Search

Temporal coding precision of bushy cells in the ventral cochlear nucleus (VCN), critical for sound localization and communication, depends on the generation of rapid and temporally precise action potentials (APs). Voltage-gated potassium (Kv) channels are critically involved in this. The bushy cells in rat VCN express Kv1.1, 1.2, 1.3, 1.6, 3.1, 4.2, and 4.3 subunits. The Kv1.1 subunit contributes to the generation of a temporally precise single AP. However, the understanding of the functions of other Kv subunits expressed in the bushy cells is limited. Here, we investigated the functional diversity of Kv subunits concerning their contributions to temporal coding. We characterized the electrophysiological properties of the Kv channels with different subunits using whole cell patch-clamp recording and pharmacological methods. The neuronal firing pattern changed from single to multiple APs only when the Kv1.1 subunit was blocked. The Kv subunits, including the Kv1.1, 1.2, 1.6, or 3.1, were involved in enhancing temporal coding by lowering membrane excitability, shortening AP latencies, reducing jitter, and regulating AP kinetics. Meanwhile, all the Kv subunits contributed to rapid repolarization and sharpening peaks by narrowing half-width and accelerating fall rate, and the Kv1.1 subunit also affected the depolarization of AP. The Kv1.1, 1.2, and 1.6 subunits endowed bushy cells with a rapid time constant and a low input resistance of membrane for enhancing spike timing precision. The present results indicate that the Kv channels differentially affect intrinsic membrane properties to optimize the generation of rapid and reliable APs for temporal coding. NEW & NOTEWORTHY This study investigates the roles of Kv channels in effecting precision using electrophysiological and pharmacological methods in bushy cells. Different Kv channels have varying electrophysiological characteristics, which contribute to the interplay between changes in the membrane properties and regulation of neuronal excitability which then improve temporal coding. We conclude that the Kv channels are specialized to promote the precise and rapid coding of acoustic input by optimizing the generation of reliable APs.

Members of the voltage-gated K + channel subfamily (Kv1), involved in regulating transmission between neurons or to muscles, are associated with human diseases and, thus, putative targets for neurotherapeutics. This applies especially to those containing Kv1.1 α subunits which become prevalent in murine demyelinated axons and appear abnormally at inter-nodes, underlying the perturbed propagation of nerve signals. To overcome this dysfunction, akin to the consequential debilitation in multiple sclerosis (MS), small inhibitors were sought that are selective for the culpable hyper-polarising K + currents. Herein, we report a new semi-podand - compound 3 - that was designed based on the modelling of its interactions with the extracellular pore region in a deduced Kv1.1 channel structure. After synthesis, purification, and structural characterisation, compound 3 was found to potently (IC 50  = 8 µM) and selectively block Kv1.1 and 1.6 channels. The tested compound showed no apparent effect on native Nav and Cav channels expressed in F-11 cells. Compound 3 also extensively and selectively inhibited MS-related Kv1.1 homomer but not the brain native Kv1.1- or 1.6-containing channels. These collective findings highlight the therapeutic potential of compound 3 to block currents mediated by Kv1.1 channels enriched in demyelinated central neurons., (Copyright © 2020. Published by Elsevier Inc.)

Urotoxin (α-KTx 6), a peptide from venom of the Australian scorpion Urodacus yaschenkoi, is the most potent inhibitor of Kv1.2 described to date (IC 50  = 160 pM). The native peptide also inhibits Kv1.1, Kv1.3 and KCa3.1 with nanomolar affinity but its low abundance in venom precluded further studies of its actions. Here we produced recombinant Urotoxin (rUro) and characterized the molecular determinants of Kv1 channel inhibition. The 3D structure of rUro determined using NMR spectroscopy revealed a canonical cysteine-stabilised α/β (CSα/β) fold. Functional assessment of rUro using patch-clamp electrophysiology revealed the importance of C-terminal amidation for potency against Kv1.1-1.3 and Kv1.5. Neutralization of the putative pore-blocking K25 residue in rUro by mutation to Ala resulted in a major decrease in rUro potency against all Kv channels tested, without perturbing the toxin's structure. Reciprocal mutations in the pore of Uro-sensitive Kv1.2 and Uro-resistant Kv1.5 channels revealed a direct interaction between Urotoxin and the Kv channel pore. Our experimental work supports postulating a mechanism of action in which occlusion of the permeation pathway by the K25 residue in Urotoxin is the basis of its Kv1 inhibitory activity. Docking analysis was consistent with occlusion of the pore by K25 and the requirement of a small, non-charged amino acid in the Kv1 channel vestibule to facilitate toxin-channel interactions. Finally, computational studies revealed key interactions between the amidated C-terminus of Urotoxin and a conserved Asp residue in the turret of Kv1 channels, offering a potential rationale for potency differences between native and recombinant Urotoxin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Somatosensory neurons have historically been classified by a variety of approaches, including structural, anatomical, and genetic markers; electrophysiological properties; pharmacological sensitivities; and more recently, transcriptional profile differentiation. These methodologies, used separately, have yielded inconsistent classification schemes. Here, we describe phenotypic differences in response to pharmacological agents as measured by changes in cytosolic calcium concentration for the rapid classification of neurons in vitro; further analysis with genetic markers, whole-cell recordings, and single-cell transcriptomics validated these findings in a functional context. Using this general approach, which we refer to as tripartite constellation analysis (TCA), we focused on large-diameter dorsal-root ganglion (L-DRG) neurons with myelinated axons. Divergent responses to the K-channel antagonist, κM-conopeptide RIIIJ (RIIIJ), reliably identified six discrete functional cell classes. In two neuronal subclasses (L1 and L2), block with RIIIJ led to an increase in [Ca] i Simultaneous electrophysiology and calcium imaging showed that the RIIIJ-elicited increase in [Ca] i corresponded to different patterns of action potentials (APs), a train of APs in L1 neurons, and sporadic firing in L2 neurons. Genetically labeled mice established that L1 neurons are proprioceptors. The single-cell transcriptomes of L1 and L2 neurons showed that L2 neurons are Aδ-low-threshold mechanoreceptors. RIIIJ effects were replicated by application of the K v 1.1 selective antagonist, Dendrotoxin-K, in several L-DRG subclasses (L1, L2, L3, and L5), suggesting the presence of functional K v 1.1/K v 1.2 heteromeric channels. Using this approach on other neuronal subclasses should ultimately accelerate the comprehensive classification and characterization of individual somatosensory neuronal subclasses within a mixed population., Competing Interests: The authors declare no competing interest.

