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Therapeutic potential of NaV1.1 activators
Opinion
Therapeutic potential of NaV1.1 activators Henrik S. Jensen*, Morten Grunnet*, and Jesper F. Bastlund Neuroscience Drug Discovery, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Copenhagen, Denmark
Sodium channel inhibitors have been developed and approved as drugs to treat a variety of indications. By contrast, sodium channel activators have not previously been considered relevant in a therapeutic setting owing to their high risk of toxicity and side effects. Here we present an opinion that selective activators of the NaV1.1 sodium channel may hold therapeutic potential for diseases such as epilepsy, schizophrenia, and Alzheimer’s disease. Central to this novel avenue of sodium channel drug discovery is that fact that NaV1.1 comprises the majority of the sodium current in specific inhibitory interneurons. Conversely, it plays only a modest role in excitatory neurons owing to the high redundancy of other types of sodium channels in these cells. We discuss the biological background and rationale and present reflections on how to identify activators of NaV1.1. Modulation of sodium channel function as a therapeutic strategy Emerging data highlight the important role of interneurons in both health and disease. Interneurons are inhibitory in nature because they synthesize and release GABA, the major inhibitory neurotransmitter in the brain. They have important roles in the central nervous system (CNS) in regulating excitability and securing synchronized activity of neuronal populations. Interneurons can be divided into different subclasses according to their neurochemical markers, connectivity, and physiological properties. In the fastspiking, parvalbumin-expressing subclass of inhibitory neurons, the sodium current is mainly carried by one type of sodium channel, namely the NaV1.1 channel. This is in contrast to excitable cells, such as hippocampal pyramidal cells, in which NaV1.1 channels are also expressed [1,2] but are co-expressed with additional subtypes of NaV1.X channels. We could thus consider excitable cells as potentially having a depolarization reserve, in analogy to the cardiac repolarization reserve [3], whereby different NaV1.X channel subtypes can substitute for each other in creating depolarizing current and thus initiating action potentials. Corresponding author: Jensen, H.S. ([email protected]). Keywords: interneurons; cognition; oscillations; epilepsy; connectivity SCN1A mutations; schizophrenia. * These authors contributed equally to this work. 0165-6147/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tips.2013.12.007
However, this depolarization reserve is not present in parvalbumin interneurons, where NaV1.1 channels are essential for action potential generation [4]. It is firmly established that sodium channels play a pivotal role in neuronal excitability, and to date, nine genes have been described (SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A, and SCN11A) that encode the voltage-gated NaV1.X channels NaV1.1– NaV1.9, respectively. In addition, a closely related Na+ channel-like protein has been cloned but not functionally expressed [5]. Voltage-gated sodium channels have been the focus of an immense body of research that includes both basic science and drug discovery activities. Inhibitors of sodium channels are currently approved and indicated for the treatment of pain, epilepsy, and cardiac arrhythmia. By contrast, activators of voltage-gated sodium channels have not been utilized as drugs, probably because complete pan-selective activation of human NaV channels induces seizures with no therapeutic window for efficacy. Sodium channel activators targeting insect channels have, however, been explored and are being used as insecticides [6]. However, because NaV1.1 is by far the most important sodium channel for initiation of action potentials in the fast-spiking subtype of GABAergic interneurons, selective activation of NaV1.1 might constitute a new potential treatment paradigm. In support of this idea, transgenic mouse models with reduced NaV1.1 expression have been associated with various diseases [7]. Furthermore, in a human context, loss-of-function mutations in the SCNA1 gene encoding NaV1.1 have been linked to a spectrum of seizure disorders [8–11]. In this opinion article, we give a brief account of the pathology associated with reduced NaV1.1 channel function and discuss how drugmediated increases in NaV1.1 function might be beneficial as a consequence of better overall interneuron function and thereby overall synchronization of neuronal networks. We also outline possible screening approaches and considerations for identifying NaV1.1 channel activators. Voltage-gated Na+ channels Voltage-gated Na+ channels are transmembrane (TM) proteins consisting of a single pore-forming a-subunit with 24 TM segments. These are divided into four relatively homogeneous domains of six TMs with the voltage sensor located in TM4 and the pore-forming domain situated between the fifth and sixth TM. The linkers between the four domains vary in length and have important functions in channel modulation, inactivation, and drug binding [12]. All voltage-gated Na+ channels are closed at the resting hyperpolarized membrane potential. Depolarization of the Trends in Pharmacological Sciences, March 2014, Vol. 35, No. 3
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Opinion membrane potential induces a conformational change in the channel that allows selective conduction of Na+ ions in an inward direction dictated by the electrochemical gradient. Voltage-gated Na+ channels are further characterized by fast inactivation that changes the channels to a non-conducting state [13]. The pore-forming a-subunit is able to constitute a fully functional conducting ion channel but biophysical properties can be affected by accessory b-subunits [14,15]. The b-subunits are one-TM proteins with immunoglobulin-like N-terminal extracellular structures. A sodium ion channel complex is typically a heterodimeric or heterotrimeric structure with a:b stoichiometry of 1:1 or 1:2 [16,17]. The voltage-gated Na+ channels NaV1.1–1.9 are all expressed in excitable tissue found in the CNS, the peripheral nervous system (PNS), and the heart [18]. The tenth member, Nax, is not as well characterized but might play a function in Na+ homeostasis [5]. Expression of NaV1.X channels is developmentally regulated, and NaV1.1, 1.2, 1.3, and 1.6 are the most predominant subtypes in CNS, with NaV1.6 being the most abundant subtype in the adult brain [18]. NaV1.4 is expressed in skeletal muscle [19] and NaV1.5 is the major cardiac sodium channel [20], whereas NaV1.7, 1.8, and 1.9 are important players in nociceptive signaling transduction owing to their presence in peripheral primary sensory afferents [21–23]. Voltage-gated Na+ channels in diseases It is generally accepted that hyperactivity of NaV1.X channels is associated with pathophysiological conditions due to inappropriate regulation of neuronal action potential firing and thereby excessive activity. Multiple NaV1.X channel inhibitors have been developed as anti-epileptic and antiarrhythmic drugs and are widely used in clinical applications. However, there are exceptions to this general notion of beneficial inhibition of NaV1.X channels. Examples are NaV1.5 loss-of-function mutations causing Brugada syndrome, a potentially life-threatening heart rhythm disorder that can develop into ventricular fibrillation and ultimately sudden cardiac arrest [24], congenital