An integrative approach to the facile functional classification of dorsal root ganglion neuronal subclasses.

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 Kv1.1 selective antagonist, Dendrotoxin-K, in several L-DRG subclasses (L1, L2, L3, and L5), suggesting the presence of functional Kv1.1/Kv1.2 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.

Differential contributions of voltage-gated potassium channel subunits in enhancing temporal coding in the bushy cells of the ventral cochlear nucleus.

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.

Development of a selective inhibitor for Kv1.1 channels prevalent in demyelinated nerves.

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 (IC50 = 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.

Genetic ablation or pharmacological inhibition of Kv1.1 potassium channel subunits impairs atrial repolarization in mice.

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.

Scorpion Potassium Channel-blocking Defensin Highlights a Functional Link with Neurotoxin.

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/ß 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.

Subtype-specific block of voltage-gated K+ channels by µ-conopeptides.

The neurotoxic cone snail peptide µ-GIIIA specifically blocks skeletal muscle voltage-gated sodium (NaV1.4) channels. The related conopeptides µ-PIIIA and µ-SIIIA, however, exhibit a wider activity spectrum by also inhibiting the neuronal NaV channels NaV1.2 and NaV1.7. Here we demonstrate that those µ-conopeptides with a broader target range also antagonize select subtypes of voltage-gated potassium channels of the KV1 family: µ-PIIIA and µ-SIIIA inhibited KV1.1 and KV1.6 channels in the nanomolar range, while being inactive on subtypes KV1.2-1.5 and KV2.1. Construction and electrophysiological evaluation of chimeras between KV1.5 and KV1.6 revealed that these toxins block KV 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 NaV channels, and the known structure of some KV channel subtypes may provide access to structural insight into the molecular interaction between µ-conopeptides and their target channels.

Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1: AN INTEGRATED TRANSCRIPTOMIC AND PROTEOMIC STUDY.

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/ß 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.

The Role of Kv1.2 Channel in Electrotaxis Cell Migration.

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.

Analog modulation of spike-evoked transmission in CA3 circuits is determined by axonal Kv1.1 channels in a time-dependent manner.

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.

Development of highly selective Kv1.3-blocking peptides based on the sea anemone peptide ShK.

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.

Coexpression of auxiliary Kvß2 subunits with Kv1.1 channels is required for developmental acquisition of unique firing properties of zebrafish Mauthner cells.

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, zKvß2b, an auxiliary ß-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 zKvß2b subunits. Finally, knockdown of zKvß2b expression in zebrafish larvae resulted in repetitive firing of M cells at 4 dpf. Taken together, these results suggest that associative expression of Kvß2 subunits with Kv1.1 channels is crucial for developmental acquisition of the unique firing properties of the M cells among homologous neurons.

Pharmacological characteristics of Kv1.1- and Kv1.2-containing channels are influenced by the stoichiometry and positioning of their α subunits.

Voltage-sensitive neuronal Kv1 channels composed of four α subunits and four associated auxiliary ß 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.

Kcna1 gene deletion lowers the behavioral sensitivity of mice to small changes in sound location and increases asynchronous brainstem auditory evoked potentials but does not affect hearing thresholds.

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.

Synthesis of the non-peptidic snail toxin 6-bromo-2-mercaptotryptamine dimer (BrMT)(2), its lower and higher thio homologs and their ability to modulate potassium ion channels.

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.

Solution structure of BTK-2, a novel hK(v)1.1 inhibiting scorpion toxin, from the eastern Indian scorpion Mesobuthus tamulus.

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-ß-ß 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.

Imiquimod enhances excitability of dorsal root ganglion neurons by inhibiting background (K(2P)) and voltage-gated (K(v)1.1 and K(v)1.2) potassium channels.

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.

Resonance in neocortical neurons and networks.

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.

Structural basis of the potency and selectivity of Urotoxin, a potent Kv1 blocker from scorpion venom.

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 (IC50 = 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/ß (CSα/ß) 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.

In Vitro electrophysiological activity of nerispirdine, a novel 4-aminopyridine derivative.

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.

Conformational exchange in the potassium channel blocker ShK.

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.