Block of GABAA Receptor Ion Channel by Penicillin: Electrophysiological and Modeling Insights Toward the Mechanism

Molecular and Cellular Neuroscience 63 (2014) 72–82

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Molecular and Cellular Neuroscience

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Block of GABAA receptor ion channel by penicillin: Electrophysiological and modeling insights toward the mechanism

Alexey V. Rossokhin ⁎, Irina N. Sharonova, Julia V. Bukanova, Sergey N. Kolbaev, Vladimir G. Skrebitsky

Research Center of Neurology, Russian Academy of Medical Sciences, 105064 Moscow, Russia article info abstract

Article history: GABAA receptors (GABAAR) mainly mediate fast inhibitory neurotransmission in the central nervous system. Dif- Received 5 June 2014 ferent classes of modulators target GABAAR properties. Penicillin G (PNG) belongs to the class of noncompetitive Revised 29 September 2014 antagonists blocking the open GABAAR and is a prototype of β-lactam antibiotics. In this study, we combined elec- Accepted 7 October 2014 trophysiological and modeling approaches to investigate the peculiarities of PNG blockade of GABA-activated Available online 8 October 2014 currents recorded from isolated rat Purkinje cells and to predict the PNG binding site. Whole-cell patch-сlamp

Keywords: recording and fast application system was used in the electrophysiological experiments. PNG block developed after channel activation and increased with membrane depolarization suggesting that the ligand binds within GABAA receptor Penicillin the open channel pore. PNG blocked stationary component of GABA-activated currents in a concentration- Isolated neurons dependent manner with IC50 value of 1.12 mM at −70 mV. The termination of GABA and PNG co-application Patch clamp was followed by a transient tail current. Protection of the tail current from bicuculline block and dependence Molecular modeling of its kinetic parameters on agonist afﬁnity suggest that PNG acts as a sequential open channel blocker that pre- Monte-Carlo energy minimization vents agonist dissociation while the channel remains blocked. We built the GABAAR models based on nAChR and GLIC structures and performed an unbiased systematic search of the PNG binding site. Monte-Carlo energy minimization was used to ﬁnd the lowest energy binding modes. We have shown that PNG binds close to the intracellular vestibule. In both models the maximum contribution to the energy of ligand–receptor interactions revealed residues located on the level of 2′,6′ and 9′ rings formed by a bundle of M2 transmembrane segments, indicating that these residues most likely participate in PNG binding. The predicted structural models support the described mechanism of PNG block. © 2014 Elsevier Inc. All rights reserved.

1. Introduction for receptor activation by GABA and the γ subunit deﬁnes sensitivity to benzodiazepines.

The γ-aminobutyric acid type A receptor (GABAAR) is a member of The GABAAR is a target for many pharmacological compounds of dif- the Cys-loop receptor family of pentameric ligand-gated ion channels, ferent classes including benzodiazepines, barbiturates, steroids, and which also comprises excitatory nicotinic acetylcholine receptors noncompetitive antagonists (Cascio, 2006; Sieghart, 2006). The last (nAChR) and 5-hydroxytryptamine receptors, as well as the inhibitory ones include β-lactam antibiotics, t-butylbicyclophosphorothionate glycine receptors (Betz, 1990; Connolly and Wafford, 2004). Cys-loop (TBPS), insecticides and picrotoxin (PTX). Despite large structural diver- receptors consist of an extracellular domain (ECD) including agonist sity, these noncompetitive antagonists are believed to bind in different binding pocket and a transmembrane domain (TMD) forming an ion positions within a common “convulsant” binding pocket in the chloride channel. The TMD of each subunit contains four transmembrane helixes channel lumen (Chen et al., 2006; Olsen, 2006). The amino acid compo- (M1–M4) and a large intracellular loop M3–M4. Five M2 segments form sition of the M2 helixes determines the ion selectivity and conductance the receptor pore. GABAARs are the major inhibitory receptors in the properties of the channel (Galzi et al., 1992; Keramidas et al., 2004). central nervous system (CNS), formed by combination of α1–6, β1–3, A classic GABAAR blocker is the convulsant PTX that inhibits γ1–3, ρ1–3, ε, π, δ or θ subunits with the predominant receptor being GABAergic transmission by channel blockade (Dillon et al., 1995; α1β2γ2 and with a subunit stoichiometry of 2:2:1 (Hevers and Newland and Cull-Candy, 1992). Accumulating evidences indicate that Luddens, 1998; Sieghart, 2006). Alpha and beta subunits are necessary a PTX binding site is located within the channel pore near the cytoplasmatic end (Buhr et al., 2001; Erkkila et al., 2008; Ffrench- Constant et al., 1993). Another well-known open channel blocker of GABA R is penicillin (Chow and Mathers, 1986; Twyman et al., 1992). ⁎ Corresponding author at: Research Center of Neurology RAMS, 105064 Moscow, A by-str. Obukha 5, Russia. While extensive efforts have been made to determine the molecular E-mail address: [email protected] (A.V. Rossokhin). mechanism of PTX inhibition of GABAAR channels, paradoxically little

http://dx.doi.org/10.1016/j.mcn.2014.10.001 1044-7431/© 2014 Elsevier Inc. All rights reserved. A.V. Rossokhin et al. / Molecular and Cellular Neuroscience 63 (2014) 72–82 73 work has been done on the mechanisms of penicillin interaction with

