The experiments were carried out on cultured Chinese hamster ovary (CHO) cells obtained from the American Type Tissue Culture Collection (ATCC, Molsheim, France) that were maintained in culture conditions as previously described (Mukhtarov et al., 2013; Maleeva et al., 2015).

For electrophysiological analysis cells were transfected with cDNAs of different subunits of glycine receptor (α1ΔIns, α2B, α3L, α1-G254A, and β). One day before transfection, cells were plated on the cover slips (12 mm in diameter) and placed inside 35-mm cell culture dishes with 2 ml of medium. Transfection was performed using the Lipofectamine 3000 protocol (Life Technology, USA). To facilitate identification of transfected cells a green fluorescent protein (GFP) was added to the transfection mixture. For expression of functional heteromeric receptors, cells were simultaneously transfected with cDNAs of α and β subunits in the ratio 1:10. Three hours after the initial exposure of cells to the cDNAs the culture medium was replaced with that containing strychnine (1 μM), which prevents spontaneous activation of GlyRs. Electrophysiological recordings were performed in the fluorescent cells 24–72 h after transfection.

Whole-cell and outside-out recordings were performed at room temperature (20–25°C) using an EPC-9 amplifier (HEKA Elektronik, Germany). Cells were continuously superfused with external solution containing (mM): NaCl 140, CaCl₂ 2, KCl 2.8, MgCl₂ 4, HEPES 20, glucose 10; pH 7.4; 320–330 mOsm. Intracellular solution used for filling recording patch pipettes contained (mM): CsCl 140, CaCl₂ 6, MgCl₂ 2, MgATP 2, NaGTP 0.4, HEPES/CsOH 10, BAPTA (tetrapotassium salt) 2; pH 7.3; 290 mOsm. Two different protocols of solutions application were used in this study. A “long protocol” was designed to obtain recordings of current induced by subsequent application of glycine (2 s)/glycine+NFA (10 s)/glycine (5 s) on the same trace; holding potential (V_hold) was fixed at −30 or +30 mV. A “ramp protocol” implies the gradual change of V_hold alternately from −80 to +80 mV and from +80 to −80 mV during 1 s, V_hold was fixed at −80 or +80 mV at the beginning and at the end of the ramp for 500 ms (Figure 2B).

Nonsaturating and subsaturating concentrations of glycine for different subunit combinations of GlyRs were chosen according to the EC₅₀ curves obtained in previous studies. It was shown that for homomeric α1 GlyRs EC₅₀ varies between 25 and 40 μM (Zhang et al., 2008; Lynagh et al., 2011; Maleeva et al., 2015), thus nonsaturating concentration of the agonist used for α1GlyRs was 30 μM, subsaturating – 100 μM. For α2 GlyRs mean value of EC₅₀ for glycine was estimated as 42 ± 2 μM (Maleeva et al., 2015), i.e., close to that for α1 GlyRs, so subsaturating concentrations of glycine were the same as for α1 GlyRs. The sensitivity of α3 GlyRs to glycine is significantly lower than of α1 and α2 GlyRs, comprising 80–150 μM (Zhang et al., 2008; Maleeva et al., 2015; Sánchez et al., 2015); according to this we used 100 μM of glycine as nonsaturating concentration of the agonist. Sensitivity of α1β GlyRs is close to that of homomeric α1 GlyRs, i.e., between 25 and 50 μM (Rundstrom et al., 1994; Shan et al., 2001; Islam and Lynch, 2012); EC₅₀ of α2β GlyRs varies between 50 and 85 μM (Pribilla et al., 1992; Miller et al., 2005; Zhang et al., 2008). It was demonstrated previously (Shan et al., 2001; Islam and Lynch, 2012) and confirmed by our experiments that mutation G254A does not change sensitivity of α1 GlyRs to glycine.

Recording pipettes were pulled from borosilicate glass capillaries (Harvard Apparatus Ltd, USA) and had resistances of 5–10 MOhm. For the rapid replacement of the solutions, the fast application system was used. Three parallel rectangular tubes (100 × 100 μm) were positioned 40–50 μm above the recorded cell. The movement of the tubes was controlled by a computer-driven fast exchange system (SF 77A Perfusion Fast-Step, Warner, USA) allowing a 10–90% solution exchange in 3–5 ms, as measured by open electrode controls (1/10 external solution/water). Cells with low input resistance (<150 MOhm) and a rapid run-down (>30% with repetitive application) were excluded from analysis.

