Disease-causing Slack potassium channel mutations produce opposite effects on excitability of excitatory and inhibitory neurons.

The KCNT1 gene encodes the sodium-activated potassium channel Slack (KCNT1, KNa1.1), a regulator of neuronal excitability. Gain-of-function mutations in humans cause cortical network hyperexcitability, seizures, and severe intellectual disability. Using a mouse model expressing the Slack-R455H mutation, we find that Na+-dependent K+ (KNa) and voltage-dependent sodium (NaV) currents are increased in both excitatory and inhibitory cortical neurons. These increased currents, however, enhance the firing of excitability neurons but suppress that of inhibitory neurons. We further show that the expression of NaV channel subunits, particularly that of NaV1.6, is upregulated and that the length of the axon initial segment and of axonal NaV immunostaining is increased in both neuron types. Our study on the coordinate regulation of KNa currents and the expression of NaV channels may provide an avenue for understanding and treating epilepsies and other neurological disorders.

New aryl and acylsulfonamides as state-dependent inhibitors of Nav1.3 voltage-gated sodium channel.

Voltage-gated sodium channels (Navs) play an essential role in neurotransmission, and their dysfunction is often a cause of various neurological disorders. The Nav1.3 isoform is found in the CNS and upregulated after injury in the periphery, but its role in human physiology has not yet been fully elucidated. Reports suggest that selective Nav1.3 inhibitors could be used as novel therapeutics to treat pain or neurodevelopmental disorders. Few selective inhibitors of this channel are known in the literature. In this work, we report the discovery of a new series of aryl and acylsulfonamides as state-dependent inhibitors of Nav1.3 channels. Using a ligand-based 3D similarity search and subsequent hit optimization, we identified and prepared a series of 47 novel compounds and tested them on Nav1.3, Nav1.5, and a selected subset also on Nav1.7 channels in a QPatch patch-clamp electrophysiology assay. Eight compounds had an IC50 value of less than 1 µM against the Nav1.3 channel inactivated state, with one compound displaying an IC50 value of 20 nM, whereas activity against the inactivated state of the Nav1.5 channel and Nav1.7 channel was approximately 20-fold weaker. None of the compounds showed use-dependent inhibition of the cardiac isoform Nav1.5 at a concentration of 30 µM. Further selectivity testing of the most promising hits was measured using the two-electrode voltage-clamp method against the closed state of the Nav1.1-Nav1.8 channels, and compound 15b displayed small, yet selective, effects against the Nav1.3 channel, with no activity against the other isoforms. Additional selectivity testing of promising hits against the inactivated state of the Nav1.3, Nav1.7, and Nav1.8 channels revealed several compounds with robust and selective activity against the inactivated state of the Nav1.3 channel among the three isoforms tested. Moreover, the compounds were not cytotoxic at a concentration of 50 µM, as demonstrated by the assay in human HepG2 cells (hepatocellular carcinoma cells). The novel state-dependent inhibitors of Nav1.3 discovered in this work provide a valuable tool to better evaluate this channel as a potential drug target.

Cerebellar spreading depolarization mediates paroxysmal movement disorder.

Paroxysmal kinesigenic dyskinesia (PKD) is the most common paroxysmal dyskinesia, characterized by recurrent episodes of involuntary movements provoked by sudden changes in movement. Proline-rich transmembrane protein 2 (PRRT2) has been identified as the major causative gene for PKD. Here, we report that PRRT2 deficiency facilitates the induction of cerebellar spreading depolarization (SD) and inhibition of cerebellar SD prevents the occurrence of dyskinetic movements. Using Ca2+ imaging, we show that cerebellar SD depolarizes a large population of cerebellar granule cells and Purkinje cells in Prrt2-deficient mice. Electrophysiological recordings further reveal that cerebellar SD blocks Purkinje cell spiking and disturbs neuronal firing of the deep cerebellar nuclei (DCN). The resultant aberrant firing patterns in DCN are tightly, temporally coupled to dyskinetic episodes in Prrt2-deficient mice. Cumulatively, our findings uncover a pivotal role of cerebellar SD in paroxysmal dyskinesia, providing a potent target for treating PRRT2-related paroxysmal disorders.