GABAA channel. Penicillin G (PNG) is a prototype of β-lactam antibiotics which are the antibacterial agents. However, administration of these drugs can cause toxic side effect on CNS. Epileptogenic properties of PNG were documented since the 1940s (Chow et al., 2005) and since then adverse effects of PNG were thoroughly investigated (Curtis et al., 1972). The widely accepted theory of the pathogenesis of convulsions induced by penicillin and related β-lactam compounds suggests that these drugs suppress GABAergic transmission in the CNS (Chow and Mathers, 1986). At millimolar concentrations, PNG causes a voltage-dependent Fig. 1. Sequence alignment of M2 segment of nACh, GAB A and GLIC receptors. Sequences open channel block of GABA R(Fujimoto et al., 1995; Pickles and A A taken from the Protein Data Bank with PDB codes: nAChR 2BG9 and GLIC 2XQ3 and from Simmonds, 1980; Twyman et al., 1992). These data suggest that PNG the UniProt database with ascension numbers GABAA_α1 P14867, GABAA_β2 P47870, enters the open pore of GABAAR and then occludes it. This theory is sup- GABAA_β1 P18505,andGABAA_γ2 P18507. Sequences aligned relative to the highly conserved Arg 0′ and Lys 0′ (GABA and nAChR) or Pro 23′ (GABA , nAChR and GLIC) ported by competition between PNG and PTX for suppression of GABAA A A receptors (Bali and Akabas, 2007). It has been demonstrated that PTX residues highlighted in bold. Last residue number is shown. Dashed and solid line rectangles underline the difference in M2 amino acid composition in the GABAA_nAChR and inhibits GABAAR by a noncompetitive, open channel block mechanism GABAA_GLIC models. (Newland and Cull-Candy, 1992; Olsen, 2006; Yoon et al., 1993). Single channel recordings have shown that PNG reduced mean life-time of the because this allows the blocker increased access to its binding site GABA-activated channels in the open state rather than changed the (Ascher et al., 1979). The influence of agonist concentration on the ex- channel conductance (Chow and Mathers, 1986; Twyman et al., 1992) tent of blockade was determined by measuring the inhibition (by fl indicating the drug in uence on the receptor gating. However, the 1 mM PNG) of currents evoked by increasing concentration of GABA exact mechanism of PNG interaction with GABAARandthesiteofPNG (Fig. 4A). The amplitudes of currents were measured at the end of binding in GABAAR pore still remain unknown. drug co-application. PNG inhibition was tested at GABA concentrations In this study, we combined electrophysiological and modeling ap- from 3 to 300 μM. The inhibition was GABA concentration-dependent, proaches to investigate the peculiarities of PNG blockade of GABA- being larger at higher concentrations of GABA (Fig. 4B). Penicillin activated currents in the isolated cerebellar Purkinje cells and to build inhibited the steady-state component of current induced by 3 μM fi the structural model of PNG binding in the pore of GABAAR. We con rm GABA to 75 ± 6.1% of control (n = 4), to 49 ± 2.7% for current induced fi that PNG is an open channel blocker and extend the previous ndings by 10 μM GABA (n = 4), and to 36 ± 6.2% (n = 4) at GABA concentra- “ ” by demonstrating that PNG acts as a sequential blocker , which pre- tions of 100 μM(Fig. 4C). The comparison of concentration–response vents the channel from closing while blocked. Such mechanism of curve for GABA in control and during co-application with 1 mM PNG “ ” block is also known as a foot-in-the door effect and was described pre- shows that the blocker both inhibited the maximal GABA current and viously for acetylcholine (Adams, 1976; Neher and Steinbach, 1978), shifted dose–response curve to the left (Fig. 4B). The concentration– NMDA (Benveniste and Mayer, 1995; Vorobjev and Sharonova, 1994) response curve for GABA experienced a leftward shift: from 15.5 μMin and GABAA receptors (Kolbaev et al., 2002). We have designed a struc- control conditions to 6.0 μMinthepresenceof1mMPNG(n=4). tural model of PNG binding in the GABAAR pore. The molecular model- The shift in GABA dose–response curves fits well to open channel – ing revealed that the maximum contribution to the energy of ligand blocking action of PNG. receptor interactions is provided by the residues located on the level of 2′,6′ and 9′ rings formed by a bundle of M2 transmembrane segments, indicating that these residues most likely participate in PNG 2.3. Penicillin block at different holding potentials binding. The proposed model indicates that the binding sites of PNG and PTX are overlapping. To determine the voltage dependence of PNG effect, PNG was co-applied with GABA at several membrane potentials. Fig. 5Aillus- μ 2. Results trates the effect of 1 mM PNG on the current elicited by 5 MGABAat −110, −90, −70, −50, −30, −10, +10, and +30 mV. The GABA cur- – 2.1. Inhibition of GABA-induced currents by penicillin rent voltage relationship was roughly linear in the absence of PNG, but showed significant inward rectification in the presence of PNG (Fig. 5B). All recorded Purkinje cells (n = 46) displayed responses to GABA Thus, the block of GABA-induced currents was greater at more positive which were modulated by PNG. Fig. 3A illustrates the inhibitory effects holding potentials. At 1 mM, PNG depressed stationary GABA current by − of penicillin on currents activated by 5 μM GABA. Penicillin (0.1– 49 ± 6% at 110 mV and by 87 ± 1% at +30 mV. 10 mM) applied together with GABA, at −70 mV suppressed the A voltage-dependent effect suggests that action of a drug is located responses to GABA, measured at steady state, in a concentration- within the channel pore of the receptor. Therefore, we analyzed the re- dependent manner. The effect of PNG developed quickly, and was easily sults according to a simple one-site blockade model (Woodhull, 1973). reversible. By itself, penicillin (up to 10 mM) had no action on resting The voltage dependence of the block is shown in Fig. 5C. The proportion currents (data not shown). Application of PNG in the absence of GABA of blocked current increased with membrane depolarization and did not change the peak response of subsequent GABA applications reached the maximum of ~90% block at +30 mV. Using Woodhull (not shown), suggesting that the compounds can only access the bind- model, we estimated the electrical depth of the PNG binding site in δ μ ing site in the open state. When GABA receptors were activated by 5 μM GABAAR = 0.39 ± 0.03 and respective KD(0) = 200 ± 20 M (see Experimental methods). GABA the IC50 value for PNG inhibition of GABA-induced currents was 1.1 ± 0.12 mM and Hill coefficient was 0.84 ± 0.10 (n =6)(Fig. 3B). 2.4. Penicillin block prevents the channel closure and agonist dissociation 2.2. Penicillin-induced inhibition is more potent at high vs low GABA concentrations The termination of GABA and PNG co-application was followed by transient increase in the inward current (“rebound” or “tail” current) The degree of block produced by a fixed concentration of an open (Fig. 3A). The onset of this current coincided with the initiation of per- channel blocker is expected to increase with agonist concentration fusion with normal extracellular solution. The amplitude and kinetics 74 A.V. Rossokhin et al. / Molecular and Cellular Neuroscience 63 (2014) 72–82

Fig. 2. PNG structure and the method of systematic unbiased search of the minimum energy ligand–receptor complexes. A. PNG structural formulae. B. PNG minimal energy conformation.