All the drugs were obtained from Tocris or Sigma–Aldrich (France). NFA (100 mM) and picrotoxin (50 mM) were first dissolved in DMSO and then diluted with the extracellular solution to the final concentrations. In the test experiments, DMSO itself had no effect on the glycine-induced current (data not shown; see also Mascia et al., 1996; Hall et al., 2004). A stock solution of glycine (1M) was prepared using MilliQ water.

Electrophysiological recordings were performed using PatchMaster (HEKA Electronic, Germany) software. To plot concentration-response curves, responses to different concentrations of glycine and NFA were fitted using a nonlinear fitting routine of the Origin 7.5 software (OriginLabs, USA) with the Hill equation: For glycine:I=Imax/(1+(EC50/[A])nH)For NFA:I=Imax/(1+([inh]/IC50)nH) where I is the normalized current amplitude induced by the agonist at concentration [A], [inh] – concentration of NFA, Imax is a maximal current induced at given cell, n_H is the Hill coefficient and EC₅₀ or IC₅₀ are the concentrations at which a half-maximum response was induced.

The fractional electrical distance from the external side of the membrane (δ) at which bound NFA blocked the current was calculated using the Woodhull equation: IC50(V)=IC50(0) exp (−δFV/RT), where IC₅₀(V) is IC₅₀ at the given membrane potential, IC₅₀(0) – at 0 mV, V – membrane potential and F, R, T have their usual meanings. δ was determined from a plot of IC₅₀ values against membrane potential.

For statistical analysis paired and unpaired t-tests were used. Data are represented as means ± SEM.

The NFA ability to modulate currents in different GlyR subtypes was determined using a whole-cell configuration of patch-clamp technique. GlyRs composed of different subunits were transiently expressed in the CHO cell line. First, we have examined the effect of varying concentrations of NFA on homomeric α1 GlyR at constant holding membrane potentials (V_hold) of +30 and −30 mV using a “long” protocol of solutions application. Ionic currents were evoked by 30 μM of glycine alone (near EC₅₀ concentration for α1 and α2 GlyRs, Maleeva et al., 2015) or mixed with different NFA concentrations. The inhibition was more pronounced at positive membrane potentials (Figure 2A). Thus, at V_hold of −30 mV, concentrations of NFA 30, 100 and 300 μM caused inhibition of α1GlyR-mediated currents by 3 ± 4, 16 ± 6, and 48 ± 9%, respectively (n = 7), while at V_hold of +30 mV currents were inhibited by 16 ± 7, 43 ± 11, and 75 ± 4%, respectively (n = 7).

To examine in detail the voltage dependence of GlyR block by NFA we have used a “ramp” protocol that allowed fast changes of the membrane potential from −80 to +80 mV (Figure 2B). Representative current-voltage dependence curves recorded during application of glycine alone or mixed with different concentrations of NFA are shown in Figure 2C. Glycine concentration of 30 μM (near EC₅₀) produced an outwardly rectifying current due to the higher probability of the open-state α1 GlyR at positive potentials (Fucile et al., 1999). While NFA exhibited rather low affinity to α1 GlyR, especially at negative potentials, we have revealed significant (p < 0.01) voltage dependence of inhibition. Figure 2E shows that at −80 mV IC₅₀ of NFA was 315 ± 30 μM, while at +80 mV it was 197 ± 18 μM (n = 10). The voltage dependence of inhibition suggests that NFA acts as an open channel blocker of α1 GlyRs.

To test this hypothesis we performed the same experiment with higher, near-saturating concentration of glycine (100 μM) that causes longer mean open time of GlyR channels. The potency of the channel block by NFA increased, particularly at positive potentials (p < 0.001). IC₅₀ of NFA at −80 mV was 270 ± 26 μM, while at +80 mV about 3-fold decrease was observed: IC₅₀ = 90±8 μM (n = 10) (Figures 2D,E). These results provide additional support to the suggestion that NFA inhibits α1 GlyR currents as an open channel blocker.