indifference to pain due to loss of function of NaV1.7 channels [25], and, most notably, loss of function of NaV1.1 channels associated with various diseases, such as familial hemiplegic migraine, familial autism, and sporadic autism spectrum disorder [8,9,26–28]. However, the best-characterized understanding of reduced NaV1.1 function and a disease phenotype stems from patients with Dravet syndrome, which represents a genetic link between the SCN1A gene (encoding NaV1.1) and a disease. Patients with Dravet syndrome are primarily characterized by multiple epilepsy phenotypes such as convulsive seizures and recurrent periods of status epilepticus. In childhood, seizures are often precipitated by fever. Following the onset of seizures, cognitive impairment, mental retardation, behavioral abnormalities, and autistic traits can occur. The febrile seizures tend to ameliorate in early childhood, but afebrile seizures can last beyond childhood [11]. In addition, mouse models of reduced NaV1.1 channel expression have revealed phenotypes such as ataxia, severe myoclonic epilepsy, autistic-like behaviors, cognitive deficits, and sudden unexpected death [4,29–31]. Thus, a number of diseases, including several in humans, have been associated with loss 114
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of function (haploinsufficiency) in NaV1.1 channels. Although the mutations identified in NaV1.1 do not share a completely identical phenotype, we speculate that an innovative approach to ameliorating symptoms associated with these diseases could involve pharmacological restoration of normal function for NaV1.1 channels. NaV1.1: Functional role and therapeutic potential Epilepsy It is established that reduced NaV1.1 channel function has a large impact on the excitability of neuronal networks because multiple heterozygous loss-of-function mutations have been associated with epilepsies such as Dravet syndrome. Importantly, the magnitude of reduced NaV1.1 channel function seems to correlate directly to the severity of the epileptic phenotype [10]. Not all Dravet patients have SCN1A mutations, although the association is strong, because an estimated 85% of patients carry SCN1A mutations [11]. As mentioned above, NaV1.1 channels carry the main sodium current in parvalbumin-containing, fastspiking GABAergic interneuron subtypes and are pivotal for the generation of action potentials and thus continuous excitability of these neurons. Consequently, selective increases in NaV1.1 channel function might hold the potential to increase the function of fast-spiking GABAergic interneurons and thus affect overall excitability in the CNS. Pharmacological activation of NaV1.1 channels is therefore an obvious suggestion as a treatment paradigm for patients with SCN1A haploinsufficiency and other disorders with impaired functionality of fast-spiking parvalbumin interneurons. NaV1.1 activators might also have therapeutic potential in the treatment of symptomatic epilepsies such as temporal lobe epilepsy, for which partial loss of fast-spiking GABAergic interneurons has been reported as an early pathological hallmark [32]. Importantly, SCN1A+/ mice have been developed that capture the epileptic phenotype of patients with SCN1A haploinsufficiency [4]. This model can aid in further exploration of NaV1.1 activators as therapeutics for epileptic syndromes. Recent experiments with induced pluripotent stem cells (iPSCs) derived from two Dravet patients revealed increased expression of NaV1.1 channels in neurons derived from differentiated iPSCs [33,34]. Whether such observations mirror the true patient phenotype or are a consequence of the iPSC differentiation procedure still remains to be addressed. In the context of these two studies, it should be noted that seizure worsening has been reported for Dravet’s patients treated with sodium with pan-selective sodium channel inhibitors [35]. However, as for all drug development, a thorough safety assessment during the development process would be required for future NaV1.1 activators, which present some challenges (see below). Cognitive deficits and Alzheimer’s disease (AD) SCN1A+/ mice also exhibit non-seizure deficits that are found in human mutation carriers, such as reduced cognitive performance [30]. This raises the question whether reduced NaV1.1 channel function directly affects cognitive processing or if the effect is secondary (i.e., a consequence of repeated seizures). Local targeted reductions in NaV1.1
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but may be related to increased activity of b-site APPcleaving enzyme (BACE), because it has been reported that BACE regulates NaV1.1 expression [39,40]. Importantly, crossing of J20 mice with NaV1.1-overexpressing mice partially normalizes the phenotype in these mice and recovers deficient gamma oscillations and cognitive performance [38], suggesting a therapeutic potential of NaV1.1 activators for cognitive disturbance associated with AD. The mechanistic importance of fast-spiking GABAergic interneurons, and thus of NaV1.1 channels, in cognition might rely on the ability to affect synchronization of fast oscillations in the gamma range (30–100 Hz) (Box 1). Importantly, it has been demonstrated that increased interneuron function and gamma oscillations have a positive effect on cognitive performance in rodents [41,42].
expression in parvalbumin-positive interneurons in the medial septum revealed impaired cognitive performance in these non-epileptic animals [36]. Thus, cognitive deficits might also be a primary consequence of reduced NaV1.1 expression. Further evidence supporting this notion was provided by a study demonstrating reduced short-term memory in apparently healthy individuals carrying SCNA1 mutations, which suggests a therapeutic potential for NaV1.1 activators as pro-cognitive therapy [37]. In relation to this, reduced NaV1.1 expression has also been reported in humans suffering from AD and in J20 mice [38], which are carriers of amyloid precursor protein (APP) mutations found in familial forms of AD. The mechanism by which NaV1.1 expression is reduced in Alzheimerrelated pathological conditions is not fully characterized,
Box 1. Impact of NaV1.1 channels on fast-spiking interneuron function and gamma oscillations Fast-spiking GABAergic interneurons (FSIs; Figure I) that are immunoreactive for the calcium-binding protein parvalbumin are crucial for synchronized high-frequency neuronal activity in the gamma range (30–100 Hz) as measured by the local field potential (LFP). This FSI class shows high expression of NaV1.1 [53] and can be further subdivided according to morphological characteristics into chandelier and basket cells. However, here we mainly consider the broader FSI class, because this is where NaV1.1 expression is reported. Extensive research documents the dominant role of FSIs in synchronization of gamma oscillations, and a reduction in
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excitatory drive to FSIs by decreased expression of NMDA or AMPA receptors selectively on FSIs reduces both capacity for synchronized gamma oscillations and cognitive performance [41,42]. In addition, gamma oscillations can be promoted by pacing FSIs utilizing optogenetic technology, whereby light-sensitive ion channels are introduced into neurons, allowing spatial and temporal control of neuronal activity in a transient manner. By contrast, optogenetically paced pyramidal cells in the gamma range do not induce gamma oscillations, supporting the paramount importance of FSIs in driving gamma oscillations [54].