ShK is a 35-residue disulfide-linked polypeptide produced by the sea anemone Stichodactyla helianthus, which blocks the potassium channels Kv1.1 and Kv1.3 with pM affinity. An analogue of ShK has been developed that blocks Kv1.3 > 100 times more potently than Kv1.1, and has completed Phase 1b clinical trials for the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis. Previous studies have indicated that ShK undergoes a conformational exchange that is critical to its function, but this has proved difficult to characterise. Here, we have used high hydrostatic pressure as a tool to increase the population of the alternative state, which is likely to resemble the active form that binds to the Kv1.3 channel. By following changes in chemical shift with pressure, we have derived the chemical shift values of the low- and high-pressure states, and thus characterised the locations of structural changes. The main difference is in the conformation of the Cys17-Cys32 disulfide, which is likely to affect the positions of the critical Lys22-Tyr23 pair by twisting the 21-24 helix and increasing the solvent exposure of the Lys22 sidechain, as indicated by molecular dynamics simulations.

Aims: Kv1.1 (KCNA1) channels contribute to the control of neuronal excitability and have been associated with epilepsy. Kv1.1 channels can associate with the cytoplasmic Kvβ1 subunit resulting in rapid inactivating A-type currents. We hypothesized that removal of channel inactivation, by modulating Kv1.1/Kvβ1 interaction with a small molecule, would lead to decreased neuronal excitability and anticonvulsant activity., Methods: We applied high-throughput screening to identify ligands able to modulate the Kv1.1-T1 domain/Kvβ1 protein complex. We then selected a compound that was characterized on recombinant Kv1.1/Kvβ1 channels by electrophysiology and further evaluated on sustained neuronal firing and on in vitro epileptiform activity using a high K + -low Ca 2+ model in hippocampal slices., Results: We identified a novel compound able to modulate the interaction of the Kv1.1/Kvβ1 complex and that produced a functional inhibition of Kv1.1/Kvβ1 channel inactivation. We demonstrated that this compound reduced the sustained repetitive firing in hippocampal neurons and was able to abolish the development of in vitro epileptiform activity., Conclusions: This study describes a rational drug discovery approach for the identification of novel ligands that inhibit Kv1.1 channel inactivation and provides pharmacological evidence that such a mechanism translates into physiological effects by reducing in vitro epileptiform activity., (© 2018 John Wiley & Sons Ltd.)

Voltage-gated Kv1.1 potassium channel α-subunits, encoded by the Kcna1 gene, have traditionally been regarded as neural-specific with no expression or function in the heart. However, recent data revealed that Kv1.1 subunits are expressed in atria where they may have an overlooked role in controlling repolarization and arrhythmia susceptibility independent of the nervous system. To explore this concept in more detail and to identify functional and molecular effects of Kv1.1 channel impairment in the heart, atrial cardiomyocyte patch-clamp electrophysiology and gene expression analyses were performed using Kcna1 knockout ( Kcna1 -/- ) mice. Specifically, we hypothesized that Kv1.1 subunits contribute to outward repolarizing K + currents in mouse atria and that their absence prolongs cardiac action potentials. In voltage-clamp experiments, dendrotoxin-K (DTX-K), a Kv1.1-specific inhibitor, significantly reduced peak outward K + currents in wild-type (WT) atrial cells but not Kcna1 -/- cells, demonstrating an important contribution by Kv1.1-containing channels to mouse atrial repolarizing currents. In current-clamp recordings, Kcna1 -/- atrial myocytes exhibited significant action potential prolongation which was exacerbated in right atria, effects that were partially recapitulated in WT cells by application of DTX-K. Quantitative RT-PCR measurements showed mRNA expression remodeling in Kcna1 -/- atria for several ion channel genes that contribute to the atrial action potential including the Kcna5, Kcnh2, and Kcnj2 potassium channel genes and the Scn5a sodium channel gene. This study demonstrates a previously undescribed heart-intrinsic role for Kv1.1 subunits in mediating atrial repolarization, thereby adding a new member to the already diverse collection of known K + channels in the heart.

K + channels containing Kv1.1 α subunits, which become prevalent at internodes in demyelinated axons, may underlie their dysfunctional conduction akin to muscle weakness in multiple sclerosis. Small inhibitors were sought with selectivity for the culpable hyper-polarizing K + currents. Modeling of interactions with the extracellular pore in a Kv1.1-deduced structure identified diaryldi(2-pyrrolyl)methane as a suitable scaffold with optimized alkyl ammonium side chains. The resultant synthesized candidate [2,2'-((5,5'(di-p-topyldiaryldi(2-pyrrolyl)methane)bis(2,2'carbonyl)bis(azanediyl)) diethaneamine·2HCl] (8) selectively blocked Kv1.1 channels (IC 50 ≈ 15 μM) recombinantly expressed in mammalian cells, induced a positive shift in the voltage dependency of K + current activation, and slowed its kinetics. It preferentially inhibited channels containing two or more Kv1.1 subunits regardless of their positioning in concatenated tetramers. In slices of corpus callosum from mice subjected to a demyelination protocol, this novel inhibitor improved neuronal conduction, highlighting its potential for alleviating symptoms in multiple sclerosis.

Low-voltage-activated K + (g KL ) and hyperpolarization-activated mixed cation conductances (g h ) mediate currents, I KL and I h , through channels of the Kv1 (KCNA) and HCN families respectively and give auditory neurons the temporal precision required for signaling information about the onset, fine structure, and time of arrival of sounds. Being partially activated at rest, g KL and g h contribute to the resting potential and shape responses to even small subthreshold synaptic currents. Resting g KL and g h also affect the coupling of somatic depolarization with the generation of action potentials. To learn how these important conductances are regulated we have investigated how genetic perturbations affect their expression in octopus cells of the ventral cochlear nucleus (VCN). We report five new findings: First, the magnitude of g h and g KL varied over more than two-fold between wild type strains of mice. Second, average resting potentials are not different in different strains of mice even in the face of large differences in average g KL and g h . Third, I KL has two components, one being α-dendrotoxin (α-DTX)-sensitive and partially inactivating and the other being α-DTX-insensitive, tetraethylammonium (TEA)-sensitive, and non-inactivating. Fourth, the loss of Kv1.1 results in diminution of the α-DTX-sensitive I KL , and compensatory increased expression of an α-DTX-insensitive, tetraethylammonium (TEA)-sensitive I KL . Fifth, I h and I KL are balanced at the resting potential in all wild type and mutant octopus cells even when resting potentials vary in individual cells over nearly 10 mV, indicating that the resting potential influences the expression of g h and g KL . The independence of resting potentials on g KL and g h shows that g KL and g h do not, over days or weeks, determine the resting potential but rather that the resting potential plays a role in regulating the magnitude of either or both g KL and g h ., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The neurotoxic cone snail peptide μ-GIIIA specifically blocks skeletal muscle voltage-gated sodium (Na V 1.4) channels. The related conopeptides μ-PIIIA and μ-SIIIA, however, exhibit a wider activity spectrum by also inhibiting the neuronal Na V channels Na V 1.2 and Na V 1.7. Here we demonstrate that those μ-conopeptides with a broader target range also antagonize select subtypes of voltage-gated potassium channels of the K V 1 family: μ-PIIIA and μ-SIIIA inhibited K V 1.1 and K V 1.6 channels in the nanomolar range, while being inactive on subtypes K V 1.2-1.5 and K V 2.1. Construction and electrophysiological evaluation of chimeras between K V 1.5 and K V 1.6 revealed that these toxins block K V channels involving their pore regions; the subtype specificity is determined in part by the sequence close to the selectivity filter but predominantly by the so-called turret domain, i.e. the extracellular loop connecting the pore with transmembrane segment S5. Conopeptides μ-SIIIA and μ-PIIIA, thus, are not specific for Na V channels, and the known structure of some K V channel subtypes may provide access to structural insight into the molecular interaction between μ-conopeptides and their target channels., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Brain development and interictal function are unaffected in many paroxysmal neurological channelopathies, possibly explained by homoeostatic plasticity of synaptic transmission. Episodic ataxia type 1 is caused by missense mutations of the potassium channel Kv1.1, which is abundantly expressed in the terminals of cerebellar basket cells. Presynaptic action potentials of small inhibitory terminals have not been characterized, and it is not known whether developmental plasticity compensates for the effects of Kv1.1 dysfunction. Here we use visually targeted patch-clamp recordings from basket cell terminals of mice harbouring an ataxia-associated mutation and their wild-type littermates. Presynaptic spikes are followed by a pronounced afterdepolarization, and are broadened by pharmacological blockade of Kv1.1 or by a dominant ataxia-associated mutation. Somatic recordings fail to detect such changes. Spike broadening leads to increased Ca(2+) influx and GABA release, and decreased spontaneous Purkinje cell firing. We find no evidence for developmental compensation for inherited Kv1.1 dysfunction.

