Sustained NPY Overexpression in the PVN Results in Obesity via Temporarily Increasing Food Intake

Increasing neuropeptide Y (NPY) signaling in the paraventricular nucleus (PVN) by recombinant adeno‐associated virus (rAAV)‐mediated overexpression of NPY in rats, results in hyperphagia and obesity in rats. To determine the importance of hyperphagia in the observed obesity phenotype, we pair‐fed a group of AAV‐NPY‐injected rats to AAV‐control‐injected rats and compared parameters of energy balance to ad libitum fed AAV‐NPY‐injected rats. For 3 weeks, AAV‐NPY‐injected rats, received the same amount of food as ad libitum‐fed rats injected with control rAAV They did not gain more body weight than these controls. When allowed access to food ad libitum, these AAV‐NPY‐injected rats increased food intake, which subsequently decreased when rats reached the same body weight as AAV‐NPY‐injected rats that were fed ad libitum for the entire study. These data indicate that overexpression of NPY in the PVN results in obesity by increasing food intake until a certain body weight is achieved.     

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. J. Cell. Physiol. 231: 1375–1384, 2016. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Molecular basis of dysfunctional Kv channels in small coronary artery smooth muscle cells of streptozotocin-induced diabetic rats

We have previously shown that diabetes impaired cAMP-mediated endothelium independent vasodilation of rat small coronary arteries. Inhibition of Kv channel activity plays an important role in the decrease of cAMP mediated vasodilation. The present study investigated the effect of streptozotocin (STZ)-induced diabetes on mRNA and protein expressions of Kv1.2 and Kv1.5 channels in vascular smooth muscle cells of rat small coronary artery using RT-PCR, Western blot and immunohistochemistry methods. STZ-induced diabetes obviously impaired mRNA expression of Kv1.2 and Kv1.5 channel. The mRNA levels of Kv1.2 channel were 0.65 +/- 0.08 and 1.02 +/- 0.17 in STZ rats and control rats, respectively (n = 7, P < 0.05). Whereas the levels of Kv1.5 channel were 0.58 +/- 0.05 and 0.94 +/- 0.13 in STZ rats and control rats, respectively (n = 7, P < 0.05). Western blotting analysis showed that protein expression of Kv1.2 channel was decreased significantly but not Kv1.5 channel. Protein expressions of Kv1.2 channel were 0.49 +/- 0.04 and 0.70 +/- 0.06 in STZ rats and control rats, respectively (n = 5, P < 0.05), but those of Kv1.5 channel were 0.61 +/- 0.12 and 0.59 +/- 0.14 in STZ rats and control rats, respectively (n = 5, P > 0.05). Immunohistochemistry identification indicated that immunological reaction of Kv1.2 channel protein was attenuated, but Kv1.5 channel protein was not altered. Positive staining intensity normalized by gray values of Kv1.2 channel were 173 +/- 13 and 131 +/- 11 in STZ rats and control rats, respectively (n = 5, P < 0.05), but those of Kv1.5 channel were 139 +/- 16 and 141 +/- 12 in STZ rats and control rats, respectively (n = 5, P > 0.05). These results suggested that impairment of cAMP-mediated endothelium independent vasodilation of rat small coronary artery by STZ-induced diabetes was resulted from decrease of mRNA and protein expressions of Kv channels, and which eventually leads to a reduced current from Kv channels.     

Structural refinement of the hERG1 pore and voltage‐sensing domains with ROSETTA‐membrane and molecular dynamics simulations

The hERG1 gene (Kv11.1) encodes a voltage‐gated potassium channel. Mutations in this gene lead to one form of the Long QT Syndrome (LQTS) in humans. Promiscuous binding of drugs to hERG1 is known to alter the structure/function of the channel leading to an acquired form of the LQTS. Expectably, creation and validation of reliable 3D model of the channel have been a key target in molecular cardiology and pharmacology for the last decade. Although many models were built, they all were limited to pore domain. In this work, a full model of the hERG1 channel is developed which includes all transmembrane segments. We tested a template‐driven de‐novo design with ROSETTA‐membrane modeling using side‐chain placements optimized by subsequent molecular dynamics (MD) simulations. Although backbone templates for the homology modeled parts of the pore and voltage sensors were based on the available structures of KvAP, Kv1.2 and Kv1.2‐Kv2.1 chimera channels, the missing parts are modeled de‐novo. The impact of several alignments on the structure of the S4 helix in the voltage‐sensing domain was also tested. Herein, final models are evaluated for consistency to the reported structural elements discovered mainly on the basis of mutagenesis and electrophysiology. These structural elements include salt bridges and close contacts in the voltage‐sensor domain; and the topology of the extracellular S5‐pore linker compared with that established by toxin foot‐printing and nuclear magnetic resonance studies. Implications of the refined hERG1 model to binding of blockers and channels activators (potent new ligands for channel activations) are discussed. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Regulatory role of the extreme C‐terminal end of the S6 inner helix in C‐terminal‐truncated Kv1.2 channel activation