Analysis of NFA action on α2 GlyR revealed two important peculiarities. Firstly, its inhibitory activity was much higher in comparison with that on α1 GlyR. Secondly, the voltage-dependence of inhibition was much more profound. Using a “long application” protocol we have demonstrated that NFA concentration as small as 10 μM inhibited currents by ~50% at MP of +30 mV (Figure 3A). The voltage dependence of α2 GlyR inhibition by NFA (10 μM) was also prominent: at MP = −80 mV glycine-evoked current comprised 89 ± 4% from the control, while at +80 mV it was as small as 43 ± 5% (n = 5) (Figure 3B).

To study in detail the voltage dependence of α2 GlyR block by NFA, we have used the same “ramp” protocol as we did for α1 GlyR. Unlike in α1 GlyRs, activation of α2 GlyRs by nonsaturating agonist concentration (30 μM) produced inwardly rectifying currents, suggesting that the open probability of α2 GlyR channel is higher at negative potentials. Another peculiarity of NFA action on α2 GlyRs was the strong voltage dependence of inhibition (Figure 3C). Thus, at −80 mV IC₅₀ of NFA was 166 ± 28 μM, while at +80 mV it decreased nearly 20 times, to 9 ± 2 μM (n = 8). Contrary to α1 GlyRs, the potency of NFA block did not increase further with elevation of glycine concentration (Figure 3D; p > 0.05). Currents induced by 100 μM of glycine were inhibited by NFA with IC₅₀ of 133 ± 20 μM at −80 mV and 9 ± 2 μM at +80 mV (n = 7).

The strong voltage dependence of the block suggests that NFA is an open channel blocker interacting with residues located deeply in the pore.

This suggestion was confirmed by results of single channel recordings performed in outside-out patches from cells expressing α2 GlyRs in the presence of glycine or mixture of glycine with NFA (Supplementary Figure 1). In control conditions, application of 5 μM glycine at V_hold −30 mV induced single channel openings (mean amplitude 2,8 pA) with rare short closings (Supplementary Figure 1A). Addition of 30 μM NFA transferred rectangular single channel pulses into bursts with high frequency interburst flickering (Supplementary Figure 1B). The effect became much more profound at elevation of the NFA concentration to 100 μM (Supplementary Figure 1C). This phenomenology is similar to earlier described phenomenology of channel blockers, for instance, action of local anesthetics on single acetylcholine-receptor channels (Neher and Steinbach, 1978) or flickering mode of NMDA receptors channel block by Mg⁺⁺ (Nowak et al., 1984) or amantadine (Blanpied et al., 2005).

Since NFA has shown distinct profiles of interaction with α1 and α2 GlyRs, we decided to explore its action on α3 GlyR with the aim to determine the critical amino acids for the blocker action. Notably, the TM2 helix in α3 GlyR is the same as in α2 GlyR. Both possess alanine in position 2′ (Figure 9A) implying that NFA may block α3 GlyR as strongly as α2 GlyR and stronger than α1 GlyR, which has glycine at position 2′.

Indeed, during “long application” protocol, α3 GlyR-mediated currents induced by 100 μM of glycine (near EC₅₀ concentration for α3 GlyRs, Maleeva et al., 2015) were blocked by NFA with the higher potency than currents mediated by α1 GlyRs (Figure 4A). 30 μM of NFA inhibited α3 GlyR-mediated currents by 32 ± 4% at −30 mV and by 62 ± 5% at +30 mV, while 300 μM NFA at −30 mV inhibited ionic currents by 86 ± 5% and by 90 ± 2% at +30 mV (n = 7). It is well documented that α3 GlyRs rapidly desensitize (Nikolic et al., 1998) and this complicates accurate estimation of the NFA blocking potency. Thus, over all the cells percentage of inhibition was measured as the ratio of the current amplitudes at the maximal inhibition (I₁) and after washout of NFA, when the glycine-induced current reaches the quasi-stationary level (I₂, Figure 4A).

Interestingly, the current/voltage dependence recorded during application of 100 μM of glycine demonstrated the outward rectification, similar to that observed for α1 GlyRs. Like for both α1 and α2 GlyRs, the efficiency of α3 GlyRs block by NFA was higher at positive potentials (Figures 4B,C). At MP of −80 mV and +80 mV, IC₅₀ of NFA was 86 ± 14 and 16 ± 6 μM, respectively (n = 7).