C. PNG pulling and rotation in the pore of GABAA receptor. Three of five subunits are shown for clarity (α1 — cyan, β2 — rose, γ2 — magenta). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) of this current were dependent on blocker concentration and holding antagonist, only during generation of this current. We have observed potential (Figs. 3A, 5A). The maximal relative amplitude of the tail cur- that application of bicuculline did not change neither the amplitude, rent measured from the level of the steady-state current was observed nor the time course of the current (Fig. 6A, right panel), while PNG when the degree of block was maximal — at PNG concentration of (5 mM) completely suppressed it (Fig. 6A, middle panel). Protection 10 mM and holding potential of +30 mV. The tail current was not ob- of tail current from bicuculline block is consistent with the hypotheses served when PNG was not applied (Fig. 6A, left panel) or was continu- that agonists remain bound in the open-blocked state. ously present in the solution (Fig. 6A, middle panel). These properties However, these experiments do not exclude the possibility that of tail current are consistent with the sequential model of open channel the decay of tail current reflects the closing kinetics of an open, block (Adams, 1976; Antonov and Johnson, 1996; Neher and Steinbach, but unliganded receptor, and therefore binding of competitive an- 1978). Sequential blockers prevent the channel from closing while tagonists has no effect on this process. According to the model of se- blocked. In this model channel closure takes place after dissociation of quential open channel block (Benveniste and Mayer, 1995), the time the blocker, which occurs via the open state. The appearance of tail cur- course of the tail current will depend on the dissociation rate con- rent is due to transition from open-blocked to the open state of the re- stant for both PNG and the agonists. Thus, a clear relationship be- ceptor (Benveniste and Mayer, 1995; Vorobjev and Sharonova, 1994). tween tail current decay kinetics and agonist affinity should be This model also implies that a sequential open channel blocker prevents observed, similar to the agonist dependence of NMDA receptor de- dissociation of the agonist while the channel is blocked. To test the hy- activation (Lester and Jahr, 1992) and NMDA receptor blockade by pothesis that GABA is trapped on the receptors during the tail current 9-aminoacridine (Benveniste and Mayer, 1995). We tested this hy- development, we applied bicuculline, a competitive GABAA receptor pothesis using three GABAAR agonists with different affinities for

Fig. 3. Block by PNG of GABA-induced currents in isolated cerebellar Purkinje cells. A. Changes in the amplitude of currents induced by 5 μM GABA during coapplication with increasing concentrations of PNG. A current trace from a whole-cell voltage clamp recording is shown. PNG concentrations are indicated on the right. Increasing concentrations of PNG led to a decrease of steady-state currents and to an increase of the amplitudes of tail currents. The onsets of traces are slightly shifted relative to each other for the best presentation of the tail currents. Co-application of GABA and PNG is indicated by a horizontal line above the current traces. The cell was held at −70 mV. B. Concentration–response analysis for PNG induced block of currents activated by 5 μM GABA. The data points are the mean steady-state currents normalized to the control current, and the error bars are standard deviations. The line is unweighted least-squares ﬁt of the data to a Hill equation (Eq. (2)). A.V. Rossokhin et al. / Molecular and Cellular Neuroscience 63 (2014) 72–82 75

2.5. Molecular modeling

In parallel with electrophysiological investigation we used a molecular modeling approach to predict the binding site of PNG in the

GABAAR pore. We performed a systematic unbiased search of the binding site pulling PNG through the pore and rotating this molecule around its long axis (see Experimental methods). In that way we found MEC of the ligand–receptor complex at each level of the pore. Fig. 7 presents the

energy proﬁle of PNG in the pore of GABAA_nAChR and GABAA_GLIC models. The minimums on the energy curve correspond to the probable

sites of PNG binding. In the case of the GABAA_nAChR model one can ex- tract two strongly pronounced minimums in the upper (9 Å) and lower (32 Å) parts of the pore. Only one minimum in the lower part of the pore

exists at the depth of 26 Å in the case of the GABAA_GLIC model. Below we will refer to them as upper and lower binding sites, respectively. Fig. 7 shows that the lower binding sites do not coincide in the

GABAA_nAChR and GABAA_GLIC models. In both models the energy of Van der Waals and electrostatic interactions changes in opposite directions at PNG moving toward the intracellular space, increases (in absolute values) in the ﬁrst case and decreases in the second one (data not shown). The growth of the energy of Van der Waals interactions correlates with a decrease of the pore lumen. When PNG is pulled deeper than the lower binding site the Van der Waals interactions attenuate and the total energy of the ligand–receptor interactions decreases (Fig. 7). The difference of the energy proﬁles in the upper part of the pore is mainly caused by electrostatic component of the energy of ligand– receptor interactions. At physiological pH PNG carboxyl group is nega-

tively charged. In the GABAA_nAChR model six positively charged lysine residues (γ2 20′ and α1, β2, γ2 24′) in the C-terminal part of M2 segments face the pore and strongly interact with PNG. In the GABAA_GLIC model ﬁve lysine residues 24′ are moved to the M2–M3 loop that atten- uates electrostatic interactions in the upper part of the pore.

The upper binding site is found only in the GABAA_nAChR model. At this site Van der Waals and electrostatic interactions approximately equally contribute to the ligand–receptor interaction energy (data not shown). The predicted structural model of PNG binding at the upper site is shown in Fig. 8 (A, B). PNG occludes the pore (Fig. 8A) and accepts