Voltage-gated potassium Kv1.2 channels play pivotal role in maintaining of resting membrane potential and, consequently, regulation of cellular excitability of neurons. Endogenously generated electric field (EF) have been proven as an important regulator for cell migration and tissue repair. The mechanisms of ion channel involvement in EF-induced cell responses are extensively studied but largely are poorly understood. In this study we generated three COS-7 clones with different expression levels of Kv1.2 channel, and confirmed their functional variations with patch clamp analysis. Time-lapse imaging analysis showed that EF-induced cell migration response was Kv1.2 channel expression level depended. Inhibition of Kv1.2 channels with charybdotoxin (ChTX) constrained the sensitivity of COS-7 cells to EF stimulation more than their motility. Immunocytochemistry and pull-down analyses demonstrated association of Kv1.2 channels with actin-binding protein cortactin and its re-localization to the cathode-facing membrane at EF stimulation, which confirms the mechanism of EF-induced directional migration. This study displays that Kv1.2 channels represent an important physiological link in EF-induced cell migration. The described mechanism suggests a potential application of EF which may improve therapeutic performance in curing injuries of neuronal and/or cardiac tissue repair, post operational therapy, and various degenerative syndromes., (© 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.)

The structural similarity between defensins and scorpion neurotoxins suggests that they might have evolved from a common ancestor. However, there is no direct experimental evidence demonstrating a functional link between scorpion neurotoxins and defensins. The scorpion defensin BmKDfsin4 from Mesobuthus martensiiKarsch contains 37 amino acid residues and a conserved cystine-stabilized α/β structural fold. The recombinant BmKDfsin4, a classical defensin, has been found to have inhibitory activity against Gram-positive bacteria such as Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteusas well as methicillin-resistant Staphylococcus aureus Interestingly, electrophysiological experiments showed that BmKDfsin4,like scorpion potassium channel neurotoxins, could effectively inhibit Kv1.1, Kv1.2, and Kv1.3 channel currents, and its IC50value for the Kv1.3 channel was 510.2 nm Similar to the structure-function relationships of classical scorpion potassium channel-blocking toxins, basic residues (Lys-13 and Arg-19) of BmKDfsin4 play critical roles in peptide-Kv1.3 channel interactions. Furthermore, mutagenesis and electrophysiological experiments demonstrated that the channel extracellular pore region is the binding site of BmKDfsin4, indicating that BmKDfsin4 adopts the same mechanism for blocking potassium channel currents as classical scorpion toxins. Taken together, our work identifies scorpion BmKDfsin4 as the first invertebrate defensin to block potassium channels. These findings not only demonstrate that defensins from invertebrate animals are a novel type of potassium channel blockers but also provide evidence of a functional link between defensins and neurotoxins., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

To realize the medicinal potential of peptide toxins, naturally occurring disulfide-rich peptides, as ion channel antagonists, more efficient pharmaceutical optimization technologies must be developed. Here, we show that the therapeutic properties of multiple cysteine toxin peptides can be rapidly and substantially improved by combining direct chemical strategies with high-throughput electrophysiology. We applied whole-molecule, brute-force, structure-activity analoging to ShK, a peptide toxin from the sea anemone Stichodactyla helianthus that inhibits the voltage-gated potassium ion channel Kv1.3, to effectively discover critical structural changes for 15× selectivity against the closely related neuronal ion channel Kv1.1. Subsequent site-specific polymer conjugation resulted in an exquisitely selective Kv1.3 antagonist (>1000× over Kv1.1) with picomolar functional activity in whole blood and a pharmacokinetic profile suitable for weekly administration in primates. The pharmacological potential of the optimized toxin peptide was demonstrated by potent and sustained inhibition of cytokine secretion from T cells, a therapeutic target for autoimmune diseases, in cynomolgus monkeys.

The lesser Asian scorpion Mesobuthus eupeus (Buthidae) is one of the most widely spread and dispersed species of the Mesobuthus genus, and its venom is actively studied. Nevertheless, a considerable amount of active compounds is still under-investigated due to the high complexity of this venom. Here, we report a comprehensive analysis of putative potassium channel toxins (KTxs) from the cDNA library of M. eupeus venom glands, and we compare the deduced KTx structures with peptides purified from the venom. For the transcriptome analysis, we used conventional tools as well as a search for structural motifs characteristic of scorpion venom components in the form of regular expressions. We found 59 candidate KTxs distributed in 30 subfamilies and presenting the cysteine-stabilized α/β and inhibitor cystine knot types of fold. M. eupeus venom was then separated to individual components by multistage chromatography. A facile fluorescent system based on the expression of the KcsA-Kv1.1 hybrid channels in Escherichia coli and utilization of a labeled scorpion toxin was elaborated and applied to follow Kv1.1 pore binding activity during venom separation. As a result, eight high affinity Kv1.1 channel blockers were identified, including five novel peptides, which extend the panel of potential pharmacologically important Kv1 ligands. Activity of the new peptides against rat Kv1.1 channel was confirmed (IC50 in the range of 1-780 nm) by the two-electrode voltage clamp technique using a standard Xenopus oocyte system. Our integrated approach is of general utility and efficiency to mine natural venoms for KTxs., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Synaptic transmission usually depends on action potentials (APs) in an all-or-none (digital) fashion. Recent studies indicate, however, that subthreshold presynaptic depolarization may facilitate spike-evoked transmission, thus creating an analog modulation of spike-evoked synaptic transmission, also called analog-digital (AD) synaptic facilitation. Yet, the underlying mechanisms behind this facilitation remain unclear. We show here that AD facilitation at rat CA3-CA3 synapses is time-dependent and requires long presynaptic depolarization (5-10 s) for its induction. This depolarization-induced AD facilitation (d-ADF) is blocked by the specific Kv1.1 channel blocker dendrotoxin-K. Using fast voltage-imaging of the axon, we show that somatic depolarization used for induction of d-ADF broadened the AP in the axon through inactivation of Kv1.1 channels. Somatic depolarization enhanced spike-evoked calcium signals in presynaptic terminals, but not basal calcium. In conclusion, axonal Kv1.1 channels determine glutamate release in CA3 neurons in a time-dependent manner through the control of the presynaptic spike waveform., (© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