The transmembrane part of the S6 inner helix of the Kv1.2 potassium channel is a pivotal part in sustaining channel activity. However, the role of its extreme C‐terminal end, which is located on the cytoplasmic side of the channel, is largely unknown. Here, we investigated the role of the extreme C‐terminal end of the S6 inner helix (the HRET region) by constructing a series of C‐terminal‐truncated mutations related to this region in the C‐terminal‐truncated Kv1.2 channel. Mutations on Thr⁴²¹ or Glu⁴²⁰ significantly altered the activation of the truncated channel. Mutations on Arg⁴¹⁹ demonstrated that neutral or basic, but not acidic amino acid, is essential at the position for the truncated channel activation, and no functional channel was observed when the channel was truncated from His⁴¹⁸. Hence, our results indicate that the extreme C‐terminal end of the S6 inner helix plays an important regulatory role in the activation of the C‐terminal‐truncated Kv1.2 channel.     

Kv 1.1 is associated with neuronal apoptosis and modulated by protein kinase C in the rat cerebellar granule cell

Previously, we reported that apoptosis of cerebellar granular neurons induced by low‐K⁺ and serum‐free (LK‐S) was associated with an increase in the A‐type K⁺ channel current (I_A), and an elevated expression of main α‐subunit of the I_A channel, which is known as Kv4.2 and Kv4.3. Here, we show, as assessed by quantitative RT‐PCR and whole‐cell recording, that besides Kv4.2 and Kv4.3, Kv1.1 is very important for I_A channel. The expression of Kv1.1 was elevated in the apoptotic neurons, while silencing Kv1.1 expression by siRNA reduced the I_A amplitude of the apoptotic neuron, and increased neuron viability. Inhibiting Kv1.1 current by dendrotoxin‐K evoked a similar effect of reduction of I_A amplitude and protection of neurons. Applying a protein kinase C (PKC) activator, phorbol ester acetate A (PMA) mimicked the LK‐S‐induced neuronal apoptotic effect, enhanced the I_A amplitude and reduced the granule cell viability. The PKC inhibitor, bisindolylmaleimide I and Gö6976 protected the cell against apoptosis induced by LK‐S. After silencing the Kv1.1 gene, the effect of PMA on the residual K⁺ current was reduced significantly. Quantitative RT‐PCR and Western immunoblot techniques revealed that LK‐S treatment and PMA increased the level of the expression of Kv1.1, in contrast, bisindolylmaleimide I inhibited Kv1.1 expression. In addition, the activation of the PKC isoform was identified in apoptotic neurons. We thus conclude that in the rat cerebellar granule cell, the I_A channel associated with apoptotic neurons is encoded mainly by the Kv1.1 gene, and that the PKC pathway promotes neuronal apoptosis by a brief modulation of the I_A amplitude and a permanent increase in the levels of expression of the Kv1.1 α‐subunit.     

Sleep in Kcna2 knockout mice

Background  

Shaker codes for a Drosophila voltage-dependent potassium channel. Flies carrying Shaker null or hypomorphic mutations sleep 3–4 h/day instead of 8–14 h/day as their wild-type siblings do. Shaker-like channels are conserved across species but it is unknown whether they affect sleep in mammals. To address this issue, we studied sleep in Kcna2 knockout (KO) mice. Kcna2 codes for Kv1.2, the alpha subunit of a Shaker-like voltage-dependent potassium channel with high expression in the mammalian thalamocortical system.     