two hydrogen bonds from Lys289 (γ2 24′). Along with Lys289, residues Lys279 (24′ α1), Lys285 (20′, γ2), Ile282 (17′ γ2) and His267 (17′ β2) provide strong contribution to the ligand–receptor interaction energy in the upper binding site (Fig. 8A, B). In our models we did not consider the interaction of the ionized residues with counter-ions because the location of the last ones is unknown. Therefore it is reasonable to suggest that some of the positively Fig. 4. The degree of penicillin effects depends on GABA concentration. A. Representative charged lysine residues facing the pore can be screened by the opposite- current traces induced by GABA at various concentrations (3, 10 and 30 μM) in control ly charged ions. To estimate the influence of the screening effect on for- (thin line) and during coapplication with 1 mM PNG (thick line). The lines above each mation of the energy minimum corresponding to the upper binding site panel indicate the drug application periods. B. PNG modifies EC50 value of GABA. Concen- tration–response curves for GABA were obtained in control (squares) and in the presence we performed three additional series of numerical experiments. We of 1 mM PNG (triangles). Data points represent average from four cells. Data are normal- built three modifications of the GABAA_nAChR model with: (1) all ized with respect to the response to 300 μM GABA in the absence of PNG. The EC value 50 six lysine residues (20′ and 24′), (2) two α1 Lys279 and one γ2 for GABA was 15.5 ± 0.2 μM in the absence of PNG and 6.0 ± 0.21 μM in the presence Lys289 at 24′ level, and (3) two β2 Lys274 and one γ2 Lys289 at of 1 mM PNG (n = 4). C. Comparison of the percent of inhibition induced 1 mM PNG of ′ sustained GABA currents evoked by different GABA concentrations. Mean values of 24 level generated in the neutral form. We found that in the models GABA current recorded during co-application with 1 mM PNG normalized to that evoked with completely or partially neutralized charges on the lysine resi- by GABA alone (control); n = 4. dues the energy minimum corresponding to the upper binding site disappears (data not shown). Lower binding site was found in both models. Predicted structural

models of PNG binding in the lower site in the GABAA_nAChR and GABAA_GLIC models are shown in Figs. 8 (C, D) and 9, respectively. At this receptor — β-alanine, GABA and muscimol, for which it was this site Van der Waals interactions prevail than the electrostatic ones demonstrated that the time course of current decay (deactivation) in the ligand stabilization (−20.1 vs −5.7 kcal/mol in GABAA_nAChR depended on agonist afﬁnity (Jones et al., 1998). The time course of and −29.3 vs −0.4 kcal/mol in GABAA_GLIC). In both models PNG oc- decay of PNG-induced tail currents is much faster when β-alanine was ap- cludes the pore binding in the extended conformation. However, in plied with PNG (Fig. 6B, left panel) than when GABA was the agonist these models PNG carboxyl group is oppositely oriented in the pore

(Fig. 6B, middle panel) and much slower when muscimol was used as (Figs. 8D, 9B). In the GABAA_GLIC model PNG accepts H-bonds from β2 receptor agonist (Fig. 6B, right panel). Thr256 (6′)andγ2 Thr271 (6′)residues. 76 A.V. Rossokhin et al. / Molecular and Cellular Neuroscience 63 (2014) 72–82

Fig. 5. Voltage-dependence of the effect of penicillin on GABA currents. A. Representative current traces induced by 5 μM GABA at different membrane potentials in control (left) and during co-application with 1 mM PNG (right). The lines above panels indicate the drug application periods. B. The current–voltage relationship for the control (5 μM GABA) currents (squares) and the currents blocked by 1 mM PNG (triangles). Responses obtained at different membrane potentials are normalized to the response at −70 mV in the absence of PNG. C. Woodhull analysis of PNG block. Normalized block is plotted versus the holding potential. The data are ﬁtted using Eq. (3) after normalizing. The fraction of the membrane ﬁeld sensing by PNG δ is 0.39 ± 0.03. The respective KD(0) is 200 ± 20 μM.

In the GABAA_GLIC model the ligand phenyl ring deeply penetrates Simultaneous wash out of the agonist and blocker entails an appearance into α1–β2 intersubunit interface and strongly interacts with α1 of the tail currents. Val260 (5′), Val257 (2′), Thr256 (1′)andβ2 Ala252 (2′)residues It is generally accepted that PNG as well as PTX acts as an open chan- (Fig. 9). In the GABAA_nAChR model the PNG phenyl ring faces the nel blocker in GABAAR. Both drugs require agonist binding and channel α1–β2 interface and residues α1 Thr265 (10′), Leu264 (9′), Thr261 opening to access their binding site (Dillon et al., 1995; Newland and (6′) and β2 Thr256 (6′) form binding pocket for this moiety (Fig. 8D). Cull-Candy, 1992). Our results are in agreement with these data. How- In both models residues α1 Leu264 (9′), Thr261 (6′)andβ2 Thr256 ever, we found an important difference between PNG and PTX blocking (6′), Ala252 (2′) strongly contribute to the ligand–receptor interactions. effects. It was shown that channel closure and agonist dissociation are

Apart from the above, residues γ2 Ser267 (2′) in the GABAA_nAChR permitted while PTX is bound in the GABAA-receptor-mediated chan- model and γ2 Leu274 (9′), α1 Val257 (2′) in the GABAA_GLIC model nel, resulting in trapping of the drug in the channel (Bali and Akabas, make a signiﬁcant contribution to the ligand–receptor energy. 2007). This characteristic of the action of PTX, which is termed “trapping channel block”, distinguishes it from “sequential block,” which prevents 3. Discussion the channel from closing while blocked (Adams, 1976; Antonov and Johnson, 1996; Neher and Steinbach, 1978). Open channel block is a process by which the ligand binds to the site We report here that for PNG, the mechanism of block is clearly dif- inside of a channel pore and blocks the ﬂow of ions through that chan- ferent from PTX. Our results are in agreement with the observation nel. Pore blocker molecules constitute a helpful tool for understanding that PNG is an open channel blocker and extend these studies by show- the general architecture of the permeation pathway and the gating ing that PNG acts as a sequential open channel blocker which prevents properties of ion channels. Our goal in this study was to examine the agonist dissociation from the open-blocked state. The appearance of