ShK, from the sea anemone Stichodactyla helianthus, is a 35-residue disulfide-rich peptide that blocks the voltage-gated potassium channel Kv1.3 at ca. 10 pM and the related channel Kv1.1 at ca. 16 pM. We developed an analog of this peptide, ShK-186, which is currently in Phase 1b-2a clinical trials for the treatment of autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. While ShK-186 displays a >100-fold improvement in selectivity for Kv1.3 over Kv1.1 compared with ShK, there is considerable interest in developing peptides with an even greater selectivity ratio. In this report, we describe several variants of ShK that incorporate p-phophono-phenylalanine at the N-terminus coupled with internal substitutions at Gln16 and Met21. In addition, we also explored the combinatorial effects of these internal substitutions with an alanine extension at the C-terminus. Their selectivity was determined by patch-clamp electrophysiology on Kv1.3 and Kv1.1 channels stably expressed in mouse fibroblasts. The peptides with an alanine extension blocked Kv1.3 at low pM concentrations and exhibited up to 2250-fold selectivity for Kv1.3 over Kv1.1. Analogs that incorporates p-phosphono-phenylalanine at the N-terminus blocked Kv1.3 with IC50s in the low pM range and did not affect Kv1.1 at concentrations up to 100 nM, displaying a selectivity enhancement of >10,000-fold for Kv1.3 over Kv1.1. Other potentially important Kv channels such as Kv1.4 and Kv1.6 were only partially blocked at 100 nM concentrations of each of the ShK analogs.

Spider venom contains a very valuable repertoire of natural resources to discover novel components for molecular diversity analyses and therapeutic applications. In this study, HWTX-XI toxins from the spider venom glands of Ornithoctonus huwena which are Kunitz-type toxins (KTTs) and were directly cloned, analyzed and functionally characterized. To date, the HWTX-XI superfamily consists of 38 members deduced from 121 high-quality expressed sequence tags, which is the largest spider KTT superfamily with significant molecular diversity mainly resulted from cDNA tandem repeats as well as focal hypermutation. Among them, HW11c40 and HW11c50 may be intermediate variants between native Kunitz toxins and sub-Kunitz toxins based on evolutionary analyses. In order to elucidate their biological activities, recombinant HW11c4, HW11c24, HW11c27 and HW11c39 were successfully expressed, further purified and functionally characterized. Both HW11c4 and HW11c27 display inhibitory activities against trypsin, chymotrypsin and kallikrein. Moreover, HW11c4 is also an inhibitor relatively specific for Kv1.1 channels. HW11c24 and HW11c39 are found to be inactive on chymotrysin, trypsin, kallikrein, thrombin and ion channels. These findings provide molecular evidence for toxin diversification of the HWTX-XI superfamily and useful molecular templates of serine protease inhibitors and ion channel blockers for the development of potentially clinical applications., (Copyright © 2014 Elsevier Inc. All rights reserved.)

HsTX1 toxin, from the scorpion Heterometrus spinnifer, is a 34-residue, C-terminally amidated peptide cross-linked by four disulfide bridges. Here we describe new HsTX1 analogues with an Ala, Phe, Val or Abu substitution at position 14. Complexes of HsTX1 with the voltage-gated potassium channels Kv1.3 and Kv1.1 were created using docking and molecular dynamics simulations, then umbrella sampling simulations were performed to construct the potential of mean force (PMF) of the ligand and calculate the corresponding binding free energy for the most stable configuration. The PMF method predicted that the R14A mutation in HsTX1 would yield a > 2 kcal/mol gain for the Kv1.3/Kv1.1 selectivity free energy relative to the wild-type peptide. Functional assays confirmed the predicted selectivity gain for HsTX1[R14A] and HsTX1[R14Abu], with an affinity for Kv1.3 in the low picomolar range and a selectivity of more than 2,000-fold for Kv1.3 over Kv1.1. This remarkable potency and selectivity for Kv1.3, which is significantly up-regulated in activated effector memory cells in humans, suggest that these analogues represent valuable leads in the development of therapeutics for autoimmune diseases.

Each neuron possesses a unique firing property, which is largely attributed to heterogeneity in the composition of voltage-gated ion channel complexes. Zebrafish Mauthner (M) cells, which are bilaterally paired giant reticulospinal neurons (RSNs) in the hindbrain and induce rapid escape behavior, generate only a single spike at the onset of depolarization. This single spiking is in contrast with the repetitive firing of the M cell's morphologically homologous RSNs, MiD2cm and MiD3cm, which are also involved in escapes. However, how the unique firing property of M cells is established and the underlying molecular mechanisms remain unclear. In the present study, we first demonstrated that the single-spiking property of M cells was acquired at 4 days postfertilization (dpf), accompanied by an increase in dendrotoxin I (DTX)-sensitive low-threshold K(+) currents, prior to which the M cell repetitively fires as its homologs. Second, in situ hybridization showed that among DTX-sensitive Kv1 channel α-subunits, zKv1.1a was unexpectedly expressed even in the homologs and the bursting M cells at 2 dpf. In contrast, zKvβ2b, an auxiliary β-subunit of Kv1 channels, was expressed only in the single-spiking M cells. Third, zKv1.1a expressed in Xenopus oocytes functioned as a low-threshold K(+) channel, and its currents were enhanced by coexpression of zKvβ2b subunits. Finally, knockdown of zKvβ2b expression in zebrafish larvae resulted in repetitive firing of M cells at 4 dpf. Taken together, these results suggest that associative expression of Kvβ2 subunits with Kv1.1 channels is crucial for developmental acquisition of the unique firing properties of the M cells among homologous neurons.

