4. Discussion

A broad number of medicinal plants have been used for centuries as therapeutic tools in TCM. However, neither the active compounds of many of these plants nor their mechanisms of action are yet well-understood. We have now studied the effect of Pm, an isosteroidal alkaloid considered one of the main bioactive molecules of Fb, on nAChRs. Remarkably, Pm decreased I_ACh in a dose-dependent manner, with an IC₅₀ in the low micromolar range (circa 3 and 1 µM for I_p and I_ss, respectively; Figure 1). These IC₅₀s are markedly lower than the values previously reported for Pm blockade of voltage-dependent potassium channels. Thus, Pm IC₅₀ for blocking Kv1.2 was 472 µM, it was 354 µM for Kv1.3 (142 µM if measured 150 ms after the current peak), and even much higher for Kv1.4 to Kv1.8 channels [10]; additionally, Pm inhibited the potassium channel hERG, with an IC₅₀ of 44 µM, most likely by enhancing its inactivation [11]. Pm also blocked the Nav1.7 channel (IC₅₀, 47 µM), demonstrating use-dependent inhibition, like the blocking mechanism of lidocaine on this channel [10]. Pm effects on hERG channels are particularly remarkable, since these channels play a key role in myocardial repolarization and, therefore, their inhibition might cause serious cardiac arrhythmias. Despite this, humans have used Fb as a therapeutic herb for centuries, being considered safe for consumption. Consequently, intake of Fb should not markedly affect the activity of hERG channels, despite that Pm, one of its main bioactive compounds, blocks these channels with an IC₅₀ of 44 µM [11]. Nevertheless, Pm has a very low oral bioavailability [9] and thus its plasma concentration after Fb intake should be fairly low. Actually, Pm content in bulbs of Fritillaria ussuriensis and thunbergii ranged from 0.58 to 1.2 mg/g and oral administration of powder from these Fritillaria plants to dogs (1 g/kg) raised Pm plasma concentration to a maximum of 100–200 nM ^[24][30]. Accordingly, a similar Pm bioavailability was reported after oral administration of Fritillaria thunbergii extracts in rats, with peak plasma concentrations of Pm of roughly 100 nM ^[25][31]. These Pm concentrations are several orders of magnitude lower than the IC₅₀s reported for sodium or potassium channels (including hERG), muscarinic receptors, or acethylcholinesterase [8]. Interestingly, submicromolar Pm concentrations elicit a significant inhibition of nAChRs (roughly 20%; Figure 1) and therefore this family of LGIC might be relevant targets of its actions. Of note, we have assessed Pm actions on muscle-type nAChRs, because they are the prototype member of this family of receptors, but Pm might have different affinities for related receptors, as the homomeric α7. In fact, α7 nAChRs are largely expressed in non-neuronal tissues, including macrophages, and exert powerful anti-inflammatory actions [16]. Moreover, other nAChR subtypes, such as α4β2 and/or α9α10, may also play a role in modulating inflammatory processes and even in chronic pain [15]. Noticeably, Pm displayed a differential affinity for different LGICs, even of the same family. Thus, whereas Pm inhibits muscle-type nAChRs with and IC₅₀ close to 1 µM, GABA_A receptors were almost not affected by Pm at concentrations up to 100 µM. Pm exerted a non-competitive inhibition on muscle-type nAChRs, since I_AChs halved when co-applying Pm, at its IC₅₀, with different ACh concentrations (Figure 2A,B). Several blockade mechanisms seem involved in this non-competitive inhibition of nAChRs by Pm, which is coherent with the multiple binding sites predicted by the docking simulations. First, open-channel blockade, as I_ACh inhibition by Pm was voltage-dependent, the more hyperpolarized the cell, the more pronounced the blockade (Figure 5). Actually, this is what would be expected for a positively charged molecule plugging the channel pore. In agreement with this, the docking assays predicted some Pm clusters located within the channel pore, both in the open and closed conformations (Figure 89). Thus, the open-channel blockade of nAChRs mediated by Pm resembles that mediated by some LAs, such as lidocaine ^[26][24] or tetracaine ^[27][26]. Nevertheless, the kinetics of open-channel blockade of nAChR elicited by Pm was rather slow. Thus, at −60 mV, the τ of open channel blockade elicited by 1 µM Pm (close to its IC₅₀) was over 2 s and even by 5 µM Pm (eliciting roughly 70% of I_ss blockade) the time constant was above 1 s (Figure 67). In contrast, at the same membrane potential, 0.7 µM tetracaine (close to its IC₅₀) blocked open nAChRs with a time course of roughly 300 ms ^[27][26]. These differences in time constants between Pm and tetracaine are most likely related to their distinct molecular sizes (Pm molecular weight is over 60% greater than that of tetracaine). Second, Pm enhanced nAChR desensitization, as evidenced by: (i) acceleration of I_ACh decay when co-applying ACh with Pm at concentrations of 0.5 µM, or above (Figure 3A₂,B₂), and (ii) shortening of the I_ACh aTtp, which correlated with the acceleration of I_ACh decay (Figure 3A₁,B₁). Likewise, lidocaine decreased the I_ACh aTtp only at concentrations that enhanced I_ACh decay ^[26][24]. Furthermore, DMA, a lidocaine analog, both sped up I_ACh decay and shortened the aTtP ^[28][25]. By contrast, diethylamine (DEA), a lidocaine analog that mainly blocks nAChR by open channel blockade neither accelerates I_ACh decay nor decreases aTtP [21], and (iii) deceleration of I_ACh deactivation, which was dependent on Pm concentration and displayed a good correlation with the rate of I_ACh decay (Figure 4A, B). The deceleration of I_ACh deactivation when Pm remained in the solution strongly supports that Pm enhanced nAChR desensitization, since desensitized nAChRs display higher affinity for the agonist ^[22][27][29][22,26,27]. Third, Pm elicited the blockade of resting (closed) nAChRs. This effect was unraveled by applying Pm before challenging the cells with ACh alone. This protocol, which allowed Pm to act only on resting (closed) nAChRs, elicited a