Direct evidence for high affinity blockade of NaV1.6 channel subtype by huwentoxin-IV spider peptide, using multiscale functional approaches.

The Chinese bird spider huwentoxin-IV (HwTx-IV) is well-known to be a highly potent blocker of NaV1.7 subtype of voltage-gated sodium (NaV) channels, a genetically validated analgesic target, and thus promising as a potential lead molecule for the development of novel pain therapeutics. In the present study, the interaction between HwTx-IV and NaV1.6 channel subtype was investigated using multiscale (from in vivo to individual cell) functional approaches. HwTx-IV was approximatively 2 times more efficient than tetrodotoxin (TTX) to inhibit the compound muscle action potential recorded from the mouse skeletal neuromuscular system in vivo, and 30 times more effective to inhibit nerve-evoked than directly-elicited muscle contractile force of isolated mouse hemidiaphragms. These results strongly suggest that the inhibition of nerve-evoked skeletal muscle functioning, produced by HwTx-IV, resulted from a toxin-induced preferential blockade of NaV1.6, compared to NaV1.4, channel subtype. This was confirmed by whole-cell automated patch-clamp experiments performed on human embryonic kidney (HEK)-293 cells overexpressing hNaV1.1-1.8 channel subtypes. HwTx-IV was also approximatively 850 times more efficient to inhibit TTX-sensitive than TTX-resistant sodium currents recorded from mouse dorsal root ganglia neurons. Finally, based on our data, we predict that blockade of the NaV1.6 channel subtype was involved in the in vivo toxicity of HwTx-IV, although this toxicity was more than 2 times lower than that of TTX. In conclusion, our results provide detailed information regarding the effects of HwTx-IV and allow a better understanding of the side-effect mechanisms involved in vivo and of channel subtype interactions resulting from the toxin activity.

Voltage-Gated Ion Channels in the PNS: Novel Therapies for Neuropathic Pain?

Neuropathic pain arises from injury to the nervous system. Conditions associated with neuropathic pain are diverse, and lesions and/or pathological changes in the central nervous system (CNS) or peripheral nervous system (PNS) can frequently, but not always, be identified. It is difficult to treat, with patients often on multiple, different classes of medications, all with appreciable adverse side effect profiles. Consequently, there is a pressing need for the development of new medications. The development of such therapeutics is predicated on a clear understanding of the relevant molecular and cellular processes that contribute to the development, and maintenance, of the neuropathic pain state. One proposed mechanism thought to contribute to the ontogeny of neuropathic pain is altered expression, trafficking, and functioning of ion channels expressed by primary sensory neurons. Here, we will focus on three voltage-gated ion channel families, CaV, HCN, and NaV, first reviewing the preclinical data and then the human data where it exists.

Partial transcriptomic profiling of toxins from the venom gland of the scorpion Parabuthus stridulus.

Since it is an apocrine secretion, scorpion venom is a complex mixture that contains a variety of low-molecular-weight basic proteins (neurotoxins), mucus, salts, as well as a large number of other constituents. Diversity of scorpion venom peptides exists also at the transcript level. Two kinds of venom peptides are typically considered: the neurotoxins and the antimicrobial peptides. We constructed a cDNA library and carried an EST (Expressed Sequence Tag) approach to overview the different peptides in the transcriptome of the telson from Parabuthus stridulus. P. stridulus are psammophilous and highly venomous scorpions endemic to Namibia (Prendini 2004) with medical relevance because of important human envenomation occurrence. We obtained 111 ESTs, 20% of them corresponding to cellular process transcripts, 7% to hypothetical proteins and 17% were sequences without good matches, but the majority of ESTs, 56%, corresponds to transcripts encoding for different venom components, including voltage-gated sodium, potassium and calcium channel toxins, antimicrobial peptides and other venom and cell proteins. To the best of our knowledge this report contains the first transcriptome analysis of genes transcribed by the venomous gland of the scorpion species P. stridulus, belonging to the family of medically important Buthidae scorpions. One hundred and eleven ESTs were analyzed, showing an important number of genes that encode for products similar to known scorpion venom components. In total, 17 unique and novel sequences were indentified. The identification and characterization of these compounds will be a good source of novel pharmacological tools for studying ion channels and the understanding of the physiological effects of toxins in P. stridulus envenomations at a molecular level.