The first synthesis of the non-peptidic snail toxin 6-bromo-2-mercaptotryptamine dimer (BrMT)2 is described, along with the preparation of its lower and higher thio homologs. The synthetic (BrMT)2 and its derivatives reported herein are all capable of slowing the activation of the Kv1.1 potassium ion channel. Only the monosulfide variant shows significant slowing of the deactivation process. This synthetic strategy can now be applied to creating a more extensive set of compounds that vary in the length of the linker connecting the two monomers, the substituents on the indole ring core, and terminal amine., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Voltage-sensitive neuronal Kv1 channels composed of four α subunits and four associated auxiliary β subunits control neuronal excitability and neurotransmission. Limited information exists on the combinations of α subunit isoforms (i.e. Kv1.1-1.6) or their positions in the oligomers, and how these affect sensitivity to blockers. It is known that TEA (tetraethylammonium) inhibits Kv1.1 channels largely due to binding a critical tyrosine (Tyr379) in the pore, whereas Val381 at the equivalent location in Kv1.2 makes it insensitive. With the eventual aim of developing blockers for therapeutic purposes, Kv1.1 and 1.2 α subunit genes were concatenated to form combinations representing those in central neurons, followed by surface expression in HEK (human embryonic kidney)-293 cells as single-chain functional proteins. Patch-clamp recordings demonstrated the influences of the ratios and positioning of these α subunits on the biophysical and pharmacological properties of oligomeric K+ channels. Raising the ratio of Kv1.1 to Kv1.2 in Kv1.2-1.2-1.1-1.2 led to the resultant channels being more sensitive to TEA and also affected their biophysical parameters. Moreover, mutagenesis of one or more residues in the first Kv1.2 to resemble those in Kv1.1 increased TEA sensitivity only when it is adjacent to a Kv1.1 subunit, whereas placing a non-interactive subunit between these two diminished susceptibility. The findings of the present study support the possibility of α subunits being precisely arranged in Kv1 channels, rather than being randomly assembled. This is important in designing drugs with abilities to inhibit particular oligomeric Kv1 subtypes, with the goal of elevating neuronal excitability and improving neurotransmission in certain diseases.

Voltage-gated K(+) (Kv) channels are known to be associated with the proliferation of several types of cancer cells, including lung adenocarcinoma cells, and certain Kv channel blockers inhibit cancer cell proliferation. In the present study, we investigated the effects of Kv channel blockers in gefitinib-resistant H460 non-small cell lung cancer (NSCLC) cells. Treatment with dendrotoxin-κ (DTX-κ), which is a Kv1.1-specific blocker, reduced H460 cell viability and arrested cells in G(1)/S transition during cell-cycle progression. We administered DTX-κ in a xenograft model using nude mice. The tumor volume was reduced by the injection of DTX-κ into the tumor tissues compared to the control group. These results indicate that DTX-κ has antitumor effects in gefitinib-resistant H460 cells through the pathway governing the G(1)/S transition both in vitro and in vivo. These findings suggest that Kv1.1 could serve as a novel therapeutic target for gefitinib-resistant NSCLC.

Neocortical networks produce oscillations that often correspond to characteristic physiological or pathological patterns. However, the mechanisms underlying the generation of and the transitions between such oscillatory states remain poorly understood. In this study, we examined resonance in mouse layer V neocortical pyramidal neurons. To accomplish this, we employed standard electrophysiology to describe cellular resonance parameters. Bode plot analysis revealed a range of resonance magnitude values in layer V neurons and demonstrated that both magnitude and phase response characteristics of layer V neocortical pyramidal neurons are modulated by changes in the extracellular environment. Specifically, increased resonant frequencies and total inductive areas were observed at higher extracellular potassium concentrations and more hyperpolarised membrane potentials. Experiments using pharmacological agents suggested that current through hyperpolarization-activated cyclic nucleotide-gated channels (I(h) ) acts as the primary driver of resonance in these neurons, with other potassium currents, such as A-type potassium current and delayed-rectifier potassium current (Kv1.4 and Kv1.1, respectively), contributing auxiliary roles. The persistent sodium current was also shown to play a role in amplifying the magnitude of resonance without contributing significantly to the phase response. Although resonance effects in individual neurons are small, their properties embedded in large networks may significantly affect network behavior and may have potential implications for pathological processes., (© 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Two voltage-dependent potassium channels, Kv1.1 (KCNA1) and Kv1.2 (KCNA2), are found to co-localize at the juxtaparanodal region of axons throughout the nervous system and are known to co-assemble in heteromultimeric channels, most likely in the form of the concatemer Kv1.1-1.2((3)) . Loss of the myelin sheath, as is observed in multiple sclerosis, uncovers the juxtaparanodal region of nodes of Ranvier in myelinated axons leading to potassium conductance, resulting in loss of nerve conduction. The selective blocking of these Kv channels is therefore a promising approach to restore nerve conduction and function. In the present study, we searched for novel inhibitors of Kv1.1-1.2((3)) by combining a virtual screening protocol and electrophysiological measurements on a concatemer Kv1.1-1.2((3)) stably expressed in Chinese hamster ovary K1 (CHO-K1) cells. The combined use of four popular virtual screening approaches (eHiTS, FlexX, Glide, and Autodock-Vina) led to the identification of several compounds as potential inhibitors of the Kv1.1-1.2((3)) channel. From 89 electrophysiologically evaluated compounds, 14 novel compounds were found to inhibit the current carried by Kv1.1-1.2((3)) channels by more than 80 % at 10 μM. Accordingly, the IC(50) values calculated from concentration-response curve titrations ranged from 0.6 to 6 μM. Two of these compounds exhibited at least 30-fold higher potency in inhibition of Kv1.1-1.2((3)) than they showed in inhibition of a set of cardiac ion channels (hERG, Nav1.5, and Cav1.2), resulting in a profile of selectivity and cardiac safety. The results presented herein provide a promising basis for the development of novel selective ion channel inhibitors, with a dramatically lower demand in terms of experimental time, effort, and cost than a sole high-throughput screening approach of large compound libraries., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Sound localization along the azimuth depends on the sensitivity of binaural nuclei in the auditory brainstem to small differences in interaural level and timing occurring within a submillisecond epoch and on monaural pathways that transmit level and timing cues with high temporal fidelity to insure their coincident arrival at the binaural targets. The soma and axons of these brainstem neurons are heavily invested with ion channels containing the low-threshold potassium channel subunit Kv1.1, which previous in vitro and in vivo studies suggest are important for regulating their high input-output correspondence and temporal synchrony. We compared awake Kcna1-null mutant (Kcna1-/-) mice lacking Kv1.1 with Kcna1+/+ mice to determine whether Kv1.1 activity contributes to sound localization and examined anesthetized mice for absolute hearing thresholds for suprathreshold differences that may be revealed in the waveforms of auditory brainstem response potentials. The awake -/- mice tested with reflex modification audiometry had reduced sensitivity to an abrupt change in the location of a broad band noise compared to +/+ mice, while anesthetized -/- mice had normal absolute thresholds for tone pips but a high level of stimulus-evoked but asynchronous background activity. Evoked potential waveforms had progressively earlier peaks and troughs in -/- mice, but the amplitude excursions between adjacent features were identical in the two groups. Their greater excitability and asynchrony in suprathreshold evoked potentials coupled with their normal thresholds suggests that a disruption in central neural processing in -/- mice and not peripheral hearing loss is responsible for their poor sound localization.