PNG-mediated inhibition of the GABAAR and to correlate electrophysio- tail currents indicates that the blocker has to exit the channel before it logical data with the structural model of PNG binding predicted by mo- can be closed, as has been described for NMDA receptor blocker such lecular modeling approach. as tacrine, 9-aminoacridine, tetrapentylammonium and adamantane There are two main mechanisms by which blockers can interact with derivatives IEM-1857 (Antonov and Johnson, 1996; Benveniste and ion channel gating, namely, “trapping” and “foot-in-the door”. In the Mayer, 1995; Sobolevsky, 2000; Sobolevsky et al., 1999; Vorobjev and ﬁrst case, channel closure and agonist dissociation can occur while Sharonova, 1994). blocker is bound, trapping the blocker within the receptor's channel In the present experiments the tail currents were evoked following until subsequent agonist binding and channel opening permit blocker termination of co-application of GABA and PNG (Fig. 3A). These tail cur- dissociation. In the second case, bound blocker does not permit the ag- rents were resistant to block by competitive GABAA receptor antagonist onist dissociation and the channel transition to the closed conformation. bicuculline and their decay kinetics were dependent on agonist afﬁnity A.V. Rossokhin et al. / Molecular and Cellular Neuroscience 63 (2014) 72–82 77

Fig. 6. Properties of the tail current induced by co-application of GABA agonists and PNG. A. Effects of modulators of GABAA receptors on the tail current triggered after termination of GABA and PNG co-application. GABAA receptor competitive antagonist bicuculline does not influence the decay of the response neither in control (left panel), nor after co-application of GABA with PNG (right panel), while PNG itself completely blocks the tail current (middle panel). For drug applications two different pipettes were used. Control responses obtained in the absence of modulator during current deactivation (black curves) are superimposed with the responses obtained in the presence of PNG or bicuculline during washout (gray curves). The bars above panels indicate the drug application periods. All records were obtained from one neuron. B. Amplitude and kinetics of tail currents depend on agonist affinity. Representative traces induced by β-alanine (ALA) (left panel), GABA (middle panel) and muscimol (MUSC) (right panel) in control (thin lines) and during co-application with 1 mM PNG (thick lines). The time constants of the tail current deactivation (single exponential fitting) after agonist + PNG co-application were 13 ms for β-alanine, 54 ms for GABA and 142 ms for muscimol (indicated on the right). The horizontal lines above panels indicate the drug application periods. All records were obtained from one neuron.

(Fig. 6). These results are in accord with a sequential model for block of Thus, PNG blocks GABAARby“foot-in-the door” mechanism. In con- GABAA receptor by PNG, in which the agonist cannot dissociate from the trast, tail currents do not appear after termination of PTX block. Bali and open-blocked state of the receptor. Akabas (2007) have shown that PTX molecule is trapped in the closed

We have observed that at low GABA concentrations (1–3 μM) 1 mM GABAAR. Even though PTX also blocks the channel, it does not interfere PNG practically does not change the amplitude of steady-state compo- with the gating mechanism, suggesting that PTX molecule can reside in nent of GABA-induced current and only prolongs it (Fig. 4). This ﬁnding its blocking site even when the channel is closed and that the channel is consistent with the properties of the sequential open channel block ob- pore is large enough to accommodate this molecule. served with single channel recording. It was shown that during the block Along with electrophysiological studies we used a molecular model- of nicotinic acetylcholine receptors by local anesthetic QX-222 the burst ing approach to predict the PNG binding site in the GABAARpore.How- length increases, but the cumulative open time within a burst remains ever, the reliability of predictions, which can be made with the help of constant in the presence of an ion channel blocker (Neher, 1983). homology models, is highly sensitive to the choice of the templates. In

Fig. 7. Energy proﬁles of PNG in the pore of GABAA_nAChR and GABAA_GLIC models. 78 A.V. Rossokhin et al. / Molecular and Cellular Neuroscience 63 (2014) 72–82

Fig. 8. Predicted structural model of PNG binding in GABAA_nAChR model. View from extracellular space (A, C) and side view (B, D) on the upper (A, B) and lower (C, D) binding sites. In A–D parts of the M2 helixes are shown. PNG and side chains of residues which strongly contributed to the ligand–receptor energy are represented by sticks. In B and D frontal subunit is not shown for clarity. H-bonds are depicted by dotted lines. Subunit α1 is in cyan, β2 is in rose, and γ2 is in magenta. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

this study, we have built homology models of GABAAR using nAChR and One of the aims of our study was to check the possibility of usage of GLIC structures as templates. Electron microscopy structure of nAChR GLIC structure as a template for homology modeling of TMD of GABAAR. obtained with 4 Å resolution (Unwin, 2005) is used traditionally for We performed an unbiased systematic search of the PNG binding modeling of GABAAR. However, in this template conformation of TMD sites in the pore of the GABAA_nAChR and GABAA_GLIC models. The corresponds to the closed state of the channel (Miyazawa et al., 2003) upper binding site was found only in the GABAA_nAChR model and some additional procedures are necessary to model the conducting (Fig. 7). Negatively charged PNG is very attractive for positively charged conformation of GABAAR(O'Mara et al., 2005). Recently, X-ray structure residues lining the pore at the extracellular vestibule. However, residues of bacterial pentameric ligand-gated receptor GLIC became available composing M2 segment are different in the homology models based on (Bocquet et al., 2009). Apparent advantages of GLIC template are an nAChR and GLIC structures (Fig. 1). Six lysine residues facing the pore open conformation of the channel and its higher resolution (2.9 Å). exist in the 20′ and 24′ rings in the GABAA_nAChR model while only

Fig. 9. Predicted structural model of PNG binding in GABAA_GLIC model. View from extracellular space (A) and side view (B) on the lower binding site. In A and B parts of the M2 helixes are shown. PNG and side chains of residues which strongly contributed to the ligand–receptor energy are represented by sticks. In B frontal subunit is not shown for clarity. H-bonds are depicted by dotted lines. Subunit α1 is in cyan, β2 is in rose, and γ2 is in magenta. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) A.V. Rossokhin et al. / Molecular and Cellular Neuroscience 63 (2014) 72–82 79

