The molecular interaction between local anesthetic/antiarrhythmic agents and voltage-gated sodium channels

Local anesthetic/antiarrhythmic agents render their therapeutic effects via suppression of ionic current through voltage-gated Na channels. Recent work to understand the molecular basis of this drug-receptor interaction has exploited the combined technologies of molecular biology and electrophysiology. Despite the complexity of the effects of site-directed mutations on Na channel function and local anesthetic action, some consistent themes are emerging. Recent studies suggest that the local anesthetic compounds actively promote channel inactivation and, in doing so, function as allosteric effectors. Although the charged moiety may enter the Na channel pore, the primary mechanism whereby local anesthetic agents reduce excitability may be to induce channel inactivation.     

Targeting voltage-gated sodium channels for treatment for chronic visceral pain

Voltage-gated sodium channels (VGSCs) play a fundamental role in controlling cellular excitability, and their abnormal activity is related to several pathological processes, including cardiac arrhythmias, epilepsy, neurodegenerative diseases, spasticity and chronic pain. In particular, chronic visceral pain, the central symptom of functional gastrointestinal disorders such as irritable bowel syndrome, is a serious clinical problem that affects a high percentage of the world population. In spite of intense research efforts and after the dedicated decade of pain control and research, there are not many options to treat chronic pain conditions. However, there is a wealth of evidence emerging to give hope that a more refined approach may be achievable. By using electronic databases, available data on structural and functional properties of VGSCs in chronic pain, particularly functional gastrointestinal hypersensitivity, were reviewed. We summarize the involvement and molecular bases of action of VGSCs in the pathophysiology of several organic and functional gastrointestinal disorders. We also describe the efficacy of VGSC blockers in the treatment of these neurological diseases, and outline future developments that may extend the therapeutic use of compounds that target VGSCs. Overall, clinical and experimental data indicate that isoform-specific blockers of these channels or targeting of their modulators may provide effective and novel approaches for visceral pain therapy.     

Noninactivating voltage-gated sodium channels in severe myoclonic epilepsy of infancy

Mutations in SCN1A, the gene encoding the brain voltage-gated sodium channel alpha(1) subunit (Na(V)1.1), are associated with at least two forms of epilepsy, generalized epilepsy with febrile seizures plus and severe myoclonic epilepsy of infancy (SMEI). We examined the functional properties of five SMEI mutations by using whole-cell patch-clamp analysis of heterologously expressed recombinant human SCN1A. Two mutations (F902C and G1674R) rendered SCN1A channels nonfunctional, and a third allele (G1749E) exhibited minimal functional alterations. However, two mutations within or near the S4 segment of the fourth repeat domain (R1648C and F1661S) conferred significant impairments in fast inactivation, including persistent, noninactivating channel activity resembling the pattern of channel dysfunction observed for alleles associated with generalized epilepsy with febrile seizures plus. Our data provide evidence for a range of SCN1A functional abnormalities in SMEI, including gain-of-function defects that were not anticipated in this disorder. Our results further indicate that a complex relationship exists between phenotype and aberrant sodium channel function in these inherited epilepsies.     

Adaptive evolution of voltage-gated sodium channels: the first 800 million years

Voltage-gated Na(+)-permeable (Nav) channels form the basis for electrical excitability in animals. Nav channels evolved from Ca(2+) channels and were present in the common ancestor of choanoflagellates and animals, although this channel was likely permeable to both Na(+) and Ca(2+). Thus, like many other neuronal channels and receptors, Nav channels predated neurons. Invertebrates possess two Nav channels (Nav1 and Nav2), whereas vertebrate Nav channels are of the Nav1 family. Approximately 500 Mya in early chordates Nav channels evolved a motif that allowed them to cluster at axon initial segments, 50 million years later with the evolution of myelin, Nav channels "capitalized" on this property and clustered at nodes of Ranvier. The enhancement of conduction velocity along with the evolution of jaws likely made early gnathostomes fierce predators and the dominant vertebrates in the ocean. Later in vertebrate evolution, the Nav channel gene family expanded in parallel in tetrapods and teleosts (～9 to 10 genes in amniotes, 8 in teleosts). This expansion occurred during or after the late Devonian extinction, when teleosts and tetrapods each diversified in their respective habitats, and coincided with an increase in the number of telencephalic nuclei in both groups. The expansion of Nav channels may have allowed for more sophisticated neural computation and tailoring of Nav channel kinetics with potassium channel kinetics to enhance energy savings. Nav channels show adaptive sequence evolution for increasing diversity in communication signals (electric fish), in protection against lethal Nav channel toxins (snakes, newts, pufferfish, insects), and in specialized habitats (naked mole rats).     