Background: Imiquimod (IQ) is known as an agonist of Toll-like receptor 7 (TLR7) and is widely used to treat various infectious skin diseases. However, it causes severe itching sensation as its side effect. The precise mechanism of how IQ causes itching sensation is unknown. A recent report suggested a molecular target of IQ as TLR7 expressed in dorsal root ganglion (DRG) neurons. However, we recently proposed a TLR7-independent mechanism, in which the activation of TLR7 is not required for the action of IQ in DRG neurons. To resolve this controversy regarding the involvement of TLR7 and to address the exact molecular identity of itching sensation by IQ, we investigated the possible molecular target of IQ in DRG neurons., Findings: When IQ was applied to DRG neurons, we observed an increase in action potential (AP) duration and membrane resistance both in wild type and TLR7-deficient mice. Based on these results, we tested whether the treatment of IQ has an effect on the activity of K(+) channels, K(v)1.1 and K(v)1.2 (voltage-gated K(+) channels) and TREK1 and TRAAK (K(2P) channels). IQ effectively reduced the currents mediated by both K(+) channels in a dose-dependent manner, acting as an antagonist at TREK1 and TRAAK and as a partial antagonist at K(v)1.1 and K(v)1.2., Conclusions: Our results demonstrate that IQ blocks the voltage-gated K(+) channels to increase AP duration and K(2P) channels to increase membrane resistance, which are critical for the membrane excitability of DRG neurons. Therefore, we propose that IQ enhances the excitability of DRG neurons by blocking multiple potassium channels and causing pruritus.

The three dimensional structure of a 32 residue three disulfide scorpion toxin, BTK-2, from the Indian red scorpion Mesobuthus tamulus has been determined using isotope edited solution NMR methods. Samples for structural and electrophysiological studies were prepared using recombinant DNA methods. Electrophysiological studies show that the peptide is active against hK(v)1.1 channels. The structure of BTK-2 was determined using 373 distance restraints from NOE data, 66 dihedral angle restraints from NOE, chemical shift and scalar coupling data, 6 constraints based on disulfide linkages and 8 constraints based on hydrogen bonds. The root mean square deviation (r.m.s.d) about the averaged co-ordinates of the backbone (N, C(α), C') and all heavy atoms are 0.81 ± 0.23Å and 1.51 ± 0.29Å respectively. The backbone dihedral angles (ϕ and ψ) for all residues occupy the favorable and allowed regions of the Ramachandran map. The three dimensional structure of BTK-2 is composed of three well defined secondary structural regions that constitute the α-β-β structural motif. Comparisons between the structure of BTK-2 and other closely related scorpion toxins pointed towards distinct differences in surface properties that provide insights into the structure-function relationships among this important class of voltage-gated potassium channel inhibiting peptides., (2011 Elsevier B.V. All rights reserved.)

Voltage-gated K(+) (Kv) channels have been considered to be a regulator of membrane potential and neuronal excitability. Recently, accumulated evidence has indicated that several Kv channel subtypes contribute to the control of cell proliferation in various types of cells and are worth noting as potential emerging molecular targets of cancer therapy. In the present study, we investigated the effects of the Kv1.1-specific blocker, dendrotoxin-κ (DTX-κ, on tumor formation induced by the human lung adenocarcinoma cell line A549 in a xenograft model. Kv1.1 mRNA and protein was expressed in A549 cells and the blockade of Kv1.1 by DTX-κ, reduced tumor formation in nude mice. Furthermore, treatment with DTX-κ significantly increased protein expression of p21(Waf1/Cip1), p27(Kip1), and p15(INK4B) and significantly decreased protein expression of cyclin D3 in tumor tissues compared to the control. These results suggest that DTX-κ has anti-tumor effects in A549 cells through the pathway governing G1-S transition.

Shaker-related Kv1 channels contain four channel-forming alpha subunits. Subfamily member Kv1.1 often occurs oligomerized with Kv1.2 alpha subunits in synaptic membranes, and so information was sought on the influence of their positions within tetramers on the channels' properties. Kv1.1 and 1.2 alpha genes were tandem linked in various arrangements, followed by expression as single-chain proteins in mammalian cells. As some concatenations reported previously seemed not to reliably position Kv1 subunits in their assemblies, the identity of expressed channels was methodically evaluated. Surface protein, isolated by biotinylation of intact transiently transfected HEK-293 cells, gave Kv1.1/1.2 reactivity on immunoblots with electrophoretic mobilities corresponding to full-length concatenated tetramers. There was no evidence of protein degradation, indicating that concatemers were delivered intact to the plasmalemma. Constructs with like genes adjacent (Kv1.1-1.1-1.2-1.2 or Kv1.2-1.2-1.1-1.1) yielded delayed-rectifying, voltage-dependent K(+) currents with activation parameters and inactivation kinetics slightly different from the diagonally positioned genes (Kv1.1-1.2-1.1-1.2 or 1.2-1.1-1.2-1.1). Pore-blocking petidergic toxins, alpha dendrotoxin, agitoxin-1, tityustoxin-Kalpha, and kaliotoxin, were unable to distinguish between the adjacent and diagonal concatamers. Unprecedentedly, external application of the pore-blocker tetraethylammonium (TEA) differentially inhibited the adjacent versus diagonal subunit arrangements, with diagonal constructs having enhanced susceptibility. Concatenation did not directly alter the sensitivities of homomeric Kv1.1 or 1.2 channels to TEA or the toxins. TEA inhibition of currents generated by channels made up from dimers (Kv1.1-1.2 and/or Kv1.2-1.1) was similar to the adjacently arranged constructs. These collective findings indicate that assembly of alpha subunits can be directed by this optimized concatenation, and that subunit arrangement in heteromeric Kv channels affects TEA affinity.

Degeneration of retinal ganglion cells (RGCs) - an important cause of visual impairment - is often modeled by optic nerve transection, which leads to apoptotic death of these central nervous system neurons. With this model, we show that specific voltage-gated K(+) channels (Kv1 family) contribute to the degeneration of rat RGCs and expression of apoptosis-related molecules in vivo. Retinal expression of Kv1.1, Kv1.2, Kv1.3 and Kv1.5 was examined by quantitative real-time reverse transcriptase-PCR and immunohistochemistry. Kv channel blockers and channel-specific short-interfering RNAs (siRNAs) were used to assess their roles in RGC degeneration. We found that (i) rat RGCs express Kv1.1, Kv1.2 and Kv1.3 (but not Kv1.5); (ii) intraocular injection of agitoxin-2 or margatoxin, potent blockers of Kv1.1, Kv1.2 and Kv1.3 channels, dose-dependently reduced the RGC degeneration; (iii) siRNAs applied to the cut optic nerve were rapidly transported throughout RGCs only, in which they reduced the expression of the cognate channel only. Our results show differential roles of the channels; siRNAs directed against Kv1.1 or Kv1.3 channels greatly reduced RGC death, whereas Kv1.2-targeted siRNAs had only a small effect, and siRNAs against Kv1.5 were without effect. (iv) Kv1.1 and Kv1.3 channels apparently contribute to cell-autonomous death of RGCs through different components of the apoptotic machinery. Kv1.1 depletion increased the antiapoptotic gene, Bcl-X(L), whereas Kv1.3 depletion reduced the proapoptotic genes, caspase-3, caspase-9 and Bad.