Thus, the sensitivity of α3 GlyRs to NFA is higher than that of α1 GlyRs and is rather close to that of α2 GlyRs.

Taking into account the voltage dependence and presumably pore-blocking mechanism of NFA action, we suggested that amino acids, which are crucial for the interaction of NFA with GlyRs, are located in the pore-lining helices. The TM2 helices are highly conserved between different α subunits of GlyRs and differ only at position 2′ where α1 subunit has glycine, while α2 and α3 subunits have alanine (Figure 9A). We suggested that this difference is the main determinant of the distinct profiles of inhibition of different GlyRs by NFA. To test this hypothesis, we performed a point mutation at position 254 of the α1 subunit exchanging glycine for alanine (Figure 5A). We expected to convert low NFA sensitivity of the α1 GlyR to high sensitivity, characteristic for α2 and α3 GlyRs.

The dose/response relationships showed that the G254A-α1 GlyR mutant sensitivity to glycine is similar to that of the wild-type α1 GlyR: the EC₅₀ was 34 ± 6 μM (n = 6, data not shown), which agrees with previous data (Shan et al., 2001). At the “long application” protocol, NFA relatively weakly inhibited G254A-α1 GlyR mediated currents induced by 30 μM glycine, but its potency was higher than that in the wild-type α1 GlyRs (Figure 5A). At V_hold of −30 mV, 30, 100, and 300 μM of NFA inhibited G254A-α1 GlyR mediated currents by 22 ± 4, 34 ± 6, and 52 ± 11%, respectively (n = 6), while at V_hold of +30 mV, the same NFA concentrations inhibited currents by 23 ± 7, 43 ± 10, and 76 ± 4%, respectively (n = 7).

Using the “ramp” protocol, we found the above effect to be more pronounced at higher potentials (Figure 5B). Notably, at +80 mV currents induced by 30 μM of glycine were inhibited by NFA with IC₅₀ of 64 ± 10 μM (n = 13; Figure 5D), which was significantly lower (p < 0.001) than for the wild-type α1 GlyR (192 ± 23 μM, n = 8). However, at negative potentials sensitivity of the G254A-α1 GlyR was close to that of wild type α1 GlyR and comprised 257 ± 25 μM (n = 13). With the increase of agonist concentration, the strength of the block did not increase significantly and comprised 58 ± 8 μM (n = 8) at MP = +80 mV.

Importantly, the NFA blocking potency for G254A-α1 GlyR was lower than that for α2 and α3 GlyRs. This suggests that amino acids beyond the TM2 helices affect the NFA action, likely by an allosteric mechanism.

To estimate the fractional depth of the NFA binding sites in the pore with respect to the external membrane surface, we used classical Woodhull analysis developed to describe the hydrogen block of sodium channels (Woodhull, 1973). We have plotted NFA IC₅₀ values against membrane potential for α1, α2, and G254A-α1 GlyRs at glycine concentrations of 30 and 100 μM (Figure 6A). The calculated values of δ correspond to the percentage of the transmembrane electric field that is sensed by the bound NFA (Figure 6B). For the NFA block of α1 GlyR, mean δ values at 30 and 100 μM of glycine were 0.16 ± 0.03 (n = 7) and 0.26 ± 0.02 (n = 10), respectively. The NFA binding site in α2 GlyR was found to be significantly deeper. With 30 and 100 μM of glycine, the sensed electric field (δ) increased to 0.65 ± 0.07 and 0.63 ± 0.07, respectively (n = 7, p < 0.001). The NFA binding site in the G254A-α1 GlyR was situated between the sites in the α1 and α2 GlyRs, at the level of ~ 30% from the extracellular side at both examined agonist concentrations (Table 1).

To estimate cooperativity of NFA inhibition of GlyRs, we determined the Hill coefficient (n_H) for α1, α2, and G254A-α1 GlyRs. The amplitude of glycine-evoked currents at different potentials was plotted against the NFA concentration and fitted with the Hill equation. At positive membrane potentials, n_H decreased with the membrane depolarization for the three GlyRs (Table 2 and Figure 6C). For α1 and α2 GlyRs n_H was significantly different at all the tested membrane potentials (−80 mV, −70 mV p < 0.001; −50 – +50 mV p < 0.01; 70, 80 mV p < 0.05), and for the G254A-α1 GlyR n_H values were between those for α1 and α2 GlyRs. Concentration dependencies of NFA action on different GlyR subunits are presented in the Supplementary Figure 2.