Antillatoxin is a marine cyanobacterial toxin that potently activates voltage-gated sodium channels

Antillatoxin (ATX) is a lipopeptide derived from the pantropical marine cyanobacterium Lyngbya majuscula. ATX is neurotoxic in primary cultures of rat cerebellar granule cells, and this neuronal death is prevented by either N-methyl-d-aspartate (NMDA) receptor antagonists or tetrodotoxin. To further explore the potential interaction of ATX with voltage-gated sodium channels, we assessed the influence of tetrodotoxin on ATX-induced Ca2+ influx in cerebellar granule cells. The rapid increase in intracellular Ca2+ produced by ATX (100 nM) was antagonized in a concentration-dependent manner by tetrodotoxin. Additional, more direct, evidence for an interaction with voltage-gated sodium channels was derived from the ATX-induced allosteric enhancement of [3H]batrachotoxin binding to neurotoxin site 2 of the alpha subunit of the sodium channel. ATX, moreover, produced a strong synergistic stimulation of [3H]batrachotoxin binding in combination with brevetoxin, which is a ligand for neurotoxin site 5 on the voltage-gated sodium channel. Positive allosteric interactions were not observed between ATX and either alpha-scorpion toxin or the pyrethroid deltamethrin. That ATX interaction with voltage-gated sodium channels produces a gain of function was demonstrated by the concentration-dependent and tetrodotoxin-sensitive stimulation of 22Na+ influx in cerebellar granule cells exposed to ATX. Together these results demonstrate that the lipopeptide ATX is an activator of voltage-gated sodium channels. The neurotoxic actions of ATX therefore resemble those of brevetoxins that produce neural insult through depolarization-evoked Na+ load, glutamate release, relief of Mg2+ block of NMDA receptors, and Ca2+ influx.     

Interaction of voltage-gated sodium channels with the extracellular matrix molecules tenascin-C and tenascin-R

The type IIA rat brain sodium channel is composed of three subunits: a large pore-forming α subunit and two smaller auxiliary subunits, β1 and β2. The β subunits are single membrane-spanning glycoproteins with one Ig-like motif in their extracellular domains. The Ig motif of the β2 subunit has close structural similarity to one of the six Ig motifs in the extracellular domain of the cell adhesion molecule contactin (also called F3 or F11), which binds to the extracellular matrix molecules tenascin-C and tenascin-R. We investigated the binding of the purified sodium channel and the extracellular domain of the β2 subunit to tenascin-C and tenascin-R in vitro. Incubation of purified sodium channels on microtiter plates coated with tenascin-C revealed saturable and specific binding with an apparent K_d of ≈15 nM. Glutathione S-transferase-tagged fusion proteins containing various segments of tenascin-C and tenascin-R were purified, digested with thrombin to remove the epitope tag, immobilized on microtiter dishes, and tested for their ability to bind purified sodium channel or the epitope-tagged extracellular domain of β2 subunits. Both purified sodium channels and the extracellular domain of the β2 subunit bound specifically to fibronectin type III repeats 1–2, A, B, and 6–8 of tenascin-C and fibronectin type III repeats 1–2 and 6–8 of tenascin-R but not to the epidermal growth factor-like domain or the fibrinogen-like domain of these molecules. The binding of neuronal sodium channels to extracellular matrix molecules such as tenascin-C and tenascin-R may play a crucial role in localizing sodium channels in high density at axon initial segments and nodes of Ranvier or in regulating the activity of immobilized sodium channels in these locations.     