one 20′ lysine remains in the GABAA_GLIC model. Residues belonging to subunit dramatically reduced the PNG blocking potency. Bali and the 21′–24′ rings move to the M2–M3 loop in the last model. This ex- Akabas (2007) established the competitive character of PNG and PTX in- plains the lack of the upper binding site in the GABAA_GLIC model. teractions at the GABAAR block. Our experimental data and structural We suggested that the upper binding site should not be detected even model allow us to suppose that PTX and PNG binding sites overlap but in the GABAA_nAChR model, since the charges at the ionizable residues not completely coincide. should be compensated by counter-ions which are vastly presented at Purkinje cells are thought to express a homogeneous population of the pore extracellular vestibule. We performed additional numerical GABAA receptors composed of α1-, β2/3-, and γ2-subunits (Laurie et al., experiments to conﬁrm this suggestion. Transformation of all or part of 1992; McKernan and Whiting, 1996). However, Kelley et al. (2013) the positively charged residues 20′ and 24′ in the unionized state resulted have observed that the β1 subunit in Purkinje cells is expressed at the in the disappearance of the energy minimum corresponding to the upper similar level as β2 one. Thus, it is quite probable that both subunit com- binding site. binations (α1β2γ2 and α1β1γ2) of GABAAR are presented in Purkinje Lower binding site was found in both models. These sites are located cells. Sequence alignment of β1 and β2 subunits has shown that M2 seg- at the different pore depths 26 and 32 Å (deﬁned according to z coordi- ments of these subunits are almost identical (Fig. 1). Only one amino nate of PNG root atom) in the GABAA_GLIC and GABAA_nAChR models, acid in 15′ position is different in M2 segment of these subunits, namely respectively (Fig. 7). Interestingly, nearly identical sets of residues at the Asn in the β2 subunit is replaced by Ser in β1 one. However, 15′ residue 2′–9′ levels contribute to the interaction with PNG at these sites. doesn't participate in PNG binding in both predicted binding modes.

The residues mainly involved in the interactions with PNG in the Therefore, modeling results obtained for GABAARofα1β2γ2 composi- GABAA_GLIC model are located on one helix turn higher than in the tion are also valid for α1β1γ2 one. GABAA_nAChR model according to the shift revealed by the structural In conclusion, in this work we performed electrophysiological and alignment of M2 segments of nAChR and GLIC (Fig. 1). According to modeling investigation of PNG blockade of GABAAR. We have shown the structural model of PNG binding in the GABAA_GLIC pore, the ligand experimentally that PNG acts as a sequential blocker, which requires a forms two H-bonds with γ2 and β2 6′ threonine residues (Fig. 9B). channel opening and after binding prevents a channel transition to the Previously it was shown (Chen et al., 2006) that some of the insecticides closed state. Thus, PNG block corresponds to a “foot-in-the door” mech- (α-endosulfan, lindane, ﬁpronil) and other noncompetitive antagonists anism. We used nAChR and GLIC structures as templates for modeling

(EBOB, TBPS, PTX and BIDN) for the GABAA receptor with widely diverse GABAAR and proposed two models of PNG binding in GABAARpore.In structure fit the 2′ to 9′ pore region forming hydrogen bonds with Thr 6′ both cases residues located on the level of 2′,6′ and 9′ rings revealed and hydrophobic interactions with Ala 2′,Thr6′ and Leu 9′,thereby the maximum contribution to the energy of ligand–receptor interac- blocking the channel. tions. The predicted structural models support the described mech- We found two PNG binding modes with similar energy of the ligand– anism of PNG block. The results also imply that GLIC can be used receptor interactions, −27.7 vs −25.8 kcal/mol for the GABAA_GLIC and for homology modeling of the pore region of GABAAR. This work GABAA_nAChR models, respectively (Figs. 8C, D and 9A,B).Inbothmodes canhelpinmappingofGABAAR for such medically important PNG binds in the extended conformation but orientation of the molecule drugs as β-lactam antibiotics. is opposite (Figs. 8D and 9B). In the GABAA_GLIC model PNG carboxyl group is oriented toward the extracellular space and located approxi- 4. Experimental methods mately at the depth of 23 Å that is 0.46 of the whole membrane depth (taking into account that the height of the TMD is about 50 Å). This 4.1. Preparation of Purkinje cells value rather closely matches the value of δ (0.39) obtained according to the Woodhull model from our experimental data. In this model δ defines All experiments were conducted in accordance with the require- the fraction of the membrane potential acting at the binding site assuming ments of the Ministry of Public Health of the Russian Federation and a uniform electric field distribution across the membrane. In contrast, in were consistent with EU directive for Use of Experimental Animals of the GABAA_nAChR model PNG charged moiety is oriented toward the the European Community. intracellular space. In the GABAA_nAChR model the lower binding site is Maximal efforts were made to minimize both the number and located deeper in the pore than in the GABAA_GLICmodelandtheprox- sufferings of used animal. Dissociated neurons were prepared from imity of the ring of positively charged Arg residues at the 0′ level can in- 14–18-day-old Wistar rats using the method described previously fluence the orientation of the carboxyl group. (Vorobjev et al., 1996). Briefly, sagittal slices of the cerebellum were in- It was established experimentally that PNG influences the receptor cubated at room temperature for 1–6 h on a mesh near the bottom of a gating reducing the mean life-time of the GABA-activated channels in beaker. The incubation solution had the following composition (in the open state (Chow and Mathers, 1986; Twyman et al., 1992). Our mM): NaCl 124, KCl 3, CaCl2 2.4, MgCl2 1.5, NaH2PO4 1.3, NaHCO3 26, model predicts that PNG protrudes its phenyl ring deep into the glucose 10, phenol red 0.01%, continuously bubbled with carbogene intersubunit interface (Fig. 9A), thus preventing transition of the recep- (5% CO2 +95%O2). One at a time, slices were transferred to the record- tor to the closed state. We can speculate that this mode of PNG molecule ing chamber and neurons were isolated by vertical vibration of a glass interaction with ion pore keeps the channel in the open-blocked state. It sphere, 0.7 mm in diameter, placed close to the surface of the slice was suggested that the channel gate in Cys-loop receptors is located at (Vorobjev, 1991). Manipulation and cell identification were performed the 9′–14′ levels (Miller and Smart, 2010). The participation of residues using an inverted microscope. Isolated Purkinje cells were distinguished at the 9′ level in the ligand stabilization at the binding site found in both from other cerebellar cells based on their large cell bodies (approxi- models should also complicate the channel transition to the closed state. mately 20 μm) and characteristic pear shape attributable to the stump Thus, our model explains the absence of “trapping” in experiments with of the apical dendrite. The solution for dissociation and recording had