mild nAChR blockade, mostly at negative potentials. Actually, at positive potentials, I_ACh only decreased by Pm pre-application when rising its concentration to 5 µM (Figure 78). In agreement with this, I_ACh inhibition by 5 µM Pm was slightly higher when Pm was pre-applied and then co-applied with ACh than when solely co-applied with ACh (Figure 78C). Our virtual docking and MD simulations used as a template the structure of Torpedo nAChRs in the open and closed conformations released by Unwin’s group ^[30][31][32,33]. However, a new structural model of the nAChR from Torpedo, at higher resolution and stabilized in the closed conformation by α-bungarotoxin, has been recently disclosed ^[32][34]. The new structural model (pdb entry 6UWZ) share large similarities with the Unwin’s model for the closed conformation, though there are certain differences between them. Mostly, they differ in the upper portion of the pore, which is more constricted in the new model, and in the δ subunit arrangement ^[33][35]. It seems that these discrepancies arise because of differences in the lipid matrix surrounding the nAChR. Actually, cholesterol interactions with the nAChR are apparently essential for stabilizing its structure and the absence of cholesterol (as in the model of Rahman et al. ^[32][34]) leads to a more compact arrangement of TM helices (displacement of helices circa 1–3 Å; ^[33][35]). Noticeably, the major lipid present in electroplax membranes rich in nAChRs is cholesterol ^[34][36] and purified nAChRs from T. marmorata and E. electricus interact preferentially with cholesterol rather than with either phospholipid monolayers or other sterols ^[35][37]. Moreover, nAChRs in native electroplax membranes are arranged as dimers, linked by their δ-subunits. This interaction between neighboring nAChRs might account for the differences in the δ-subunit between both structural models since dimers were reduced to monomeric receptors in the Rahman’s model. In fact, we chose for our structural studies Unwin’s models because of: (i) the structures for the open and closed conformations are available, (ii) the nAChR is present in their original membrane, and (iii) we have significant experience correlating structural and functional results using these commonly accepted models; actually, Unwin’s models have so far provided a coherent correlation with our functional results ^{[21][28][27][36]}[21,25,26,28]. The virtual docking assays predicted Pm binding to the nAChR at different sites of the TMD and ECD in the open conformation. Most Pm clusters were located at the TMD, at inter- and intra-subunit crevices, although some of them located into the channel pore (Figure 89A,C). The binding energies estimated for these clusters were rather high (from −9.3 to −12.87 kcal/mol; see Supplementary Table S1), pointing out that Pm has a high affinity for the nAChR. Remarkably, MD simulations of nAChRs in the open conformation indicate that Pm binding to the nAChR either at the TMD (i.e., cluster 6) or at the ECD (i.e., cluster 14) markedly decreased both the volume and the number of water molecules at the hydrophobic gate region of the channel pore (Figure 911). Furthermore, virtual docking and MD assays pointed out that Pm also interacts with the nAChR in the resting conformation, binding to residues located at both the TMD and the ECD (Figure 89B,D and Figure 911E,F). Interestingly, the structural changes of the nAChR induced by Pm, as predicted by docking and MD assays, are in good agreement with the functional changes elicited by Pm on this receptor. Actually, the structural and functional results can be correlated as follows: (i) the high binding energies computed accounted for the high potency of Pm blocking nAChRs, (ii) Pm interaction with residues located within the channel pore should trigger the open-channel blockade, (iii) Pm binding to different sites at the nAChR might explain both its heterogeneity of actions on nAChRs and the effect-dependence on Pm concentration, and (iv) Pm binding to the nAChR in the closed conformation might underlie the blockade of resting nAChR. Consequently, the good correlation between structural simulations and electrophysiological results strongly suggests that Pm actually blocks nAChRs by the different aforementioned mechanisms. Since Fb has been used as therapeutic herb for thousands of years, there is a strong support for its beneficial effects and its weak (or lack) of toxicity. However, neither the identity of all its bioactive compounds nor their mechanisms of action are yet well known. Now, we report here that Pm, considered one of the main bioactive compounds from Fritillaria, exerts a powerful inhibition of muscle-type nAChRs, which, as far as we know, is the first report demonstrating that Pm might modulate LGICs, besides acting on other targets as voltage-dependent channels or metabotropic receptors. It remains to be unraveled if Pm might modulate other nAChRs, including the homomeric α7, which is broadly expressed in immune cells and has been related to powerful anti-inflammatory actions [16]. Furthermore, both mecamylamine, a non-competitive antagonist of α7 nAChRs, and 1-ethyl-4-(3-(bromo)phenyl)piperazine, which promotes α7 desensitization, reduce pro-inflammatory responses ^[37][38]. We have now demonstrated that Pm inhibits muscle-type nAChRs and it can be hypothesized that it might modulate, though in a different way or with a different potency, either α7 or other nAChRs. In this regard, lidocaine exerted similar inhibitory actions on muscle-type nAChRs ^[26][24] and on neuronal nAChRs expressed in autonomic-ganglia neurons ^[38][39]. Alternatively, it could be that different bioactive compounds from Fb accounted for its anti-inflammatory actions, as anthocyanin pigments, which are flavonoids present in Fb ^[39][40]. Noticeably, some flavonoids act as positive allosteric modulators of α7 nAChRs, although without affecting desensitization [6], and their enhancement of α7 activity has been proposed as a therapeutic strategy for inflammatory disorders ^[40][41].