Peptide and small molecule inhibitors of HECT-type ubiquitin ligases

The human genome encodes several hundred E3 ubiquitin ligases containing RING domains, and around 28 containing HECT domains. These enzymes catalyze the transfer of ubiquitin from E2 enzyme thioesters to a huge range of substrates and play crucial roles in many cellular functions. This makes them attractive potential therapeutic targets. However, they have proven difficult to inhibit: very few good inhibitors exist for RING domain ligases, and none have been described for HECT ligases. Here we show that bicyclic peptides isolated by phage display [Heinis C, Rutherford T, Freund S, Winter G (2009) Nat Chem Biol. 5(7):502–507] can target the E2 binding sites on the HECT domains of Smurf2, Nedd4, Mule/Huwe1, and WWP1, and thus act as specific inhibitors of these enzymes in vitro. By screening for displacement of one of these peptides from Smurf2, we were able to identify a small molecule, heclin (HECT ligase inhibitor), which inhibits several HECT ligases in tissue culture cells. In vitro, heclin does not block E2 binding but causes a conformational change that results in oxidation of the active site Cys. This demonstrates that HECT domains are potentially druggable and provides molecules that may be of experimental use. Heclin kills HEK293 cells growing in culture, consistent with an essential role for HECT ligase activity in mammalian cells.Ubiquitin ligases are involved in almost every important function of cells, including the removal of misfolded proteins, the regulation of signaling pathways, DNA repair, the cell cycle, and apoptosis (1–4). This broad range of functions is mediated by a large number of E3 ubiquitin ligases, which recognize a similarly large range of substrates. Ubiquitination begins with the ATP-dependent formation of a ubiquitin (Ub)-E2 enzyme thioester intermediate by the E1 enzyme. The E3 enzymes then transfer ubiquitin from this intermediate to a lysine residue on the substrate (3). There are two main classes of E3: the RING domain ligases and their relatives, of which there are several hundred in humans, and the HECT domain ligases, which number around 28 (5–7). Because of their diversity and numerous functions and the fact that some ligases are frequently overexpressed in cancer cells, ubiquitin ligases have attracted much attention as possible targets for therapeutic intervention (2, 5, 6, 8).Despite this interest, it has proven challenging to make small molecule inhibitors of ubiquitin ligases. The action of these enzymes involves primarily protein–protein interactions among them, the Ub-E2 conjugate, and the substrate. For the RING enzymes, this is simultaneous, and the Ub moiety is transferred directly from E2 to substrate; for the HECT ligases, the interaction is sequential, with a thioester-linked Ub-E3 intermediate forming transiently. Such protein–protein interactions are considered difficult to block with small molecules. Added to this difficulty has been the complexity of the ubiquitin system, with ligases having many substrates and many proteins being substrates for multiple ligases (6, 9). Despite these problems, peptide and small molecule inhibitors have been successfully obtained that specifically target the substrate binding site of Mdm2, a RING ligase that acts on p53, and consequently activate the p53 pathway (10, 11). However, only a few other inhibitors of RING ligases have been published (12, 13), and none have been published that inhibit HECT ligases in vivo.Here we describe both peptide-based specific inhibitors of individual HECT ligases and a small molecule inhibitor with broad specificity for these enzymes. Using a previously described phage display method (14), we isolated bicyclic peptides (bicycles) that bind specifically to the HECT domains of Smurf2, Nedd4, WWP1, and Mule/Huwe1. We found that these frequently targeted the E2 binding site and inhibited ligase activity in vitro. We then used Alpha screening technology (15) to identify molecules that disrupted the bicycle–Smurf2 interaction. We identified one compound, termed heclin (HECT ligase inhibitor), that inhibits a range of HECT ligases in vitro and in tissue culture cells, with IC₅₀s in the low micromolar range. In vitro, heclin does not prevent E2 binding but induces a conformational change that results in spontaneous oxidation of the active site cysteine. Heclin allows clear distinction between RING and HECT-mediated ubiquitination, and on prolonged exposure, heclin can kill growing cells.     

Electrostatic contributions of aromatic residues in the local anesthetic receptor of voltage-gated sodium channels

Antiarrhythmics, anticonvulsants, and local anesthetics target voltage-gated sodium channels, decreasing excitability of nerve and muscle cells. Channel inhibition by members of this family of cationic, hydrophobic drugs relies on the presence of highly conserved aromatic residues in the pore-lining S6 segment of the fourth homologous domain of the channel. We tested whether channel inhibition was facilitated by an electrostatic attraction between lidocaine and pi electrons of the aromatic rings of these residues, namely a cation-pi interaction. To this end, we used the in vivo nonsense suppression method to incorporate a series of unnatural phenylalanine derivatives designed to systematically reduce the negative electrostatic potential on the face of the aromatic ring. In contrast to standard point mutations at the same sites, these subtly altered amino acids preserve the wild-type voltage dependence of channel activation and inactivation. Although these phenylalanine derivatives have no effect on low-affinity tonic inhibition by lidocaine or its permanently charged derivative QX-314 at any of the substituted sites, high-affinity use-dependent inhibition displays substantial cation-pi energetics for 1 residue only: Phe1579 in rNa(V)1.4. Replacement of the aromatic ring of Phe1579 by cyclohexane, for example, strongly reduces use-dependent inhibition and speeds recovery of lidocaine-engaged channels. Channel block by the neutral local anesthetic benzocaine is unaffected by the distribution of pi electrons at Phe1579, indicating that our aromatic manipulations expose electrostatic contributions to channel inhibition. These results fine tune our understanding of local anesthetic inhibition of voltage-gated sodium channels and will help the design of safer and more salutary therapeutic agents.     