These data suggest that the cooperativity of NFA block strongly depends on the GlyR subtype.

In the adult CNS of vertebrates, the predominant GlyR subtype is heteromeric α1β (Lynch, 2004). Several inhibitors, e.g., picrotoxin (Pribilla et al., 1992; Wang et al., 2006) and ginkgolides (Kondratskaya et al., 2005) have different affinity in heteromeric and homomeric GlyRs. To estimate the NFA sensitivity to heteromeric GlyRs we studied its interaction with α1β and α2β GlyRs. The concentration of the agonist was chosen according to the previous studies, demonstrating that 30 μM of glycine is below EC₅₀ for α1β and α2β glycine receptors (Rundstrom et al., 1994; Miller et al., 2005).

To prove formation of functional heteromeric GlyRs, we implemented a widely used picrotoxin (PTX) test: PTX blocks αXβ GlyRs much weaker than homomeric αX GlyRs (Pribilla et al., 1992; Shan et al., 2001). In our preparations, under application of 20 μM of PTX amplitudes of α1 and α2 GlyR -mediated currents were, respectively, 27 ± 3% (n = 12) and 3 ± 1% (n = 9) of the control. Heteromeric GlyRs were inhibited much weaker: the current in α1β GlyR was 75 ± 2% (n = 15) and in α2β GlyR it was 41 ± 4% (n = 7) from the control (Figures 7A–C).

Estimation of NFA IC₅₀ at different membrane potentials has shown that incorporation of the β subunit does not change significantly (p > 0.05) the sensitivity of α1 GlyR to NFA: at +80 mV IC₅₀ of NFA was 150 ± 14 μM (n = 5) (Figure 8A). Analysis of I/V curves also revealed a similarity to α1 GlyRs: currents mediated by α1β GlyRs (30 μM of glycine) were outwardly rectifying (data not shown).

Surprisingly, α2β GlyRs demonstrated the outwardly rectifying currents, contrary to inwardly rectifying α2 GlyRs (Figures 8B,C). In comparison to α2 GlyRs NFA stronger inhibited heteromeric α2β receptors at negative potentials being less effective at positive ones. This resulted in slightly weaker voltage dependence of the block vs. homomeric GlyRs: at −80 and +80 mV, IC₅₀ was 107 ± 37 and 20 ± 8 μM, respectively (n = 5, Figure 8D). In general, the β subunit does not have a strong impact on the interaction of NFA with GlyRs.

A systematic search for possible binding sites of NFA in different regions of the large GlyR proteins was beyond goals of this study. Here we focused on the pore region where NFA is likely to bind. Several arguments support this proposition. First, the voltage-dependence of the NFA action (Figures 2–6) strongly suggests that the compound binds within the membrane. Second, the anionic NFA molecules would enjoy favorable electrostatic interactions within the anion-selective pore. Third, results of single channels experiments demonstrated dramatical increase of the open state fluctuations caused by NFA application. Fourth, upon the channel activation, the α2 GlyR appears to open for a longer time than α1 GlyR (Takahashi et al., 1992; Morales et al., 1994). The longer openings would give NFA molecules more chances to reach their binding site(s) within the pore. This may explain why α2 GlyR is more sensitive to the NFA block than α1 GlyR.

Figure 9B shows computed energy (kcal/mol) of NFA interaction with α1 and α2 GlyR models and Figure 10A shows superposition of 74 snapshots with NFA at different levels of the α2 GlyR pore. The α1 and α2 GlyR models differ only at level 2′ where they have, respectively, the rings of Gly254 and Ala254 residues, but the computed energy values are rather different at each level (Figure 9B). The cause is the NFA molecule size (~13 Å between most remote atoms, see Figure 1B), which is bigger than, e.g., the distance of 5.8 Å between C^β atoms of the same TM2 helix at levels 2′ and 6′. Since the preferable orientation of NFA in the pore is unknown, the NFA molecule was allowed to rotate during MC-minimizations at each step. As a result, once the energetically favorable orientation was found at some level, the probability to find the energetically better orientation at the same level become small. A bottom line from a large number of independent runs with different seed random numbers might yield smoother trajectories, but such computations, which would require large computational resources, were beyond goals of our study.