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Figure I. The depolarizing currents of action potentials in parvalbumin-expressing FSIs are mainly mediated by the sodium channel NaV1.1. (A) It is thought that under normal physiological conditions, gamma oscillations are mainly generated by local excitatory signals from pyramidal cells (green) to FSIs (blue), which in turn send inhibitory signals back to pyramidal cells. Activated interneurons can efficiently back-propagate synchronizing inhibitory signals to multiple pyramidal cells. In fact, it has been estimated that a single FSI has afferent or efferent contact with more than 1500 pyramidal cells [55]. In the absence of pathology, during synchronized gamma oscillations, pyramidal cells fire action potentials phase-locked to the field oscillation (here, top of the oscillation), whereas FSIs fire subsequent action potentials phaselocked to the field oscillation (here, descending part of the oscillation; lower panel). (B) It is hypothesized that during pathological conditions such as loss-of-function mutations of NaV1.1 channels or decreased FSI function, the phase-locked firing pattern of pyramidal cells and FSI is compromised such that the firing pattern of both cellular types is more diffuse. However, the reduced inhibitory tone (e.g., in SCN1A haploinsufficiency) leads to hyperexcitability and increased firing of pyramidal cells. Consequently, reduced phase-locking of the firing pattern leads to lower-amplitude gamma oscillations. Interestingly, evoked gamma oscillations of lower amplitude seems to be particularly evident in schizophrenia. (C) It is hypothesized that an NaV1.1 activator recovers decreased function of FSIs and thus re-establishes the highly phase-locked firing pattern between pyramidal cells and FSIs, thereby reducing hyperexcitability and allowing normal-amplitude gamma oscillations.
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Opinion Schizophrenia Growing evidence also suggests a deficit in fast-spiking GABAergic interneurons and gamma oscillations as an important pathophysiological feature in schizophrenia. Reductions in parvalbumin levels have been found in postmortem tissues from patients suffering from schizophrenia [43] and in animal models of schizophrenia [44], Moreover, decreased axon terminal density of the chandelier subtype of interneurons positive for GABA membrane transporter (GAT-1) has also been found in postmortem studies of schizophrenic patients [45]. Collectively this points to decreased function of fast-spiking GABAergic interneurons in schizophrenia. It could be hypothesized that compromised function of fast-spiking GABAergic interneurons would reduce high-frequency oscillations in the gamma range. Reduced gamma oscillations have repeatedly been reported in schizophrenia, particularly during processing of sensory stimuli [46]. Taken together with the extensive literature on deficient cognitive performance in schizophrenia, this raises the possibility that recovery of interneuron function by a NaV1.1 activator might have beneficial effects on cognitive performance in schizophrenic patients. Pharmacological tools for NaV1.1 channels To the best of our knowledge, no claims or descriptions have been made for specific and selective NaV1.1 activators to date. However, different classes of general sodium channel activators are well known, including the pyrethroid insecticides (as exemplified by allethrin and deltamethrin [6]), alkaloid-based toxins including veratridine and batrachotoxin, and a number of peptide toxins isolated from diverse organisms such as scorpions, sea anemones, and spiders [47]. These classes of activators are known for their propensity to induce toxic effects in mammals and do not represent starting points for drug development or readily usable tools for validating the concept of NaV1.1 activation in a systemic setting. Nevertheless, the effects of non-selective sodium channel activators have been tested on brain slices to examine how these may affect neuronal firing rates. Low micromolar concentrations of veratridine dramatically increase the intrinsic neuronal excitability of hippocampal CA1 neurons [48] and b-pompilidotoxin affects firing rates of hippocampal pyramidal interneurons [49]. These data imply that different activation mechanisms of sodium channels can increase neuronal excitability. However, to avoid general overexcitation and epileptiform activity, as well as adverse cardiac effects, selectivity to NaV1.1 will be of paramount importance. Within the pyrethroid class of compounds, differentiation between NaV subtypes has been described: NaV1.2 and NaV1.7 are considered relatively insensitive to pyrethroid modulation, in contrast to the higher sensitivity of NaV1.3, NaV1.6, and NaV1.8 subtypes [6]. Inhibitors of sodium channels have long been used as therapeutic agents indicated for the treatment of epilepsy, pain, arrhythmia, anesthesia, bipolar disorders, migraine, post hepatic and trigeminal neuralgia, and Parkinson’s disease (PD). Blocking of sodium channels may be conceptually more simple and straightforward than activation of channel function because inhibition can be obtained via a simple pore block. Activation must rely on an ability to 116
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interfere with gating to increase channel opening probability or to prevent or decrease channel inactivation. However, most inhibitors of sodium channels differ from simple pore blockers such as tetrodotoxin (TTX) by virtue of their greater affinity for the inactivate channel conformation over the resting- and open-state conformation (a phenomenon termed state dependence). This is believed to be important for separation of efficacy from side effects for molecules such as TTX [50]. Furthermore, subtype selectivity of 100-fold has been reported for channel inhibitors [51], proving that subtype selectivity can be obtained and could potentially also be feasible for activators. Screening considerations for identification of NaV1.1 channel activators A broad range of different positive modulators of macroscopic sodium currents can be envisioned: increasing the peak current, increasing the activation kinetics, slowing the inactivation kinetics, left-shifting the voltage dependence of activation causing channels to activate at more negative potentials, shifting the voltage dependence of inactivation allowing more window current [52], and increasing the sustained current (Figure 1). Because current data suggest that NaV1.1 is the major sodium channel in fast-spiking GABAergic interneurons, it would appear rational that the same functional selectivity (i.e., preferential modulation of more active neurons over less active neurons as hypothesized for state-dependent channel inhibitors [50]) could also play a role for NaV1.1 activators. However, this is still speculative. In addition, owing to the lack of data for the effects of diverse activator profiles on neuronal function, qualification of what profile may be most effective will be speculative (Figure 2). From a theoretical perspective, it could be argued that affecting the activation properties or the voltage sensor would be beneficial because sequence discrepancy between NaV1.X channels is highest in this area, thereby suggesting potential selective activation of
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Figure 1. Hypothetical activator profiles. Multiple possible activator profiles for sodium channels can be envisioned. The figure gives a hypothetical representation of compounds that (A) potentiate the peak current, (B) slow the inactivation kinetics, (C) increase the activation kinetics, and (D) increase the sustained current. Control traces are shown in red and traces for the hypothetical compounds in blue. The scale bar corresponds to 1 ms. Red,control; blue, hypothetical activator.
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Figure 2. Hypothetical effects of compounds on NaV1.1 channel properties. (A) Drugs interacting with the voltage sensor may induce a left-shift in the voltage-dependent activation of NaV1.1 relative to a control. The I–V plot shows the peak current amplitude of NaV1.1 as a function of the holding potential. Such a drug would induce channel activation at lower membrane potentials and thus lower the threshold for action potential firing. (B) Change in voltage-dependent activation but not fast inactivation by a hypothetical compound shown as a Boltzmann plot of the change in the fraction of channels activated at different voltages. (C) Voltage dependence of activation and fast inactivation depicted by as a hypothetical Boltzmann plot in the presence of a compound slowing fast inactivation of NaV1.1 without affecting the voltage-dependent activation. Such a compound would extend the voltage range of sodium channel re-activation without inactivation (i.e., broader window current) and longer channel opening, favoring depolarization. (D) Theoretical compound effect on the sustained (persistent) current of NaV1.1 caused by incomplete inactivation. Such a compound effect would also be expected to increase neuronal excitability. Red, control; blue, hypothetical activator.