Summary 1. The non-selective K(+) channel blocker 4-aminopyridine (4-AP) has shown clinical efficacy in the treatment of neurological disorders such as multiple sclerosis. The clinical usefulness of 4-AP is hampered by its ability to produce seizures. Nerispirdine, an analogue of 4-AP, is currently under clinical investigation for the treatment of multiple sclerosis. In contrast with 4-AP, nerispirdine is not proconvulsant, suggesting mechanistic differences between the two drugs. 2. Using whole-cell patch-clamp electrophysiology, we compared the effects of 4-AP and nerispirdine on the cloned human K(+) channels K(v)1.1 and K(v)1.2, expressed in Chinese hamster ovary cells, and on voltage-dependent Na(+) channels recorded from human SH-SY5Y cells. 3. Nerispirdine inhibited K(v)1.1 and K(v)1.2 with IC(50) values of 3.6 and 3.7 micromol/L, respectively. 4-Aminopyridine was approximately 50-fold less potent at blocking these channels. Nerispirdine also inhibited voltage-dependent Na(+) channel currents recorded from human SH-SY5Y cells with an IC(50) of 11.9 micromol/L when measured from a -70 mV holding potential. In contrast, 4-AP had no effect on Na(+) channel currents. 4. The results demonstrate that nerispirdine, like 4-AP, can inhibit axonal K(+) channels and that this mechanism may underlie the ability of the drug to enhance neuronal conduction. Unlike 4-AP, nerispirdine can also inhibit neuronal Na(+) channels, a mechanism that may explain why nerispirdine lacks proconvulsant activity.

Currently, there is an unmet clinical need for novel immunosuppressive agents for long-term prevention of kidney transplant rejection as alternatives to the nephrotoxic calcineurin inhibitor cyclosporine (CsA). Recent studies have shown that K(+) channels have a crucial role in T-lymphocyte activity. We investigated whether combined blockade of the T-cell K(+) channels K(Ca)3.1 and K(v)1.3, both of which regulate calcium signaling during lymphocyte activation, is effective in prevention of rejection of kidney allografts from Fisher rats to Lewis rats. All recipients were initially treated with CsA (5 mg/kg d) for 7 days. In rats with intact allograft function, treatment was continued for 10 days with either CsA (5 mg/kg d), or a combination of TRAM-34 (K(Ca)3.1 inhibitor; 120 mg/kg d) plus Stichodactyla helianthus toxin (ShK, K(v)1.3 inhibitor; 80 microg/kg 3 times daily), or vehicle alone. Kidney sections were stained with periodic acid-Schiff or hematoxylin-eosin and histochemically for markers of macrophages (CD68), T-lymphocytes (CD43), or cytotoxic T-cells (CD8). Our results showed that treatment with TRAM-34 and ShK reduced total interstitial mononuclear cell infiltration (-42%) and the number of CD43+ T-cells (-32%), cytotoxic CD8+ T-cells (-32%), and CD68+ macrophages (-26%) in allografts when compared to vehicle treatment alone. Efficacy of TRAM-34/ShK treatment was comparable with that of CsA. In addition, no visible organ damage or other discernible adverse effects were observed with this treatment. Thus, selective blockade of T-lymphocyte K(Ca)3.1 and K(v)1.3 channels may represent a novel alternative therapy for prevention of kidney allograft rejection.

In this paper were described the purification, the sequencing, and the immunological and biological characterization of a new Kaliotoxin analog, Aam-KTX, from the venom of the scorpion Androctonus amoreuxi. The toxin effects on three cloned Kv channels (Kv1.1, Kv1.2, and Kv1.3) were investigated in Xenopus oocytes using electrophysiology experiments. The Aam-KTX preference for Kv1.3 channel versus Kv1.2 was expected (EC(50) values, 1.1+/-0.02 and 10.4+/-1.5 nM, respectively) but its total inefficacy on Kv1.1 was very surprising. 3D molecular modeling of Aam-KTX brought putative answers to this difference in selectivity.

Background: This study's aim was to investigate the interaction of amino-amide local anesthetics with human Kv1.1 potassium channels. These channels were chosen because of their proven physiologic role. By using a homolog series of local anesthetics with different lengths of the N-substituent, it was intended to elucidate the role of lipophilic interactions with Kv1.1. The use of stereoisomers allowed testing of the role of polar drug actions., Methods: Human Kv1.1 channels were measured with the patch clamp technique. Concentration-response data were described by Hill functions. Gating changes were described by Boltzmann functions., Results: Inhibition of Kv1.1 channels by dextrobupivacaine, bupivacaine, levobupivacaine, dextroropivacaine, levoropivacaine, and mepivacaine was concentration dependent, reversible, stereoselective, voltage dependent, and frequency independent. The IC(50) values were (mean +/- SEM) 41 +/- 3 microm (n = 20), 56 +/- 3 microm (n = 26), 76 +/- 8 microm (n = 24), 135 +/- 29 microm (n = 23), 313 +/- 32 microm (n = 25), and 1,451 +/- 351 microm (n = 23), respectively. The midpoint of current activation was shifted into the hyperpolarizing direction. The inhibitory potency as well as the potency to induce gating changes correlated with the number of CH(2) groups in the side chain of the drugs (r > 0.9, P < 0.05)., Conclusions: Kv1.1 channels constitute an important biophysical model for elucidating molecular mechanisms underlying local anesthetic drug effects. Inhibition likely results from an open state-dependent blocking mechanism. Interaction of local anesthetics with the ion channel protein is determined by lipophilic drug properties.

The low selectivity of Kv1 peptide inhibitors for specific isoforms makes them poor candidates for the development of theraputics. Using combined approaches, we showed that the Kv1 turret is the critical determinant for ADWX-1 peptide inhibitor selectivity of Kv1.3 over Kv1.1. Mutation of Kv1.1 turret residues to match the sequence of Kv1.3 lead to increased inhibition of Kv1.1 activity. These studies may lead to improvements in peptide inhibitor drug development.