Bldesin,the first functionally characterized pathogenic fungus defensin with Kv1.3 channel and chymotrypsin inhibitory activities

Fungus defensin is a kind of important natural peptide resource, such as plectasin from the soil fungus Pseudoplectania nigrella with potential application in the antimicrobial peptide lead drug discovery. Here, a fungus defensin named Bldesin with Kv1.3 channel and serine protease inhibitory activities was first explored. By GST‐Bldesin fusion expression and enterokinase cleaving strategy, recombinant Bldesin was obtained successfully. Antimicrobial assays showed that Bldesin had potent activity against Gram‐positive Staphylococcus aureus, but had no effect on Gram‐negative Escherichia coli. Electrophysiological experiments showed that Bldesin had Kv1.3 channel inhibitory activity. Serine protease inhibitory associated experiments showed that Bldesin had unique chymotrypsin protease inhibitory, elastase protease inhibitory, and serine protease‐associated coagulation inhibitory activities. To the best of our knowledge, Bldesin is the first functionally characterized pathogenic fungus defensin with Kv1.3 channel and chymotrypsin inhibitory activities and highlighted novel pharmacological effects of fungus‐derived defensin peptides.     

Potassium channel gene therapy can prevent neuron death resulting from necrotic and apoptotic insults

Necrotic insults such as seizure are excitotoxic. Logically, membrane hyperpolarization by increasing outwardly conducting potassium channel currents should attenuate hyperexcitation and enhance neuron survival. Therefore, we overexpressed a small-conductance calcium-activated (SK2) or voltage-gated (Kv1.1) channel via viral vectors in cultured hippocampal neurons. We found that SK2 or Kv1.1 protected not only against kainate or glutamate excitotoxicity but also increased survival after sodium cyanide or staurosporine. In vivo overexpression of either channel in dentate gyrus reduced kainate-induced CA3 lesions. In hippocampal slices, the kainate-induced increase in granule cell excitability was reduced by overexpression of either channel, suggesting that these channels exert their protective effects during hyperexcitation. It is also important to understand any functional disturbances created by transgene overexpression alone. In the absence of insult, overexpression of Kv1.1, but not SK2, reduced baseline excitability in dentate gyrus granule cells. Furthermore, while no behavioral disturbances during spatial acquisition in the Morris water maze were observed with overexpression of either channel, animals overexpressing SK2, but not Kv1.1, exhibited a memory deficit post-training. This difference raises the possibility that the means by which these channel subtypes protect may differ. With further development, potassium channel vectors may be an effective pre-emptive strategy against necrotic insults.     

Homeostatic regulation of Kv1.2 potassium channel trafficking by cyclic AMP

The Shaker family potassium channel, Kv1.2, is a key determinant of membrane excitability in neurons and cardiovascular tissue. Kv1.2 is subject to multiple forms of regulation and therefore integrates cellular signals involved in the homeostasis of excitability. The cyclic AMP/protein kinase A (PKA) pathway enhances Kv1.2 ionic current; however, the mechanisms for this are not fully known. Here we show that cAMP maintains Kv1.2 homeostasis through opposing effects on channel trafficking. We found that Kv1.2 is regulated by two distinct cAMP pathways, one PKA-dependent and the other PKA-independent. PKA inhibitors elevate Kv1.2 surface levels, suggesting that basal levels of cAMP control steady-state turnover of the channel. Elevation of cAMP above basal levels also increases the amount of Kv1.2 at the cell surface. This effect is not blocked by PKA inhibitors, but is blocked by inhibition of Kv1.2 endocytosis. We conclude that Kv1.2 levels at the cell surface are kept in dynamic balance by opposing effects of cAMP.     