NaV1.1. However, at this stage the feasibility of such an approach is only speculative. Pivotal experiments to address this issue must investigate which of the different activator profiles would have highest propensity to restore interneuron firing, because this will determine the disease-alleviating properties of an NaV1.1 activator. In the context of identifying novel activators of sodium channels, recent advances in automated electrophysiology equipment (such as the Molecular Device IonWorks Barracuda, the Sophion Qube, and the Nanion Synchropath 384) have allowed electrophysiological screening campaigns of several thousand compounds with reasonable speed and cost. Given the infancy of the NaV1.1 activator field and the low level of data guiding how specific activator profiles may be linked to efficacy, a rational screening approach could involve detection of many diverse profiles using a voltage protocol facilitating identification of compounds with as diverse functional profiles as possible (Figure 2). This would allow identification of compounds
for subsequent experimental qualification of the activator profile with the most efficient effect on the function of fastspiking interneurons. Concluding remarks We discussed growing evidence that increasing fast-spiking GABAergic interneuron function might represent a therapeutic strategy for diverse CNS diseases, and therefore pharmacological activators of NaV1.1 channels could have large therapeutic potential in the treatment of epilepsy, cognitive dysfunction associated with AD, and schizophrenia. For this vision to be fulfilled, a number of key questions remain to be pursued. Is it possible to obtain NaV1.1 selective compounds and if so what degree of selectivity is necessary to prevent general hyperexcitability? For a given selective NaV1.1 compound, what is the maximal therapeutic window for overactive interneurons and potential sedative effects? What other parameters add to adverse effects and thereby reduce the therapeutic 117
Opinion applicability of NaV1.1 activators? Such key questions, together with more typical developmental assessment of the druggability of NaV1.1 channel activators, are still to be answered before we know if activation of these channels constitutes a novel and innovative therapeutic approach. References 1 Chen, C. et al. (2004) Mice lacking sodium channel beta1 subunits display defects in neuronal excitability, sodium channel expression, and nodal architecture. J. Neurosci. 24, 4030–4042 2 Ogiwara, I. et al. (2013) Nav1.1 haploinsufficiency in excitatory neurons ameliorates seizure-associated sudden death in a mouse model of Dravet syndrome. Hum. Mol. Genet. 22, 4784–4804 3 Roden, D.M. (1998) Taking the ‘idio’ out of ‘idiosyncratic’: predicting torsades de pointes. Pacing Clin. Electrophysiol. 21, 1029–1034 4 Yu, F.H. et al. (2006) Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. Nat. Neurosci. 9, 1142–1149 5 Catterall, W.A. et al. (2005) International Union of Pharmacology. XLVII. Nomenclature and structure–function relationships of voltage-gated sodium channels. Pharmacol. Rev. 57, 397–409 6 Soderlund, D.M. (2012) Molecular mechanisms of pyrethroid insecticide neurotoxicity: recent advances. Arch. Toxicol. 86, 165–181 7 Oakley, J.C. et al. (2011) Insights into pathophysiology and therapy from a mouse model of Dravet syndrome. Epilepsia 52 (Suppl. 2), 59–61 8 Claes, L.R. et al. (2009) The SCN1A variant database: a novel research and diagnostic tool. Hum. Mutat. 30, E904–E920 9 Lossin, C. (2009) A catalog of SCN1A variants. Brain Dev. 31, 114–130 10 Catterall, W.A. et al. (2010) Nav1.1 channels and epilepsy. J. Physiol. 588, 1849–1859 11 Guerrini, R. (2012) Dravet syndrome: the main issues. Eur. J. Paediatr. Neurol. 16 (Suppl. 1), S1–S4 12 Catterall, W.A. (2012) Voltage-gated sodium channels at 60: structure, function and pathophysiology. J. Physiol. 590, 2577–2589 13 Vassilev, P.M. et al. (1988) Identification of an intracellular peptide segment involved in sodium channel inactivation. Science 241, 1658– 1661 14 Noda, M. et al. (1986) Expression of functional sodium channels from cloned cDNA. Nature 322, 826–828 15 Goldin, A.L. et al. (1986) Messenger RNA coding for only the alpha subunit of the rat brain Na channel is sufficient for expression of functional channels in Xenopus oocytes. Proc. Natl. Acad. Sci. U.S.A. 83, 7503–7507 16 Isom, L.L. et al. (1992) Primary structure and functional expression of the beta 1 subunit of the rat brain sodium channel. Science 256, 839–842 17 Isom, L.L. et al. (1995) Structure and function of the beta 2 subunit of brain sodium channels, a transmembrane glycoprotein with a CAM motif. Cell 83, 433–442 18 Mantegazza, M. et al. (2010) Voltage-gated sodium channels as therapeutic targets in epilepsy and other neurological disorders. Lancet Neurol. 9, 413–424 19 Hirn, C. et al. (2008) Nav1.4 deregulation in dystrophic skeletal muscle leads to Na+ overload and enhanced cell death. J. Gen. Physiol. 132, 199–208 20 Vincent, G.M. (1998) The molecular genetics of the long QT syndrome: genes causing fainting and sudden death. Annu. Rev. Med. 49, 263–274 21 Black, J.A. et al. (1996) Spinal sensory neurons express multiple sodium channel alpha-subunit mRNAs. Brain Res. Mol. Brain Res. 43, 117–131 22 Akopian, A.N. et al. (1996) A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons. Nature 379, 257–262 23 Dib-Hajj, S.D. et al. (1998) NaN, a novel voltage-gated Na channel, is expressed preferentially in peripheral sensory neurons and downregulated after axotomy. Proc. Natl. Acad. Sci. U.S.A. 95, 8963–8968 24 Yan, G.X. and Antzelevitch, C. (1999) Cellular basis for the Brugada syndrome and other mechanisms of arrhythmogenesis associated with ST-segment elevation. Circulation 100, 1660–1666 25 Cox, J.J. et al. (2006) An SCN9A channelopathy causes congenital inability to experience pain. Nature 444, 894–898 26 Weiss, L.A. et al. (2003) Sodium channels SCN1A, SCN2A and SCN3A in familial autism. Mol. Psychiatry 8, 186–194