The potassium channel Kv1.3 is an attractive pharmacological target for immunomodulation of T cell-mediated autoimmune diseases. Potent and selective blockers of Kv1.3 are potential therapeutics for treating these diseases. Here we describe the design of a new peptide inhibitor that is potent and selective for Kv1.3. Three residues (Gly(11), Ile(28), and Asp(33)) of a scorpion toxin BmKTX were substituted by Arg(11), Thr(28), and His(33), resulting in a new peptide, named ADWX-1. The ADWX-1 peptide blocked Kv1.3 with picomolar affinity (IC(50), 1.89 pM), showing a 100-fold increase in activity compared with the native BmKTX toxin. The ADWX-1 also displayed good selectivity on Kv1.3 over related Kv1.1 and Kv1.2 channels. Furthermore, alanine-scanning mutagenesis was carried out to map the functional residues of ADWX-1 in blocking Kv1.3. Moreover, computational simulation was used to build a structural model of the ADWX-1-Kv1.3 complex. This model suggests that all mutated residues are favorable for both the high potency and selectivity of ADWX-1 toward Kv1.3. While Arg(11) of ADWX-1 interacts with Asp(386) in Kv1.3, Thr(28) and His(33) of ADWX-1 locate right above the selectivity filter-S6 linker of Kv1.3. Together, our data indicate that the specific ADWX-1 peptide would be a viable lead in the therapy of T cell-mediated autoimmune diseases, and the successful design of ADWX-1 suggests that rational design based on the structural model of the peptide-channel complex should accelerate the development of diagnostic and therapeutic agents for human channelopathies.

Background: Kuntiz-type toxins (KTTs) have been found in the venom of animals such as snake, cone snail and sea anemone. The main ancestral function of Kunitz-type proteins was the inhibition of a diverse array of serine proteases, while toxic activities (such as ion-channel blocking) were developed under a variety of Darwinian selection pressures. How new functions were grafted onto an old protein scaffold and what effect Darwinian selection pressures had on KTT evolution remains a puzzle., Principal Findings: Here we report the presence of a new superfamily of ktts in spiders (TARANTULAS: Ornithoctonus huwena and Ornithoctonus hainana), which share low sequence similarity to known KTTs and is clustered in a distinct clade in the phylogenetic tree of KTT evolution. The representative molecule of spider KTTs, HWTX-XI, purified from the venom of O. huwena, is a bi-functional protein which is a very potent trypsin inhibitor (about 30-fold more strong than BPTI) as well as a weak Kv1.1 potassium channel blocker. Structural analysis of HWTX-XI in 3-D by NMR together with comparative function analysis of 18 expressed mutants of this toxin revealed two separate sites, corresponding to these two activities, located on the two ends of the cone-shape molecule of HWTX-XI. Comparison of non-synonymous/synonymous mutation ratios (omega) for each site in spider and snake KTTs, as well as PBTI like body Kunitz proteins revealed high Darwinian selection pressure on the binding sites for Kv channels and serine proteases in snake, while only on the proteases in spider and none detected in body proteins, suggesting different rates and patterns of evolution among them. The results also revealed a series of key events in the history of spider KTT evolution, including the formation of a novel KTT family (named sub-Kuntiz-type toxins) derived from the ancestral native KTTs with the loss of the second disulfide bridge accompanied by several dramatic sequence modifications., Conclusions/significance: These finding illustrate that the two activity sites of Kunitz-type toxins are functionally and evolutionally independent and provide new insights into effects of Darwinian selection pressures on KTT evolution, and mechanisms by which new functions can be grafted onto old protein scaffolds.

The paraventricular nucleus (PVN) of the hypothalamus is important for the neural regulation of cardiovascular function. Nitric oxide (NO) increases synaptic GABA release to presympathetic PVN neurons through the cyclic guanosine monophosphate (cGMP)/protein kinase G signaling pathway. However, the downstream signaling mechanisms underlying the effect of NO on synaptic GABA release remain unclear. In this study, whole-cell voltage-clamp recordings were performed on retrograde-labeled spinally projecting PVN neurons in rat brain slices. Bath application of the NO precursor l-arginine or the NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly increased the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in labeled PVN neurons. A specific antagonist of cyclic ADP ribose, 8-bromo-cyclic ADP ribose (8-Br-cADPR), had no significant effect on l-arginine-induced potentiation of mIPSCs. Surprisingly, blocking of voltage-gated potassium channels (Kv) with 4-aminopyridine or alpha-dendrotoxin eliminated the effect of l-arginine on mIPSCs in all labeled PVN neurons tested. The membrane permeable cGMP analog mimicked the effect of l-arginine on mIPSCs, and this effect was blocked by alpha-dendrotoxin. Furthermore, the specific Kv channel blocker for Kv1.1 (dendrotoxin-K) or Kv1.2 (tityustoxin-Kalpha) abolished the effect of l-arginine on mIPSCs in all neurons tested. SNAP failed to inhibit the firing activity of labeled PVN neurons in the presence of dendrotoxin-K, Kalpha. Additionally, the immunoreactivity of Kv1.1 and Kv1.2 subunits was colocalized extensively with synaptophysin in the PVN. These findings suggest that NO increases GABAergic input to PVN presympathetic neurons through a downstream mechanism involving the Kv1.1 and Kv1.2 channels at the nerve terminals.

Background: Kv1.1 and Kv7.2/7.3 channels control excitability of neuronal cells. As hyperexcitability is a sign of neuropathic pain, epilepsy, and anxiety disorders, these channels may be important molecular targets of amitriptyline that cause pharmacological as well as toxicological effects by altering neuronal excitability. Since the molecular mechanisms underlying these effects of amitriptyline have not been fully elucidated, we aimed to characterize the interaction of amitriptyline with human Kv1.1 and Kv7.2/7.3 channels. We also intended to establish the interaction of amitriptyline with the Kv7.2/7.3 channel opener, retigabine., Methods: Kv1.1 and Kv7.2/7.3 channels were expressed in human embryonic kidney cells and in Chinese hamster ovary cells. The effects of amitriptyline and retigabine were studied with the patch-clamp technique., Results: Amitriptyline inhibited Kv1.1 and Kv7.2/7.3 channels in a concentration-dependent and reversible manner. The IC50-value was 22 +/- 3 microM (n = 33) and 10 +/- 1 microM (n = 40), respectively. Deactivating inward currents of Kv7.2/7.3 channels were inhibited with an IC50-value of 4.2 +/- 0.6 microM (n = 32). Inhibition of Kv7.2/7.3 channels by amitriptyline reversibly depolarized the resting membrane potential. Retigabine reversed both the inhibitory action of amitriptyline on Kv7.2/7.3 channels as well as the depolarization of the membrane potential., Conclusions: Since amitriptyline inhibited Kv1.1 and Kv7.2/7.3 channels only at toxicologically relevant plasma concentrations, our results suggest a role for these channels in the neuroexcitatory side effects of amitriptyline. As the inhibitory effects of amitriptyline were reversed by retigabine, a combination of amitriptyline and retigabine could be of additional benefit in the therapy of neuropathic pain.