Substituted 4-phenyl-2-aminoimidazoles and 4-phenyl-4,5-dihydro-2-aminoimidazoles as voltage-gated sodium channel modulators.

Voltage-gated sodium channels play an integral part in neurotransmission and their dysfunction is frequently a cause of various neurological disorders. On the basis of the structure of marine alkaloid clathrodin, twenty eight new analogs were designed, synthesized and tested for their ability to block human NaV1.3, NaV1.4 and NaV1.7 channels, as well as for their selectivity against human cardiac isoform NaV1.5, using automated patch clamp electrophysiological assay. Several compounds exhibited promising activities on different NaV channel isoforms in the medium micromolar range and some of the compounds showed also moderate isoform selectivities. The most promising results were obtained for the NaV1.3 channel, for which four compounds were found to possess IC50 values lower than 15 µM. All of the active compounds bind to the open-inactivated states of the channels and therefore act as state-dependent modulators. The obtained results validate the approach of using natural products driven chemistry for drug discovery starting points and represent a good foundation for future design of selective NaV modulators.

Two tarantula venom peptides as potent and differential Na(V) channels blockers.

Voltage dependent sodium (Na(V)) channels are large membrane spanning proteins which lie in the basis of action potential generation and propagation in excitable cells and hence are essential mediators of neuronal signaling. Inhibition of Na(V) channel activity is one of the core mechanisms to treat conditions related to neuronal hyperexcitability, such as epilepsy in the clinic. Na(V) channel blockers are also extensively used to locally inhibit action potential generation and related pain perceptions in the form of local anesthetics. Here we describe the isolation, biochemical characterization, synthesis and in vitro characterization of two potent Na(V) channel blockers from the venom of the Paraphysa scrofa (Phrixotrichus auratus) tarantula spider. Both Voltage sensor toxin 3 (VSTx-3, κ-theraphotoxin-Gr4a) and GTx1-15 (Toxin Gtx1-15), were originally isolated from the venom of the related tarantula Grammostola rosea and described as K(V) and Ca(V) channel blockers, respectively. In our hands, GTx1-15 was shown to be a potent inhibitor of tetrodotoxin (TTX)-sensitive channels (IC50 0.007 µM for hNa(V)1.7 and 0.12 µM for hNa(V)1.3 channels), with very little effect on TTX-resistant (Na(V)1.5 and NaV1.8) channels. VSTx-3 was demonstrated to be a potent, TTX-sensitive sodium channel blocker and especially, potent blocker of Na(V)1.8 channels (IC50 0.19 µM for hNa(V)1.3, 0.43 µM for hNa(V)1.7 and 0.77 µM for hNa(V)1.8 channels). Such potent inhibitors with differential selectivity among Na(V) channel isoforms may be used as tools to study the roles of the different channels in processes related to hyperexcitability and as lead compounds to treat pathological pain conditions.

Immunoglobulinfree light chains reduce in an antigen-specific manner the rate of rise of action potentials of mouse non-nociceptive dorsal root ganglion neurons.

Plasma B cells secrete immunoglobulinfree light chains (IgLC) which by binding to mast cells can mediate hypersensitivity responses and are involved in several immunological disorders. To investigate the effects of antigen-specific IgLC activation, intracellular recordings were made from cultured murine dorsal root ganglion (DRG) neurons, which can specifically bind IgLC. The neurons were sensitized with IgLC for 90min and subsequently activated by application of the corresponding antigen (DNP-HSA). Antigen application induced a decrease in the rate of rise of the action potentials of non-nociceptive neurons (MANOVA, p=2.10(-6)), without affecting the resting membrane potential or firing threshold. The action potentials of the nociceptive neurons (p=0.57) and the electrical excitability of both types of neurons (p>0.35) were not affected. We conclude that IgLC can mediate antigen-specific responses by reducing the rate of rise of action potentials in non-nociceptive murine DRG neurons. We suggest that antigen-specific activation of IgLC-sensitized non-nociceptive DRG neurons may contribute to immunological hypersensitivity responses and neuroinflammation.