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27 Dichgans, M. et al. (2005) Mutation in the neuronal voltage-gated sodium channel SCN1A in familial hemiplegic migraine. Lancet 366, 371–377 28 O’Roak, B.J. et al. (2011) Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations. Nat. Genet. 43, 585–589 29 Kalume, F. et al. (2013) Sudden unexpected death in a mouse model of Dravet syndrome. J. Clin. Invest. 123, 1798–1808 30 Han, S. et al. (2012) Autistic-like behaviour in Scn1a+/ mice and rescue by enhanced GABA-mediated neurotransmission. Nature 489, 385–390 31 Kalume, F. et al. (2007) Reduced sodium current in Purkinje neurons from Nav1.1 mutant mice: implications for ataxia in severe myoclonic epilepsy in infancy. J. Neurosci. 27, 11065–11074 32 Andrioli, A. et al. (2007) Quantitative analysis of parvalbuminimmunoreactive cells in the human epileptic hippocampus. Neuroscience 149, 131–143 33 Liu, Y. et al. (2013) Dravet syndrome patient-derived neurons suggest a novel epilepsy mechanism. Ann. Neurol. 74, 128–139 34 Jiao, J. et al. (2013) Modeling Dravet syndrome using induced pluripotent stem cells (iPSCs) and directly converted neurons. Hum. Mol. Genet. 22, 4241–4252 35 Brunklaus, A. et al. (2012) Prognostic, clinical and demographic features in SCN1A mutation-positive Dravet syndrome. Brain 135, 2329–2336 36 Bender, A.C. et al. (2013) Focal Scn1a knockdown induces cognitive impairment without seizures. Neurobiol. Dis. 54, 297–307 37 Papassotiropoulos, A. et al. (2011) A genome-wide survey of human short-term memory. Mol. Psychiatry 16, 184–192 38 Verret, L. et al. (2012) Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model. Cell 149, 708–721 39 Kim, D.Y. et al. (2011) Reduced sodium channel Nav1.1 levels in BACE1-null mice. J. Biol. Chem. 286, 8106–8116 40 Kim, D.Y. et al. (2007) BACE1 regulates voltage-gated sodium channels and neuronal activity. Nat. Cell Biol. 9, 755–764 41 Carlen, M. et al. (2012) A critical role for NMDA receptors in parvalbumin interneurons for gamma rhythm induction and behavior. Mol. Psychiatry 17, 537–548 42 Fuchs, E.C. et al. (2007) Recruitment of parvalbumin-positive interneurons determines hippocampal function and associated behavior. Neuron 53, 591–604 43 Hashimoto, T. et al. (2003) Gene expression deficits in a subclass of GABA neurons in the prefrontal cortex of subjects with schizophrenia. J. Neurosci. 23, 6315–6326 44 Pratt, J.A. et al. (2008) Modelling prefrontal cortex deficits in schizophrenia: implications for treatment. Br. J. Pharmacol. 153 (Suppl. 1), S465–S470 45 Pierri, J.N. et al. (1999) Alterations in chandelier neuron axon terminals in the prefrontal cortex of schizophrenic subjects. Am. J. Psychiatry 156, 1709–1719 46 Basar, E. (2013) A review of gamma oscillations in healthy subjects and in cognitive impairment. Int. J. Psychophysiol. 90, 11–117 47 Catterall, W.A. et al. (2007) Voltage-gated ion channels and gating modifier toxins. Toxicon 49, 124–141 48 Otoom, S. et al. (1998) Veratridine-treated brain slices: a cellular model for epileptiform activity. Brain Res. 789, 150–156 49 Miyawaki, T. et al. (2002) Differential effects of novel wasp toxin on rat hippocampal interneurons. Neurosci. Lett. 328, 25–28 50 Nardi, A. et al. (2012) Advances in targeting voltage-gated sodium channels with small molecules. ChemMedChem 7, 1712–1740 51 England, S. and de Groot, M.J. (2009) Subtype-selective targeting of voltage-gated sodium channels. Br. J. Pharmacol. 158, 1413–1425 52 Attwell, D. et al. (1979) The steady state TTX-sensitive (‘window’) sodium current in cardiac Purkinje fibres. Pflugers Arch. 379, 137–142 53 Ogiwara, I. et al. (2007) Nav1.1 localizes to axons of parvalbuminpositive inhibitory interneurons: a circuit basis for epileptic seizures in mice carrying an Scn1a gene mutation. J. Neurosci. 27, 5903–5914 54 Cardin, J.A. et al. (2009) Driving fast-spiking cells induces gamma rhythm and controls sensory responses. Nature 459, 663–667 55 Gulyas, A.I. et al. (1999) Total number and ratio of excitatory and inhibitory synapses converging onto single interneurons of different types in the CA1 area of the rat hippocampus. J. Neurosci. 19, 10082– 10097
Therapeutic potential of NaV1.1 activators Henrik S. Jensen*, Morten Grunnet*, and Jesper F. Bastlund Neuroscience Drug Discovery, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Copenhagen, Denmark
Sodium channel inhibitors have been developed and approved as drugs to treat a variety of indications. By contrast, sodium channel activators have not previously been considered relevant in a therapeutic setting owing to their high risk of toxicity and side effects. Here we present an opinion that selective activators of the NaV1.1 sodium channel may hold therapeutic potential for diseases such as epilepsy, schizophrenia, and Alzheimer’s disease. Central to this novel avenue of sodium channel drug discovery is that fact that NaV1.1 comprises the majority of the sodium current in specific inhibitory interneurons. Conversely, it plays only a modest role in excitatory neurons owing to the high redundancy of other types of sodium channels in these cells. We discuss the biological background and rationale and present reflections on how to identify activators of NaV1.1. Modulation of sodium channel function as a therapeutic strategy Emerging data highlight the important role of interneurons in both health and disease. Interneurons are inhibitory in nature because they synthesize and release GABA, the major inhibitory neurotransmitter in the brain. They have important roles in the central nervous system (CNS) in regulating excitability and securing synchronized activity of neuronal populations. Interneurons can be divided into different subclasses according to their neurochemical markers, connectivity, and physiological properties. In the fastspiking, parvalbumin-expressing subclass of inhibitory neurons, the sodium current is mainly carried by one type of sodium channel, namely the NaV1.1 channel. This is in contrast to excitable cells, such as hippocampal pyramidal cells, in which NaV1.1 channels are also expressed [1,2] but are co-expressed with additional subtypes of NaV1.X channels. We could thus consider excitable cells as potentially having a depolarization reserve, in analogy to the cardiac repolarization reserve [3], whereby different NaV1.X channel subtypes can substitute for each other in creating depolarizing current and thus initiating action potentials. Corresponding author: Jensen, H.S. ([email protected]). Keywords: interneurons; cognition; oscillations; epilepsy; connectivity SCN1A mutations; schizophrenia. * These authors contributed equally to this work. 0165-6147/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tips.2013.12.007
