiated the EPSC in our experiments. Potentiation by cyclothiazide of recombinant veretebrate receptor responses in Xenopus oocytes showed selectivity for AMPA versus kainate receptors. In contrast, concanavalin A strongly potentiated responses at kainate but not AMPA receptors (Partin et al., 1993). It should be noted that the mechanism of action of CTZ on C. vicina iGlurs and AMPARs is not identical. If CTZ reduces desensitization, the EPSC should become longer. But it is not the case. Careful kinetic analysis of CTZ action on vertebrate AMPARs was performed by Partin et al. (1996). According to their results, CTZ induces dual effects: increase of agonist affinity and reduction of the onset rate of desensitization. We hypothesize that on C. vicina iGlurs CTZ only increases agonist affinity without affecting desensitization rate. In the present work we studied pharmacological characteristics of ionotropic glutamate receptors from C. vicina neuro-muscular junction and compared receptor properties with those of vertebrate receptors. The channel pore was well-characterized using a homologous series of channel blockers as molecular probes. We found close similarity between the ion channels of C. vicina iGlurs and vertebrate AMPARs. However, glutamate receptors in Calliphora neuro-muscular junction are not homogeneous, and according to their sensitivity to channel blockers they can be subdivided into high- and low-sensitive populations. Molecular origin of low sensitivity remains unclear. Properties of the recognition domain of C. vicina iGlurs make them markedly different from all known ionotropic glutamate receptors. In the present work we just outlined the problem; solution will require serious additional efforts. Further analysis is expected to reveal molecular determinants of drug action on the recognition domain. This will contribute to our understanding of functioning of the ionotropic glutamate receptors and of molecular evolution in these proteins. Acknowledgements This work is supported by RFBR grants 08-04-00326 and 04-00617, grant for Russian scientific schools 4821.2008.4 and by a grant from program “Molecular and Cellular Biology”, RAS. References Bahring, R., Mayer, M.L., 1998. An analysis of philanthotoxin block for recombinant rat GluR6(Q) glutamate receptor channels. J. Physiol. 509, 635–650. Bolshakov, K.V., Tikhonov, D.B., Gmiro, V.E., Magazanik, L.G., 2000. Different arrangement of hydrophobic and nucleophilic components of channel binding sites in Nmethyl-D-aspartate and AMPA receptors of rat brain is revealed by channel blockade. Neurosci. Lett. 291, 101–104. Bolshakov, K.V., Gmiro, V.E., Tikhonov, D.B., Magazanik, L.G., 2003. Determinants of trapping block of N-methyl-D-aspartate receptor channels. J. Neurochem. 87, 56–65. Bolshakov, K.V., Kim, K.H., Potapjeva, N.N., Gmiro, V.E., Tikhonov, D.B., Usherwood, P.N., Mellor, I.R., Magazanik, L.G., 2005. Design of antagonists for NMDA and AMPA receptors. Neuropharmacology 49, 144–155. Brierley, M.J., Yeoman, M.S., Benjamin, P.R., 1997. Glutamate is the transmitter for N2v retraction phase interneurons of the Lymnaea feeding system. J. Neurophysiol. 78, 3408–3414. Bruce, M., Bukownik, R., Eldefrawi, A.T., Eldefrawi, M.E., Goodnow Jr., R., Kallimopoulos, T., Konno, K., Nakanishi, K., Niwa, M., Usherwood, P.N., 1990. Structure–activity relationships of analogues of the wasp toxin philanthotoxin: non-competitive antagonists of quisqualate receptors. Toxicon 28, 1333–1346. DiAntonio, A., 2006. Glutamate receptors at the Drosophila neuromuscular junction. Int. Rev. Neurobiol 75, 165–179. DiAntonio, A., Petersen, S.A., Heckmann, M., Goodman, C.S., 1999. Glutamate receptor expression regulates quantal size and quantal content at the Drosophila neuromuscular junction. J. Neurosci. 19, 3023–3032. Dierkes, P.W., Hochstrate, P., Schlue, W.R., 1996. Distribution and functional properties of glutamate receptors in the leech central nervous system. J. Neurophysiol. 75, 2312–2321.
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