Bcl-2 decreases voltage-gated K+ channel activity and enhances survival in vascular smooth muscle cells

Cell shrinkage is an incipient hallmark of apoptosis in a variety of cell types. The apoptotic volume decrease has been demonstrated to attribute, in part, to K+ efflux; blockade of plasmalemmal K+ channels inhibits the apoptotic volume decrease and attenuates apoptosis. Using combined approaches of gene transfection, single-cell PCR, patch clamp, and fluorescence microscopy, we examined whether overexpression of Bcl-2, an anti-apoptotic oncoprotein, inhibits apoptosis in pulmonary artery smooth muscle cells (PASMC) by diminishing the activity of voltage-gated K+ (Kv) channels. A human bcl-2 gene was infected into primary cultured rat PASMC using an adenoviral vector. Overexpression of Bcl-2 significantly decreased the amplitude and current density of Kv currents (I(Kv)). In contrast, the apoptosis inducer staurosporine (ST) enhanced I(Kv). In bcl-2-infected cells, however, the ST-induced increase in I(Kv) was completely abolished, and the ST-induced apoptosis was significantly inhibited compared with cells infected with an empty adenovirus (-bcl-2). Blockade of Kv channels in control cells (-bcl-2) by 4-aminopyridine also inhibited the ST-induced increase in I(Kv) and apoptosis. Furthermore, overexpression of Bcl-2 accelerated the inactivation of I(Kv) and downregulated the mRNA expression of the pore-forming Kv channel alpha-subunits (Kv1.1, Kv1.5, and Kv2.1). These results suggest that inhibition of Kv channel activity may serve as an additional mechanism involved in the Bcl-2-mediated anti-apoptotic effect on vascular smooth muscle cells.     

MiR‐30c protects diabetic nephropathy by suppressing epithelial‐to‐mesenchymal transition in db/db mice

Epithelial‐to‐mesenchymal transition (EMT) plays a significant role in tubulointerstitial fibrosis, which is a hallmark of diabetic nephropathy. Thus, identifying the mechanisms of EMT activation could be meaningful. In this study, loss of miR‐30c accompanied with increased EMT was observed in renal tubules of db/db mice and cultured HK2 cells exposed to high glucose. To further explore the roles of miR‐30c in EMT and tubulointerstitial fibrosis, recombinant adeno‐associated viral vector was applied to manipulate the expression of miR‐30c. In vivo study showed that overexpression of miR‐30c suppressed EMT, attenuated renal tubulointerstitial fibrosis and reduced proteinuria, serum creatinine, and BUN levels. In addition, Snail1 was identified as a direct target of miR‐30c by Ago2 co‐immunoprecipitation, luciferase reporter, and Western blot assays. Downregulating Snail1 by siRNA reduced high glucose‐induced EMT in HK2 cells, and miR‐30c mimicked the effects. Moreover, miR‐30c inhibited Snail1‐TGF‐β1 axis in tubular epithelial cells undergoing EMT and thereby impeded the release of TGF‐β1; oppositely, knockdown of miR‐30c enhanced the secretion of TGF‐β1 from epitheliums and significantly promoted proliferation of fibroblasts and fibrogenesis of myofibroblasts, aggravated tubulointerstitial fibrosis, and dysfunction of diabetic nephropathy. These results suggest a protective role of miR‐30c against diabetic nephropathy by suppressing EMT via inhibiting Snail1‐TGF‐β1 pathway.     

Length-dependent regulation of the Kv1.2 channel activation by its C-terminus

The cytoplasmic C-terminus plays regulatory roles in the gating of many ion channels. However, lack of structural information on the C-terminus prevents the elucidation of how the C-terminal domain interacts with the gating machinery to exert its effects on the channel gating. In this report, we investigated the regulatory role of the C-terminus with functional study and structural modeling of a succession of C-terminal truncations of the Kv1.2 and Kv1.2₄₂₇-KcsA_112-160 chimeric channels. Functional study demonstrated a length-dependent shift of the activation curves for the C-terminal truncations of the Kv1.2 channel. Structural modeling indicated that the C-terminus of one subunit could dynamically interact with the S4–S5 linker of a neighboring subunit and the probability of interaction was dependent on the length of the C-terminal truncated Kv1.2 channels. In contrast, no length-dependent shift of the activation curve and probability of interaction between C-terminus and the neighboring S4–S5 linker were observed for the truncations of the Kv1.2-KcsA chimeric channel, suggesting that the native C-terminus of the Kv1.2 channel is essential for the interaction. Furthermore, surface plasmon resonance measurements indicated that there is direct interaction between the C-terminal domain and the S4–S5 linker of the Kv1.2 channel. These results imply that the dynamic interaction of the C-terminus with the S4–S5 linker from a neighboring subunit of the Kv1.2 channel provides a mechanism for its C-terminus to regulate the channel activation.     