However, this depolarization reserve is not present in parvalbumin interneurons, where NaV1.1 channels are essential for action potential generation [4]. It is firmly established that sodium channels play a pivotal role in neuronal excitability, and to date, nine genes have been described (SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A, and SCN11A) that encode the voltage-gated NaV1.X channels NaV1.1– NaV1.9, respectively. In addition, a closely related Na+ channel-like protein has been cloned but not functionally expressed [5]. Voltage-gated sodium channels have been the focus of an immense body of research that includes both basic science and drug discovery activities. Inhibitors of sodium channels are currently approved and indicated for the treatment of pain, epilepsy, and cardiac arrhythmia. By contrast, activators of voltage-gated sodium channels have not been utilized as drugs, probably because complete pan-selective activation of human NaV channels induces seizures with no therapeutic window for efficacy. Sodium channel activators targeting insect channels have, however, been explored and are being used as insecticides [6]. However, because NaV1.1 is by far the most important sodium channel for initiation of action potentials in the fast-spiking subtype of GABAergic interneurons, selective activation of NaV1.1 might constitute a new potential treatment paradigm. In support of this idea, transgenic mouse models with reduced NaV1.1 expression have been associated with various diseases [7]. Furthermore, in a human context, loss-of-function mutations in the SCNA1 gene encoding NaV1.1 have been linked to a spectrum of seizure disorders [8–11]. In this opinion article, we give a brief account of the pathology associated with reduced NaV1.1 channel function and discuss how drugmediated increases in NaV1.1 function might be beneficial as a consequence of better overall interneuron function and thereby overall synchronization of neuronal networks. We also outline possible screening approaches and considerations for identifying NaV1.1 channel activators. Voltage-gated Na+ channels Voltage-gated Na+ channels are transmembrane (TM) proteins consisting of a single pore-forming a-subunit with 24 TM segments. These are divided into four relatively homogeneous domains of six TMs with the voltage sensor located in TM4 and the pore-forming domain situated between the fifth and sixth TM. The linkers between the four domains vary in length and have important functions in channel modulation, inactivation, and drug binding [12]. All voltage-gated Na+ channels are closed at the resting hyperpolarized membrane potential. Depolarization of the Trends in Pharmacological Sciences, March 2014, Vol. 35, No. 3
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Opinion membrane potential induces a conformational change in the channel that allows selective conduction of Na+ ions in an inward direction dictated by the electrochemical gradient. Voltage-gated Na+ channels are further characterized by fast inactivation that changes the channels to a non-conducting state [13]. The pore-forming a-subunit is able to constitute a fully functional conducting ion channel but biophysical properties can be affected by accessory b-subunits [14,15]. The b-subunits are one-TM proteins with immunoglobulin-like N-terminal extracellular structures. A sodium ion channel complex is typically a heterodimeric or heterotrimeric structure with a:b stoichiometry of 1:1 or 1:2 [16,17]. The voltage-gated Na+ channels NaV1.1–1.9 are all expressed in excitable tissue found in the CNS, the peripheral nervous system (PNS), and the heart [18]. The tenth member, Nax, is not as well characterized but might play a function in Na+ homeostasis [5]. Expression of NaV1.X channels is developmentally regulated, and NaV1.1, 1.2, 1.3, and 1.6 are the most predominant subtypes in CNS, with NaV1.6 being the most abundant subtype in the adult brain [18]. NaV1.4 is expressed in skeletal muscle [19] and NaV1.5 is the major cardiac sodium channel [20], whereas NaV1.7, 1.8, and 1.9 are important players in nociceptive signaling transduction owing to their presence in peripheral primary sensory afferents [21–23]. Voltage-gated Na+ channels in diseases It is generally accepted that hyperactivity of NaV1.X channels is associated with pathophysiological conditions due to inappropriate regulation of neuronal action potential firing and thereby excessive activity. Multiple NaV1.X channel inhibitors have been developed as anti-epileptic and antiarrhythmic drugs and are widely used in clinical applications. However, there are exceptions to this general notion of beneficial inhibition of NaV1.X channels. Examples are NaV1.5 loss-of-function mutations causing Brugada syndrome, a potentially life-threatening heart rhythm disorder that can develop into ventricular fibrillation and ultimately sudden cardiac arrest [24], congenital indifference to pain due to loss of function of NaV1.7 channels [25], and, most notably, loss of function of NaV1.1 channels associated with various diseases, such as familial hemiplegic migraine, familial autism, and sporadic autism spectrum disorder [8,9,26–28]. However, the best-characterized understanding of reduced NaV1.1 function and a disease phenotype stems from patients with Dravet syndrome, which represents a genetic link between the SCN1A gene (encoding NaV1.1) and a disease. Patients with Dravet syndrome are primarily characterized by multiple epilepsy phenotypes such as convulsive seizures and recurrent periods of status epilepticus. In childhood, seizures are often precipitated by fever. Following the onset of seizures, cognitive impairment, mental retardation, behavioral abnormalities, and autistic traits can occur. The febrile seizures tend to ameliorate in early childhood, but afebrile seizures can last beyond childhood [11]. In addition, mouse models of reduced NaV1.1 channel expression have revealed phenotypes such as ataxia, severe myoclonic epilepsy, autistic-like behaviors, cognitive deficits, and sudden unexpected death [4,29–31]. Thus, a number of diseases, including several in humans, have been associated with loss 114
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of function (haploinsufficiency) in NaV1.1 channels. Although the mutations identified in NaV1.1 do not share a completely identical phenotype, we speculate that an innovative approach to ameliorating symptoms associated with these diseases could involve pharmacological restoration of normal function for NaV1.1 channels. NaV1.1: Functional role and therapeutic potential Epilepsy It is established that reduced NaV1.1 channel function has a large impact on the excitability of neuronal networks because multiple heterozygous loss-of-function mutations have been associated with epilepsies such as Dravet syndrome. Importantly, the magnitude of reduced NaV1.1 channel function seems to correlate directly to the severity of the epileptic phenotype [10]. Not all Dravet patients have SCN1A mutations, although the association is strong, because an estimated 85% of patients carry SCN1A mutations [11]. As mentioned above, NaV1.1 channels carry the main sodium current in parvalbumin-containing, fastspiking GABAergic interneuron subtypes and are pivotal for the generation of action potentials and thus continuous excitability of these neurons. Consequently, selective increases in NaV1.1 channel function might hold the potential to increase the function of fast-spiking GABAergic interneurons and thus affect overall excitability in the CNS. Pharmacological activation of NaV1.1 