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Background and purpose Sodium channel inhibitors can be used to treat hyperexcitability-related diseases, including epilepsies, pain syndromes, neuromuscular disorders and cardiac arrhythmias. The applicability of these drugs is limited by their nonspecific effect on physiological function. They act mainly by sodium channel block and in addition by modulation of channel kinetics. While channel block inhibits healthy and pathological tissue equally, modulation can preferentially inhibit pathological activity. An ideal drug designed to target the sodium channels of pathological tissue would act predominantly by modulation. Thus far, no such drug has been described. Experimental approach Patch-clamp experiments with ultra-fast solution exchange and photolabeling-coupled electrophysiology were applied to describe the unique mechanism of riluzole on Nav1.4 sodium channels. In silico docking experiments were used to study the molecular details of binding. Key results We present evidence that riluzole acts predominantly by non-blocking modulation. We propose that, being a relatively small molecule, riluzole is able to stay bound to the binding site, but nonetheless stay off the conduction pathway, by residing in one of the fenestrations. We demonstrate how this mechanism can be recognized. Conclusions and implications Our results identify riluzole as the prototype of this new class of sodium channel inhibitors. Drugs of this class are expected to selectively prevent hyperexcitability, while having minimal effect on cells firing at a normal rate from a normal resting potential.

Present-day people from England and Wales have more ancestry derived from early European farmers (EEF) than did people of the Early Bronze Age1. To understand this, here we generated genome-wide data from 793 individuals, increasing data from the Middle to the Late Bronze Age and Iron Age in Britain by 12-fold, and western and central Europe by 3.5-fold. Between 1000 and 875 BC, EEF ancestry increased in southern Britain (England and Wales) but not northern Britain (Scotland) due to incorporation of migrants who arrived at this time and over previous centuries, and who were genetically most similar to ancient individuals from France. These migrants contributed about half the ancestry of people of England and Wales from the Iron Age, thereby creating a plausible vector for the spread of early Celtic languages into Britain. These patterns are part of a broader trend of EEF ancestry becoming more similar across central and western Europe in the Middle to the Late Bronze Age, coincident with archaeological evidence of intensified cultural exchange2-6. There was comparatively less gene flow from continental Europe during the Iron Age, and the independent genetic trajectory in Britain is also reflected in the rise of the allele conferring lactase persistence to approximately 50% by this time compared to approximately 7% in central Europe where it rose rapidly in frequency only a millennium later. This suggests that dairy products were used in qualitatively different ways in Britain and in central Europe over this period., This work was funded in part by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 834087; the COMMIOS Project to I.A.). M.N. was supported by the Croatian Science Fund grant (HRZZ IP-2016-06-1450). P.V., M.Dobeš and Z.V. were supported by the Ministry of Culture of the Czech Republic (DKRVO 2019-2023/7.I.c, 00023272). M.E. was supported by Czech Academy of Sciences award Praemium Academiae. M.Dobisíková and A.Danielisová were supported by the grant RVO 67985912 of the Institute of Archaeology of the Czech Academy of Sciences. M.G.B.F. was funded by The Leverhulme Trust via a Doctoral Scholarship scheme awarded to M.Pala and M.B.R. Support to M.Legge came from the South, West & Wales Doctoral Training Partnership. M.G.’s osteological analyses were funded by Culture Vannin. A.S.-N. was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. T.H., T.S. and K.K.’s work was supported by a grant from the Hungarian Research, Development and Innovation Office (project number: FK128013). We acknowledge support for radiocarbon dating and stable isotope analyses as well as access to skeletal material from Manx National Heritage and A. Fox. Dating analysis was funded by Leverhulme Trust grant RPG-388. M.G.T. and I.B. were supported by a Wellcome Trust Investigator Award (project 100713/Z/12/Z). I.O. was supported by a Ramón y Cajal grant from Ministerio de Ciencia e Innovación, Spanish Government (RYC2019-027909-I). The research directed at Harvard was funded by NIH grants GM100233 and HG012287, by John Templeton Foundation grant 61220, by a gift from Jean-François Clin, and by the Allen Discovery Center program, a Paul G. Allen Frontiers Group advised program of the Paul G. Allen Family Foundation. D.R. is also an Investigator of the Howard Hughes Medical Institute.