RING finger protein 121 facilitates the degradation and membrane localization of voltage-gated sodium channels

Following their synthesis in the endoplasmic reticulum (ER), voltage-gated sodium channels (Na_V) are transported to the membranes of excitable cells, where they often cluster, such as at the axon initial segment of neurons. Although the mechanisms by which Na_V channels form and maintain clusters have been extensively examined, the processes that govern their transport and degradation have received less attention. Our entry into the study of these processes began with the isolation of a new allele of the zebrafish mutant alligator, which we found to be caused by mutations in the gene encoding really interesting new gene (RING) finger protein 121 (RNF121), an E3-ubiquitin ligase present in the ER and cis-Golgi compartments. Here we demonstrate that RNF121 facilitates two opposing fates of Na_V channels: (i) ubiquitin-mediated proteasome degradation and (ii) membrane localization when coexpressed with auxiliary Na_Vβ subunits. Collectively, these results indicate that RNF121 participates in the quality control of Na_V channels during their synthesis and subsequent transport to the membrane.Voltage-gated sodium channels (Na_V) are large (∼230 kDa) multipass transmembrane proteins (1). The Na_V channel family is comprised of nine members (Na_V1.1–Na_V1.9), whose activity typically underlies the rising phase of action potentials in excitable cells. In excitable cells, Na_V channels form complexes with auxiliary β subunits (Na_Vβ_1–4) in the Golgi apparatus (2), a process that enhances the kinetics and membrane localization of Na_V channels (3, 4). In addition to these roles, several Na_Vβ subunits also function as cell adhesion molecules independent of Na_V channels (5). At the axon initial segment (AIS) and nodes of Ranvier of neurons, Na_V channels form clusters that facilitate the generation and propagation of action potentials. Although the molecular basis of Na_V clustering at these sites has been extensively studied (6), the transport of Na_V channels to these sites has been less explored. For instance, to date, only the annexin II light chain (p11) has been shown to associate with and facilitate the transport of Na_V1.8 to the plasma membrane (7). Furthermore, subsequent efforts revealed that p11 acts only on Na_V1.8 (8). Thus, the transport of other Na_V channels remains unclear.In zebrafish, several studies have explored the contribution of Na_V channels and their auxiliary Na_Vβ subunits through the use of forward and reverse genetics. In brief, impairments in Na_V1.1, Na_V1.6a, and Na_Vβ_1b have been shown to diminish touch-evoked escape responses and Na_V channel activity in Rohon–Beard (RB) sensory neurons (9–11). In addition, two other mutants identified in forward genetic screens have been shown to affect Na_V channel activity indirectly. The first, pigu, arises from a mutation in a GPI-transamidase necessary for the proper localization of Na_V channels (12). Although the genetic locus of the second mutation, macho (13, 14), has yet to be identified, rough mapping indicates that it lies within a region lacking both Na_V channels and auxiliary Na_Vβ subunits. Collectively, these results indicate that the characterization of touch-unresponsive zebrafish mutants is an efficient strategy to gain insight into the trafficking and function of Na_V channels.In this study, we identified a touch-unresponsive zebrafish mutant (mi500), which was found to be a new allele of the molecularly unidentified motor mutant alligator (13). Electrophysiological analysis revealed that Na_V channel activity was severely diminished throughout the sensorimotor circuit in mutants. Further characterization uncovered that Na_V channels were not localized at the AIS in mutant RBs, but instead seem to be accumulated within the endoplasmic reticulum (ER) and cis-Golgi compartments. Meiotic mapping and sequence analysis showed that the alligator locus encodes really interesting new gene (RING) finger protein 121 (RNF121), an ER- and cis-Golgi–resident E3-ubiquitin ligase that mediates the ubiquitination of Na_V1.6. We found that RNF121 promotes the degradation and membrane transport of Na_V1.6. Furthermore, overexpression of Na_V1.6 worsened the touch response in rnf121-knockdown larvae, suggesting that an excess amount of Na_V exerts proteotoxicity. These findings suggest that the proper transport of Na_V channels is attributable to RNF121-mediated quality control of Na_V channels within the ER and Golgi apparatus.     