channels is therefore an obvious suggestion as a treatment paradigm for patients with SCN1A haploinsufficiency and other disorders with impaired functionality of fast-spiking parvalbumin interneurons. NaV1.1 activators might also have therapeutic potential in the treatment of symptomatic epilepsies such as temporal lobe epilepsy, for which partial loss of fast-spiking GABAergic interneurons has been reported as an early pathological hallmark [32]. Importantly, SCN1A+/ mice have been developed that capture the epileptic phenotype of patients with SCN1A haploinsufficiency [4]. This model can aid in further exploration of NaV1.1 activators as therapeutics for epileptic syndromes. Recent experiments with induced pluripotent stem cells (iPSCs) derived from two Dravet patients revealed increased expression of NaV1.1 channels in neurons derived from differentiated iPSCs [33,34]. Whether such observations mirror the true patient phenotype or are a consequence of the iPSC differentiation procedure still remains to be addressed. In the context of these two studies, it should be noted that seizure worsening has been reported for Dravet’s patients treated with sodium with pan-selective sodium channel inhibitors [35]. However, as for all drug development, a thorough safety assessment during the development process would be required for future NaV1.1 activators, which present some challenges (see below). Cognitive deficits and Alzheimer’s disease (AD) SCN1A+/ mice also exhibit non-seizure deficits that are found in human mutation carriers, such as reduced cognitive performance [30]. This raises the question whether reduced NaV1.1 channel function directly affects cognitive processing or if the effect is secondary (i.e., a consequence of repeated seizures). Local targeted reductions in NaV1.1
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but may be related to increased activity of b-site APPcleaving enzyme (BACE), because it has been reported that BACE regulates NaV1.1 expression [39,40]. Importantly, crossing of J20 mice with NaV1.1-overexpressing mice partially normalizes the phenotype in these mice and recovers deficient gamma oscillations and cognitive performance [38], suggesting a therapeutic potential of NaV1.1 activators for cognitive disturbance associated with AD. The mechanistic importance of fast-spiking GABAergic interneurons, and thus of NaV1.1 channels, in cognition might rely on the ability to affect synchronization of fast oscillations in the gamma range (30–100 Hz) (Box 1). Importantly, it has been demonstrated that increased interneuron function and gamma oscillations have a positive effect on cognitive performance in rodents [41,42].
expression in parvalbumin-positive interneurons in the medial septum revealed impaired cognitive performance in these non-epileptic animals [36]. Thus, cognitive deficits might also be a primary consequence of reduced NaV1.1 expression. Further evidence supporting this notion was provided by a study demonstrating reduced short-term memory in apparently healthy individuals carrying SCNA1 mutations, which suggests a therapeutic potential for NaV1.1 activators as pro-cognitive therapy [37]. In relation to this, reduced NaV1.1 expression has also been reported in humans suffering from AD and in J20 mice [38], which are carriers of amyloid precursor protein (APP) mutations found in familial forms of AD. The mechanism by which NaV1.1 expression is reduced in Alzheimerrelated pathological conditions is not fully characterized,
Box 1. Impact of NaV1.1 channels on fast-spiking interneuron function and gamma oscillations Fast-spiking GABAergic interneurons (FSIs; Figure I) that are immunoreactive for the calcium-binding protein parvalbumin are crucial for synchronized high-frequency neuronal activity in the gamma range (30–100 Hz) as measured by the local field potential (LFP). This FSI class shows high expression of NaV1.1 [53] and can be further subdivided according to morphological characteristics into chandelier and basket cells. However, here we mainly consider the broader FSI class, because this is where NaV1.1 expression is reported. Extensive research documents the dominant role of FSIs in synchronization of gamma oscillations, and a reduction in
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excitatory drive to FSIs by decreased expression of NMDA or AMPA receptors selectively on FSIs reduces both capacity for synchronized gamma oscillations and cognitive performance [41,42]. In addition, gamma oscillations can be promoted by pacing FSIs utilizing optogenetic technology, whereby light-sensitive ion channels are introduced into neurons, allowing spatial and temporal control of neuronal activity in a transient manner. By contrast, optogenetically paced pyramidal cells in the gamma range do not induce gamma oscillations, supporting the paramount importance of FSIs in driving gamma oscillations [54].
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Figure I. The depolarizing currents of action potentials in parvalbumin-expressing FSIs are mainly mediated by the sodium channel NaV1.1. (A) It is thought that under normal physiological conditions, gamma oscillations are mainly generated by local excitatory signals from pyramidal cells (green) to FSIs (blue), which in turn send inhibitory signals back to pyramidal cells. Activated interneurons can efficiently back-propagate synchronizing inhibitory signals to multiple pyramidal cells. In fact, it has been estimated that a single FSI has afferent or efferent contact with more than 1500 pyramidal cells [55]. In the absence of pathology, during synchronized gamma oscillations, pyramidal cells fire action potentials phase-locked to the field oscillation (here, top of the oscillation), whereas FSIs fire subsequent action potentials phaselocked to the field oscillation (here, descending part of the oscillation; lower panel). (B) It is hypothesized that during pathological conditions such as loss-of-function mutations of NaV1.1 channels or decreased FSI function, the phase-locked firing pattern of pyramidal cells and FSI is compromised such that the firing pattern of both cellular types is more diffuse. However, the reduced inhibitory tone (e.g., in SCN1A haploinsufficiency) leads to hyperexcitability and increased firing of pyramidal cells. Consequently, reduced phase-locking of the firing pattern leads to lower-amplitude gamma oscillations. Interestingly, evoked gamma oscillations of lower amplitude seems to be particularly evident in schizophrenia. (C) It is hypothesized that an NaV1.1 activator recovers decreased function of FSIs and thus re-establishes the highly phase-locked firing pattern between pyramidal cells and FSIs, thereby reducing hyperexcitability and allowing normal-amplitude gamma oscillations.