Background and purpose: Ivabradine is clinically administered to lower the heart rate, proposedly by inhibiting hyperpolarization-activated cyclic nucleotide-gated cation channels in the sinoatrial node. Recent evidence suggests that voltage-gated sodium channels (VGSC) are inhibited within the same concentration range. VGSCs are expressed within the sinoatrial node and throughout the conduction system of the heart. A block of these channels thus likely contributes to the established and newly raised clinical indications of ivabradine. We, therefore, investigated the pharmacological action of ivabradine on VGSCs in sufficient detail in order to gain a better understanding of the pro- and anti-arrhythmic effects associated with the administration of this drug.Experimental Approach: Ivabradine was tested on VGSCs in native cardiomyocytes isolated from mouse ventricles and the His-Purkinje system and on human Nav1.5 in a heterologous expression system. We investigated the mechanism of channel inhibition by determining its voltage-, frequency-, state-, and temperature-dependence, complemented by a molecular drug docking to the recent Nav1.5 cryoEM structure. Automated patch-clamp experiments were used to investigate ivabradine-mediated changes in Nav1.5 inactivation parameters and inhibition of different VGSC isoforms.Key results: Ivabradine inhibited VGSCs in a voltage- and frequency-dependent manner, but did not alter voltage-dependence of activation and fast inactivation, nor recovery from fast inactivation. Cardiac (Nav1.5), neuronal (Nav1.2), and skeletal muscle (Nav1.4) VGSC isoforms were inhibited by ivabradine within the same concentration range, as were sodium currents in native cardiomyocytes isolated from the ventricles and the His-Purkinje system. Molecular drug docking suggested an interaction of ivabradine with the classical local anesthetic binding site.Conclusion and Implications: Ivabradine acts as an atypical inhibitor of VGSCs. Inhibition of VGSCs likely contributes to the heart rate lowering effect of ivabradine, in particular at higher stimulation frequencies and depolarized membrane potentials, and to the observed slowing of intra-cardiac conduction. Inhibition of VGSCs in native cardiomyocytes and across channel isoforms may provide a potential basis for the anti-arrhythmic potential as observed upon administration of ivabradine.

Standard high throughput screening projects using automated patch-clamp instruments often fail to grasp essential details of the mechanism of action, such as binding/unbinding dynamics and modulation of gating. In this study, we aim to demonstrate that depth of analysis can be combined with acceptable throughput on such instruments. Using the microfluidics-based automated patch clamp, IonFlux Mercury, we developed a method for a rapid assessment of the mechanism of action of sodium channel inhibitors, including their state-dependent association and dissociation kinetics. The method is based on a complex voltage protocol, which is repeated at 1 Hz. Using this time resolution we could monitor the onset and offset of both channel block and modulation of gating upon drug perfusion and washout. Our results show that the onset and the offset of drug effects are complex processes, involving several steps, which may occur on different time scales. We could identify distinct sub-processes on the millisecond time scale, as well as on the second time scale. Automated analysis of the results allows collection of detailed information regarding the mechanism of action of individual compounds, which may help the assessment of therapeutic potential for hyperexcitability-related disorders, such as epilepsies, pain syndromes, neuromuscular disorders, or neurodegenerative diseases.

Hyperexcitability-related diseases include epilepsies, pain syndromes, neuromuscular disorders, and cardiac arrhythmias. Sodium channel inhibitors can be used to treat these conditions, however, their applicability is limited by their nonspecific effect on physiological function. They act by channel block (obstructing ion conduction, since the binding site is within the channel pore), and by modulation (delaying recovery from the non-conducting inactivated state). Channel block inhibits healthy and pathological tissue equally, while modulation can preferentially inhibit pathological activity. Therefore, an ideal sodium channel inhibitor drug would act by modulation alone. Unfortunately, thus far no such drug has been known to exist. Here we present evidence that riluzole acts by this “ideal” mechanism, “non-blocking modulation” (NBM). We propose that, being a relatively small molecule, riluzole is able to stay bound to the binding site, but nonetheless stay off the conduction pathway, by residing in one of the “fenestrations” (cavities connecting the central cavity to the membrane phase). Using precisely timed UV pulses to photolabel specific conformations of the channel, we show that association to the local anesthetic binding site requires prior inactivation. We discuss why kinetics of binding is crucial for selective inhibition of pathological activity, and how the NBM mechanism can be recognized using a special voltage- and drug application-protocol. Our results identify riluzole as the prototype of this new class of sodium channel inhibitors. Drugs of this class are expected to selectively prevent hyperexcitability, while having minimal effect on cells firing at a normal rate from a normal resting potential.

We investigated the mode of action of PNU-120596, a type II positive allosteric modulator of the rat α7 nicotinic acetylcholine receptor expressed by GH4C1 cells, using patch-clamp and fast solution exchange. We made two important observations: first, while PNU-120596 rapidly associated to desensitized receptors, it had at least hundredfold lower affinity to resting conformation, therefore at 10 μM concentration it dissociated from resting receptors; and second, binding of PNU-120596 slowed down dissociation of choline molecules from the receptor radically. We propose that when agonist concentration is transiently elevated in the continuous presence of the modulator (as upon the neuronal release of acetylcholine in a modulator-treated animal) these two elements together cause occurrence of a cycle of events: Binding of the modulator is limited in the absence of the agonist. When the agonist is released, it binds to the receptor, and induces desensitization, thereby enabling modulator binding. Modulator binding in turn traps the agonist within its binding site for a prolonged period of time. Once the agonist finally dissociated, the modulator can also dissociate without re-binding, and the receptor assumes its original resting conformation. In kinetic simulations this “trapped agonist cycle” mechanism did not require that the orthosteric and allosteric ligands symmetrically modify each other's affinity, only the modulator must decrease agonist accessibility, and the agonist must induce a conformation that is accessible to the modulator. This mechanism effectively prolongs and amplifies the effect of the agonist.