PNG. the following composition: NaCl 150, KCl 5, CaCl2 2.7, MgCl2 2.0, PTX blocks GABAAR according to “trapping” mechanism (Dillon HEPES 10, pH adjusted to 7.4 with NaOH. et al., 1995; Newland and Cull-Candy, 1992). Therefore, PTX binding site must be below the channel activation gate (Bali and Akabas, 4.2. Whole-cell recording 2007). A large number of experimental studies indicate that residues of the 2′ (Buhr et al., 2001; Ffrench-Constant et al., 1993; Zhang et al., Voltage-clamp recording was obtained using the whole-cell conﬁgu- 1994)and6′ (Erkkila et al., 2008; Sedelnikova et al., 2006)levelsstrong- ration of the patch-clamp technique (Hamill et al., 1981). Glass recording ly contribute to PTX stabilization at the binding site. Sugimoto et al. patch pipettes were prepared from ﬁlament-containing borosilicate tubes

(2002) have shown that mutation of the residue of 6′ level in the β2 using a two-stage puller. The electrodes, having resistance of 2–2.5 MΩ, 80 A.V. Rossokhin et al. / Molecular and Cellular Neuroscience 63 (2014) 72–82

were filled with recording solution of the following composition (in mM): the model of GABAAR to its TMD. As a prototype for GABAAR building, CsCl 100, CsF 40, CaCl2 0.2, MgCl2 4, HEPES 10, EGTA 5, ATP-Na 4 (pH ad- we used the electron microscopy (EM) structure of nicotinic acetylcho- justed to 7.2 with CsOH). Recordings were carried out at room tempera- line receptor from Torpedo marmorata (nAChR) (Miyazawa et al., 2003) ture (20–23 °C) using an EPC 7 patch-clamp amplifier. Currents were and X-ray structure of prokaryotic pentameric ligand-gated receptor filtered at 3 kHz, sampled at 10 kHz, and stored on a computer disk. from Gloeobacter violaceus (GLIC) (Bocquet et al., 2009). In GLIC Cells were held at a membrane potential of −70 mV, and I–V there are no Cys residues forming SS-bond in the Cys-loop of ECD, relationships were generated with test potentials from −110 mV to but the overall architecture of GLIC is similar to that of Cys-loop re- +30 mV by 20 mV intervals. ceptors (Corringer et al., 2010). Furthermore GLIC was crystallized in a putatively open state and at a higher resolution of 2.9 Å against 4.3. Drug solutions and drug application 4 Å for nAChR (Bocquet et al., 2009). Thus, GLIC appears to be a suit-

able candidate to build the GABAAR model and for probing pore Amodiﬁcation of the fast perfusion system was used for solution ex- block events in detail. change (Vorobjev et al., 1996). Isolated Purkinje cells were ﬁrst patch Since TMD conformation of nAChR was obtained for the closed state clamped and then lifted into the application system, where they were of the channel (Miyazawa et al., 2003), for study the open channel block continuously perfused with control bath solution. The substances we used the GABAAR model proposed by O'Mara et al. (2005) in which were applied through а glass capillary ~ 0.2 mm in diameter located the pore was gently enlarged to conducting state using molecular dy- within 0.2 mm from the cell. Solution exchange time was measured at namics simulations. Further in the text we will refer to the models the open electrode tip by switching between solutions of different os- built on the base of nAChR and GLIC structures as the GABAA_nAChR molarities. The time constant of a rise of this current was 4.5 ± 0.1 ms and GABAA_GLIC models, respectively.

(n = 20). For activation of GABAAR, in most experiments GABA was Alignment of M2 segment of nACh, GABAA and GLIC receptors is applied for periods of 0.5–1s,at40–60 s intervals. Stock solutions of shown in Fig. 1. To facilitate the comparison of different subunits, a com- 10 mM GABA and 1 M PNG were prepared in water. Stock serial dilu- mon numbering system is used for M2 amino acid positions, starting tions provided the ﬁnal concentrations given below. All reagents were with 0′, a highly conserved positively charged Arg residue in anionic obtained from Sigma. channels or Lys residues in cationic channels located near the M2 intracellular end. In GLIC cationic amino acids are absent at the N-terminal 4.4. Data analysis part of M2, therefore, we aligned its M2 residues according to conserved

Pro 23′. This alignment predicts that Asn223 is located at 0′ position in Whole-cell records were analyzed off-line being exported as text GLIC (Fig. 1). Start of M2 helix in nAChR falls on residue 1′. Moving out- ﬁles to Prism (GraphPad Software, San Diego, CA) for further analy- wards through the pore, positions 2′,6′,9′,13′,17′,20′ and 24′ face the sis. Agonist concentration–response curve was ﬁt by the least-square pore, according to the EM structure of nAChR (Miyazawa et al., 2003). method to However, X-ray structure of GLIC reveals that the N-terminal residue of M2 helix falls on residue −3′ (Bocquet et al., 2009). In GLIC the residues = ¼ ðÞ½= nH= þ ðÞ½= = nH ; ð Þ − ′ ′ ′ ′ ′ ′ ′ ′ I Imax A EC50 1 A EC50 1 in positions 2 ,2,6,9,13,17 and 20 facethepore,whileresidue24 enters the M2–M3 loop. Thus, the amino acid composition of M2 segments in the GABA _nAChR and GABA _GLIC models differs. where I is the peak current evoked by agonist concentration [A], Imax is the A A peak current evoked by a maximal agonist concentration (300 μM GABA),