Synthetic small molecule furin inhibitors derived from 2,5-dideoxystreptamine

Furin plays a crucial role in embryogenesis and homeostasis and in diseases such as Alzheimer's disease, cancer, and viral and bacterial infections. Thus, inhibition of furin may provide a feasible and promising approach for therapeutic intervention of furin-mediated disease mechanisms. Here, we report on a class of small molecule furin inhibitors based on 2,5-dideoxystreptamine. Derivatization of 2,5-dideoxystreptamine by the addition of guanidinylated aryl groups yielded a set of furin inhibitors with nanomolar range potency against furin when assayed in a biochemical cleavage assay. Moreover, a subset of these furin inhibitors protected RAW 264.7 macrophage cells from toxicity caused by furin-dependent processing of anthrax protective antigen. These inhibitors were found to behave as competitive inhibitors of furin and to be relatively specific for furin. Molecular modeling revealed that these inhibitors may target the active site of furin as they showed site occupancy similar to the alkylating inhibitor decanoyl-Arg-Val-Lys-Arg-CH(2)Cl. The compounds presented here are bona fide synthetic small molecule furin inhibitors that exhibit potency in the nanomolar range, suggesting that they may serve as valuable tools for studying furin action and potential therapeutics agents for furin-dependent diseases.     

Targeting the voltage sensor of Kv7.2 voltage-gated K+ channels with a new gating-modifier

The pore and gate regions of voltage-gated cation channels have been often targeted with drugs acting as channel modulators. In contrast, the voltage-sensing domain (VSD) was practically not exploited for therapeutic purposes, although it is the target of various toxins. We recently designed unique diphenylamine carboxylates that are powerful Kv7.2 voltage-gated K⁺ channel openers or blockers. Here we show that a unique Kv7.2 channel opener, NH29, acts as a nontoxin gating modifier. NH29 increases Kv7.2 currents, thereby producing a hyperpolarizing shift of the activation curve and slowing both activation and deactivation kinetics. In neurons, the opener depresses evoked spike discharges. NH29 dampens hippocampal glutamate and GABA release, thereby inhibiting excitatory and inhibitory postsynaptic currents. Mutagenesis and modeling data suggest that in Kv7.2, NH29 docks to the external groove formed by the interface of helices S1, S2, and S4 in a way that stabilizes the interaction between two conserved charged residues in S2 and S4, known to interact electrostatically, in the open state of Kv channels. Results indicate that NH29 may operate via a voltage-sensor trapping mechanism similar to that suggested for scorpion and sea-anemone toxins. Reflecting the promiscuous nature of the VSD, NH29 is also a potent blocker of TRPV1 channels, a feature similar to that of tarantula toxins. Our data provide a structural framework for designing unique gating-modifiers targeted to the VSD of voltage-gated cation channels and used for the treatment of hyperexcitability disorders.     

Multimeric nature of voltage-gated proton channels

Voltage-gated potassium channels are comprised of four subunits, and each subunit has a pore domain and a voltage-sensing domain (VSD). The four pore domains assemble to form one single central pore, and the four individual VSDs control the gate of the pore. Recently, a family of voltage-gated proton channels, such as H(V) or voltage sensor only protein (VSOP), was discovered that contain a single VSD but no pore domain. It has been assumed that VSOP channels are monomeric and contain a single VSD that functions as both the VSD and the pore domain. It remains unclear, however, how a protein that contains only a VSD and no pore domain can conduct ions. Using fluorescence measurements and immunoprecipitation techniques, we show here that VSOP channels are expressed as multimeric channels. Further, FRET experiments on constructs with covalently linked subunits show that VSOP channels are dimers. Truncation of the cytoplasmic regions of VSOP reduced the dimerization, suggesting that the dimerization is caused mainly by cytoplasmic protein-protein interactions. However, these N terminus- and C terminus-deleted channels displayed large proton currents. Therefore, we conclude that even though VSOP channels are expressed mainly as dimers in the cell membrane, single VSOP subunits could function independently as proton channels.     

The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule

IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4μ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.