The alpha7 nicotinic acetylcholine receptor (nAChR) has some peculiar kinetic properties. From the literature of α7 nAChR-mediated currents we concluded that experimentally measured kinetic properties reflected properties of the solution exchange system, rather than genuine kinetic properties of the receptors. We also concluded that all experimentally measured EC50 values for agonists must inherently be inaccurate. The aim of this study was to assess the undistorted kinetic properties of α7 nAChRs, and to construct an improved kinetic model, which can also serve as a basis of modeling the effect of the positive allosteric modulator PNU-120596, as it is described in the accompanying paper. Agonist-evoked currents were recorded from GH4C1 cells stably transfected with pCEP4/rat α7 nAChR using patch-clamp and fast solution exchange. We used two approaches to circumvent the problem of insufficient solution exchange rate: extrapolation and kinetic modeling. First, using different solution exchange rates we recorded evoked currents, and extrapolated their amplitude and kinetics to instantaneous solution exchange. Second, we constructed a kinetic model that reproduced concentration-dependence and solution exchange rate-dependence of receptors, and then we simulated receptor behavior at experimentally unattainably fast solution exchange. We also determined open probabilities during choline-evoked unmodulated and modulated currents using nonstationary fluctuation analysis. The peak open probability of 10 mM choline-evoked currents was 0.033 ± 0.006, while in the presence of choline (10 mM) and PNU-120596 (10 μM), it was increased to 0.599 ± 0.058. Our kinetic model could adequately reproduce low open probability, fast kinetics, fast recovery and solution exchange rate-dependent kinetics.

We investigated the kinetics of choline evoked currents, and the effect of the positive modulator PNU 120596 on alpha7 nicotinic acetylcholine receptors expressed by GH4C1 cells (obtained from Siena Biotech SpA) in whole-cell and outside-out patch-clamp experiments. using a theta-tube system for rapid application of agonists, we measured the dependence of current kinetics on the solution exchange rate. By extrapolation and by kinetic modeling we estimated the intrinsic kinetics of the receptor: what would be the amplitude and kinetics of choline-evoked currents at instantaneous agonist application. In the presence of PNU 120596 the single channel mean open time is drastically prolonged, this allowed us to determine the ratio of simultaneously open channels using nonstationary fluctuation analysis. By determining the approximate number of channels in a patch, we could determine the peak open probability of 10 mM choline-evoked currents in the absence of the modulator (0.0333 ± 0.0056), as well as the open probability in the presence of 10 mM choline and 10 μM PNU 120596 (0.632 ± 0.065). We performed kinetic experiments to determine the affinity of PNU 120596 to three different conformational states of the receptor: resting state, desensitized state and a slowly developing second desensitized state. We found that PNU 120596 was ineffective at both the resting and the slow desensitized states, while it bound to the desensitised state and re-activated the receptors. We investigated the nature of cooperativity between the agonist and the modulator. We found that while the agonist increases the apparent affinity of the modulator only by inducing desensitized conformation (which is preferred by the modulator), the modulator induces a true increase of agonist affinity probably by allosterically affecting the conformation of the agonist binding site itself.

Riluzole is a persistent-selective sodium channel inhibitor (SCI), which has a therapeutic potential to treat several neurological and psychiatric disorders. SCIs form a large and diverse group, which includes widely different modes of action. Individual SCIs may be very potent in one protocol, but ineffective in another. We have developed a method for testing the “personality” of SCIs, i.e., we assess their potency under different voltage protocols. The pattern of their relative potencies gives a characteristic fingerprint which shows correlation with specific chemical properties of molecules, and may also predict their therapeutic profile. We have identified distinct modes of action for specific groups of SCIs. Riluzole was found to have a unique “personality” type; therefore, in this study we performed a detailed analysis of its mode of action. The classic SCIs lidocaine and carbamazepine as well as the persistent-selective ranolazine were used as reference compounds. We observed a paradoxical inverse use-dependence and an apparent transient facilitation on sodium channels in the presence of riluzole (but not of other drugs) when currents were evoked by short depolarizations: in such protocols, riluzole appeared to be ineffective. On the other hand, in protocols with prolonged moderate depolarizations the drug was remarkably potent; suggesting that it strongly enhanced closed state inactivation. Recovery from fast inactivation was significantly impeded by riluzole, while recovery from slow inactivated state was - remarkably - even slightly accelerated. As a possible mechanism we propose that riluzole has an exceptionally fast binding kinetics, a high affinity for pre-open closed- and fast inactivated states, while a low affinity for slow inactivated state.