EC50 is the concentration giving half the maximal current, and nH is the Hill coefficient. 4.6. Model building To quantify the inhibitory effects of PNG upon the current induced by a constant GABA concentration the following equation was used: The starting backbone geometry of the GABAA_nAChR and GABAA_GLIC models was taken from nAChR and GLIC structures, re- = ¼ = þ ðÞ½= nH Þ; ð Þ spectively. The side chain torsions in those residues, which were identi- I Icont 1 1 PNG IC50 2 cal in a template and corresponding model, were assigned starting values as in the template. The all-trans conformations were used as where I is the GABA current at a given concentration of blocker, Icont is starting approximations for side chains of residues that mismatched be- the peak current evoked by GABA application without blocker, IC50 is tween GABA R and template. Monte-Carlo (MC) energy minimization the concentration of the ligand producing a half-maximal block of A method (Li and Scheraga, 1987) was applied to optimize the GABA R GABA-mediated responses, and nH is the Hill coefficient. A models and the ligand–receptor complexes. Energy was minimized in Voltage dependence of the blocking effect was estimated in the the space of generalized coordinates using ZMM program (http:// paradigm of “Woodhull model” (Woodhull, 1973) using following www.zmmsoft.com). Nonbonded interactions were calculated using relationship: AMBER force field (Weiner et al., 1984) with a cutoff distance of 9 Å. = ¼ =ð þ ðÞ½= ðÞ ðÞδ = ; ð Þ Electrostatic interactions were calculated with solvent exposure- and I Icont 1 1 PNG KD 0 exp z FVM RT 3 distance-dependent dielectric function (Garden and Zhorov, 2010) where I and Icont are the GABA currents at a given membrane potential without cutoff. MC-minimization (MCM) was performed in two stages. VM in the presence of blocker and control conditions respectively. At the first stage, the energy was MC-minimized with constraints. Cα KD(0) is the dissociation constant at VM = 0 mV, δ is the electrical atoms in the model were constrained to corresponding positions in depth of the binding site, z is the electrical charge of PNG (−1), F, R is the template with the help of pins. A pin is a flat-bottom penalty func- the Faraday and universal gas constant respectively and T is the ambient tion (Brooks et al., 1985) that allows penalty-free deviations of the re- temperature (F / RT = 0.04 mV−1). The curve fitwasperformedusing spective atom up to 1 Å from the template and imposes an energy the program Prism (GraphPad Software). Data values are presented as penalty for larger deviations. After the constrained MCM trajectory con- mean ± S.E.M. verged, all constraints were removed and the model was refined by the unconstrained MCM procedure. The difference between structures 4.5. Homology modeling found in the constrained and unconstrained trajectories indicated whether the constrained search yielded a stable structure. MCM trajec-

In this study, we built the model of open GABAARwithα1β2γ2 sub- tory was terminated when 10,000 consecutive energy minimizations unit combination. Since PNG binds within the pore region we restricted did not improve the energy of the apparent global minimum. A.V. Rossokhin et al. / Molecular and Cellular Neuroscience 63 (2014) 72–82 81

4.7. Ligand docking Buhr, A., Wagner, C.A., Fuchs, K., Sieghart, W., Sigel, E., 2001. Two novel residues in M2 of the g-aminobutyric acid type A receptor affecting gating by GABA and picrotoxin affinity. J. Biol. Chem. 276, 7775–7781. ZMM program was used to build the model of the PNG molecule. Cascio, M., 2006. Modulating inhibitory ligand-gated ion channels. AAPS J. 8, E353–E361. Geometry of this compound was optimized using an HGRID (Hot Chen, L., Durkin, K.A., Casida, J.E., 2006. Structural model for gamma-aminobutyric acid receptor noncompetitive antagonist binding: widely diverse structures fit the same GRID) procedure that submits a large number of MCM trajectories site. Proc. Natl. Acad. Sci. U. S. A. 103, 5185–5190. from randomly generated starting points and collects low-energy struc- Chow, P., Mathers, D., 1986. Convulsant doses of penicillin shorten the lifetime of GABA- tures found in each trajectory. Bond angles at all heavy atoms of the li- induced channels in cultured central neurones. Br. J. Pharmacol. 88, 541–547. gand were allowed to vary during energy minimization. The atomic Chow, K.M., Hui, A.C., Szeto, C.C., 2005. Neurotoxicity induced by beta-lactam antibiotics: from bench to bedside. European J. Clin. Microbiol. Infect. Dis.Off. Public. Eur. Soc. Clin. charges of the ligand were calculated by the AM1 method (Dewar Microbiol. 24, 649–653. et al., 1985) using MOPAC program (http://openmopac.net). Connolly, C.N., Wafford, K.A., 2004. The Cys-loop superfamily of ligand-gated ion channels: – Since PNG is an open channel blocker we performed the docking of theimpactofreceptorstructureonfunction.Biochem.Soc.Trans.32,529 534. fi Corringer, P.J., Baaden, M., Bocquet, N., Delarue, M., Dufresne, V., Nury, H., Prevost, M., Van the ligand within the pore region of GABAAR. MCM protocol allows nd- Renterghem, C., 2010. 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Enhancement by GABA of the as- sociation rate of picrotoxin and tert-butylbicyclophosphorothionate to the rat cloned ing site we performed an unbiased systematic search of the lowest α1β 2γ2 GABAA receptor subtype. Br. J. Pharmacol. 115, 539–545. energy binding modes. We pulled PNG along the pore axis, which coin- Erkkila, B.E., Sedelnikova, A.V., Weiss, D.S., 2008. Stoichiometric pore mutations of the cides with the Z axis of the Cartesian coordinate system, with a step of GABAAR reveal a pattern of hydrogen bonding with picrotoxin. Biophys. J. 94, 4299–4306. 1 Å. A position of the drug along the pore was determined by z coordi- Ffrench-Constant, R.H., Rocheleau, T.A., Steichen, J.C., Chalmers, A.E., 1993. Apoint nate of its root atom — nitrogen in the β-lactam group (Fig. 2A). Z axis mutation in a Drosophila GABA receptor confers insecticide resistance. Nature 363, zero corresponds to the pore entrance from the extracellular space. 449–451. 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Defining affinity with the In this way the grid of starting points covering all inner surfaces of GABAA receptor. J. Neurosci. Off. J. Soc. Neurosci. 18, 8590–8604. Kelley, M.H., Ortiz, J., Shimizu, K., Grewal, H., Quillinan, N., Herson, P.S., 2013. Alterations the receptor pore was formed. In each point of the grid the ligand– in Purkinje cell GABAA receptor pharmacology following oxygen and glucose depri- receptor complex was MC-minimized and the energy of the ligand– vation and cerebral ischemia reveal novel contribution of beta1-subunit-containing receptor interaction was calculated. The ligand–receptor energy receptors. Eur. J. Neurosci. 37, 555–563. fi extracted from the energetically best structure found at each level was Keramidas, A., Moorhouse, A.J., Scho eld, P.R., Barry, P.H., 2004. Ligand-gated ion channels: mechanisms underlying ion selectivity. Prog. Biophys. Mol. 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