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Opinion Schizophrenia Growing evidence also suggests a deficit in fast-spiking GABAergic interneurons and gamma oscillations as an important pathophysiological feature in schizophrenia. Reductions in parvalbumin levels have been found in postmortem tissues from patients suffering from schizophrenia [43] and in animal models of schizophrenia [44], Moreover, decreased axon terminal density of the chandelier subtype of interneurons positive for GABA membrane transporter (GAT-1) has also been found in postmortem studies of schizophrenic patients [45]. Collectively this points to decreased function of fast-spiking GABAergic interneurons in schizophrenia. It could be hypothesized that compromised function of fast-spiking GABAergic interneurons would reduce high-frequency oscillations in the gamma range. Reduced gamma oscillations have repeatedly been reported in schizophrenia, particularly during processing of sensory stimuli [46]. Taken together with the extensive literature on deficient cognitive performance in schizophrenia, this raises the possibility that recovery of interneuron function by a NaV1.1 activator might have beneficial effects on cognitive performance in schizophrenic patients. Pharmacological tools for NaV1.1 channels To the best of our knowledge, no claims or descriptions have been made for specific and selective NaV1.1 activators to date. However, different classes of general sodium channel activators are well known, including the pyrethroid insecticides (as exemplified by allethrin and deltamethrin [6]), alkaloid-based toxins including veratridine and batrachotoxin, and a number of peptide toxins isolated from diverse organisms such as scorpions, sea anemones, and spiders [47]. These classes of activators are known for their propensity to induce toxic effects in mammals and do not represent starting points for drug development or readily usable tools for validating the concept of NaV1.1 activation in a systemic setting. Nevertheless, the effects of non-selective sodium channel activators have been tested on brain slices to examine how these may affect neuronal firing rates. Low micromolar concentrations of veratridine dramatically increase the intrinsic neuronal excitability of hippocampal CA1 neurons [48] and b-pompilidotoxin affects firing rates of hippocampal pyramidal interneurons [49]. These data imply that different activation mechanisms of sodium channels can increase neuronal excitability. However, to avoid general overexcitation and epileptiform activity, as well as adverse cardiac effects, selectivity to NaV1.1 will be of paramount importance. Within the pyrethroid class of compounds, differentiation between NaV subtypes has been described: NaV1.2 and NaV1.7 are considered relatively insensitive to pyrethroid modulation, in contrast to the higher sensitivity of NaV1.3, NaV1.6, and NaV1.8 subtypes [6]. Inhibitors of sodium channels have long been used as therapeutic agents indicated for the treatment of epilepsy, pain, arrhythmia, anesthesia, bipolar disorders, migraine, post hepatic and trigeminal neuralgia, and Parkinson’s disease (PD). Blocking of sodium channels may be conceptually more simple and straightforward than activation of channel function because inhibition can be obtained via a simple pore block. Activation must rely on an ability to 116
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interfere with gating to increase channel opening probability or to prevent or decrease channel inactivation. However, most inhibitors of sodium channels differ from simple pore blockers such as tetrodotoxin (TTX) by virtue of their greater affinity for the inactivate channel conformation over the resting- and open-state conformation (a phenomenon termed state dependence). This is believed to be important for separation of efficacy from side effects for molecules such as TTX [50]. Furthermore, subtype selectivity of 100-fold has been reported for channel inhibitors [51], proving that subtype selectivity can be obtained and could potentially also be feasible for activators. Screening considerations for identification of NaV1.1 channel activators A broad range of different positive modulators of macroscopic sodium currents can be envisioned: increasing the peak current, increasing the activation kinetics, slowing the inactivation kinetics, left-shifting the voltage dependence of activation causing channels to activate at more negative potentials, shifting the voltage dependence of inactivation allowing more window current [52], and increasing the sustained current (Figure 1). Because current data suggest that NaV1.1 is the major sodium channel in fast-spiking GABAergic interneurons, it would appear rational that the same functional selectivity (i.e., preferential modulation of more active neurons over less active neurons as hypothesized for state-dependent channel inhibitors [50]) could also play a role for NaV1.1 activators. However, this is still speculative. In addition, owing to the lack of data for the effects of diverse activator profiles on neuronal function, qualification of what profile may be most effective will be speculative (Figure 2). From a theoretical perspective, it could be argued that affecting the activation properties or the voltage sensor would be beneficial because sequence discrepancy between NaV1.X channels is highest in this area, thereby suggesting potential selective activation of
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Figure 1. Hypothetical activator profiles. Multiple possible activator profiles for sodium channels can be envisioned. The figure gives a hypothetical representation of compounds that (A) potentiate the peak current, (B) slow the inactivation kinetics, (C) increase the activation kinetics, and (D) increase the sustained current. Control traces are shown in red and traces for the hypothetical compounds in blue. The scale bar corresponds to 1 ms. Red,control; blue, hypothetical activator.
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Figure 2. Hypothetical effects of compounds on NaV1.1 channel properties. (A) Drugs interacting with the voltage sensor may induce a left-shift in the voltage-dependent activation of NaV1.1 relative to a control. The I–V plot shows the peak current amplitude of NaV1.1 as a function of the holding potential. Such a drug would induce channel activation at lower membrane potentials and thus lower the threshold for action potential firing. (B) Change in voltage-dependent activation but not fast inactivation by a hypothetical compound shown as a Boltzmann plot of the change in the fraction of channels activated at different voltages. (C) Voltage dependence of activation and fast inactivation depicted by as a hypothetical Boltzmann plot in the presence of a compound slowing fast inactivation of NaV1.1 without affecting the voltage-dependent activation. Such a compound would extend the voltage range of sodium channel re-activation without inactivation (i.e., broader window current) and longer channel opening, favoring depolarization. (D) Theoretical compound effect on the sustained (persistent) current of NaV1.1 caused by incomplete inactivation. Such a compound effect would also be expected to increase neuronal excitability. Red, control; blue, hypothetical activator.
NaV1.1. However, at this stage the feasibility of such an approach is only speculative. Pivotal experiments to address this issue must investigate which of the different activator profiles would have highest propensity to restore interneuron firing, because this will determine the disease-alleviating properties of an NaV1.1 activator. In the context of identifying novel activators of sodium channels, recent advances in automated electrophysiology equipment (such as the Molecular Device IonWorks Barracuda, the Sophion Qube, and the Nanion Synchropath 384) have allowed electrophysiological screening campaigns of several thousand compounds with reasonable speed and cost. Given the infancy of the NaV1.1 activator field and the low level of data guiding how specific activator profiles may be linked to efficacy, a rational screening approach could involve detection of many diverse profiles using a voltage protocol facilitating identification of compounds with as diverse functional profiles as possible (Figure 2). This would allow identification of compounds
for subsequent experimental qualification of the activator profile with the most efficient effect on the function of fastspiking interneurons. Concluding remarks We discussed growing evidence that increasing fast-spiking GABAergic interneuron function might represent a therapeutic strategy for diverse CNS diseases, and therefore pharmacological activators of NaV1.1 channels could have large therapeutic potential in the treatment of epilepsy, cognitive dysfunction associated with AD, and schizophrenia. For this vision to be fulfilled, a number of key questions remain to be pursued. Is it possible to obtain NaV1.1 selective compounds and if so what degree of selectivity is necessary to prevent general hyperexcitability? For a given selective NaV1.1 compound, what is the maximal therapeutic window for overactive interneurons and potential sedative effects? What other parameters add to adverse effects and thereby reduce the therapeutic 117
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