The NFA interaction energy with the α1 and α2 GlyR models differs not only around level 2′, but also at the levels where the pore-facing residues are identical. At most of the levels, the energy difference between the two models does not exceed 2 kcal/mol, but it reaches 5 kcal/mol at level 6′ (Thr258). Despite the differences, the two trajectories have several common features as described below. First, there are rather deep minima at the cytoplasmic side of the pore, which are due to electrostatic interactions between NFA and Arg252 (Figure 10B). Second, there are energy minima at level 6′, which are due to H-bonds between NFA and Thr258 residues and hydrophobic interactions of NFA with the intracellularly-oriented side of the Leu261 ring. Third, there are energy minima at level 13′, which are due to H-bonds between NFA and Thr265 and favorable hydrophobic interactions of NFA with the extracellularly-oriented side of the Leu261 ring. Fourth, there are energy maxima at level 9′, which are due to certain repulsion between NFA and Leu261 residues that form the open pore constriction. However, even at this level the NFA-channel energy remains favorable (around −10 kcal/mol), partially because the ligand carboxylic group can accept up to four H-bonds from Thr258, Thr264, and Thr265 (Figure 10C). Fifth, relatively large energies at the extracellular part of the pore (levels 17′ to 20′) are due to two reasons. (i) The pore at these levels is too wide and NFA cannot simultaneously interact with three helices as it does at lower levels. (ii) Levels 13′ and 17′ are both hydrophilic (Figure 9A) and predominantly hydrophobic NFA cannot form multiple favorable contacts with the pore-facing residues.

Based on these results we suggest that there are more than one site along the pore where NFA can bind and block the ion permeation. The most likely locations of the NFA binding sites are at the opposite sides of the Leu261 ring, which forms the open pore constriction. And the most likely mechanism of the current block is the combination of the hydrophobic and steric block: NFA exposes its hydrophobic groups to the pore lumen, binds above and/or below the Leu261 ring and thus precludes permeation of the hydrated chloride ion. This block may be enforced by the electrostatic repulsion of the negatively charged carboxylate group of NFA and negatively charged chloride ions.

Our data on the Hill coefficient and the fractional electrical distance of the NFA binding site suggest two peculiarities of NFA action on GlyRs. Firstly, two NFA molecules preferably block permeation through the α1 GlyR channel, whereas the α2 GlyR channel is blocked by a single NFA molecule. Secondly, the NFA binding site in the α2 GlyR is located deeper than in the α1 GlyR. These experimental data provide important constraints to further elaborate models of NFA-bound α1 and α2 GlyRs.

The α2 GlyR block by a single NFA molecule bound deeply inside the pore can be illustrated by the binding mode found without constraints (Figures 10D–F). In this mode, NFA binds between hydrophobic rings Ala254 and Leu261 and accepts an H-bond from Thr258. This and similar binding modes collected in the MC-minimizations correspond to the energy minima at the left part of the energy profile (Figure 9B).

The energy profile of NFA in the α1 GlyR shows that NFA-channel interactions at levels −2′ to 6′ are energetically less preferable than in the α2 GlyR. A reason is the ring of Gly254 residues in α1 GlyR, which does not form as favorable van der Walls and hydrophobic contacts with NFA as the ring of Ala254 residues in α2 GlyR does. In particular, water molecules that hydrate the pore-exposed hydrophilic backbone atoms of Gly254 residues would destabilize binding of the hydrophobic NFA.

A complex α1 GlyR with a single NFA bound above the ring of Leu261 residues, at level 13′ (Thr265) corresponds to the wide energy minima (Figure 9B). We docked the second NFA molecules to this complex and MC-minimizing the energy. The pore easily accommodated two NFA molecules at this level and calculations predicted several possible low-energy binding modes of the two ligands. One of these is shown at Figures 10G–I, where two NFA molecules form favorable hydrophobic contacts with each other and the extracellularly-oriented side of the Leu261 ring, thus blocking the ion permeation, whereas their carboxylate groups accept H-bonds from the Thr265 residues.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.