Intracellular domains interactions and gated motions of IKS potassium channel subunits

Voltage‐gated K⁺ channels co‐assemble with auxiliary β subunits to form macromolecular complexes. In heart, assembly of Kv7.1 pore‐forming subunits with KCNE1 β subunits generates the repolarizing K⁺ current I_KS. However, the detailed nature of their interface remains unknown. Mutations in either Kv7.1 or KCNE1 produce the life‐threatening long or short QT syndromes. Here, we studied the interactions and voltage‐dependent motions of I_KS channel intracellular domains, using fluorescence resonance energy transfer combined with voltage‐clamp recording and in vitro binding of purified proteins. The results indicate that the KCNE1 distal C‐terminus interacts with the coiled‐coil helix C of the Kv7.1 tetramerization domain. This association is important for I_KS channel assembly rules as underscored by Kv7.1 current inhibition produced by a dominant‐negative C‐terminal domain. On channel opening, the C‐termini of Kv7.1 and KCNE1 come close together. Co‐expression of Kv7.1 with the KCNE1 long QT mutant D76N abolished the K⁺ currents and gated motions. Thus, during channel gating KCNE1 is not static. Instead, the C‐termini of both subunits experience molecular motions, which are disrupted by the D76N causing disease mutation.     

Central leptin gene therapy blocks ovariectomy-induced adiposity

Objective: In this study, we tested the hypothesis that insufficiency of leptin restraint in the hypothalamus is responsible for promoting weight gain and adiposity after ovariectomy (ovx). Whether increasing leptin transgene expression can overcome the diminution in leptin restraint was evaluated in ovx rats. Research Methods and Procedures: Enhanced leptin or green fluorescent protein (GFP; control) transgene expression was induced by a single intracerebroventricular injection of recombinant adeno‐associated viral vector encoding either leptin gene (rAAV‐lep) or GFP gene (rAAV‐GFP; control) in acutely and chronically ovx rats. Body weight and food intake responses were monitored weekly. White adipose tissue (WAT) mass and serum levels of WAT‐derived hormones, leptin, and adiponectin were analyzed at termination of the experiments. Results and Discussion: An increase in leptin transgene expression in the hypothalamus initiated soon after ovx blocked hyperphagia and body weight gain and markedly suppressed WAT mass and adipokines, leptin, and adiponectin. Similar suppression of weight gain and adiposity and serum leptin and adiponectin levels after intracerebroventricular rAAV‐lep injection in chronically ovx rats were observed concomitant with unchanged daily food intake. These findings are consistent with the hypothesis that in the absence of ovarian steroids, the existent insufficiency of leptin restraint at the hypothalamic level can be overcome with ectopic leptin expression, thereby reinstating central control on weight and adiposity.     

RPTPmu and protein tyrosine phosphorylation regulate K(+) channel mRNA expression in adult cardiac myocytes

Previously, we reported that cell-cell contact regulates K(+) channel mRNA expression in cultured adult rat cardiac myocytes. Here we show that exposing cardiac myocytes to tyrosine kinase inhibitors (genistein, tyrphostin A25), but not inactive analogs, prevents downregulation of Kv1.5 mRNA and upregulation of Kv4.2 mRNA normally observed when they are cultured under low-density conditions. Furthermore, cardiac myocytes cocultured with cells that endogenously (Mv 1 Lu) or heterologously (Chinese hamster ovary cells) express the receptor-type protein tyrosine phosphatase mu (RPTPmu) display Kv1.5 mRNA levels paralleling that which was observed in myocytes cultured under high-density conditions and in intact tissue. In contrast, myocytes cocultured with control cells failed to produce this response. Finally, it is shown that Kv4.2 mRNA expression is unaffected by RPTPmu. These findings reveal that multiple tyrosine phosphorylation-dependent mechanisms control cardiac myocyte K(+) channel genes. Furthermore, we conclude that RPTPmu specifically regulates cardiac myocyte Kv1.5 mRNA expression. Thus this receptor protein tyrosine phosphatase may be important in responses to pathological conditions associated with the loss of cell-cell interactions in the heart.     

Leptin Expression in Hypothalamic PVN Reverses Dietary Obesity and Hyperinsulinemia but Stimulates Ghrelin*

Objective: In order to circumvent the multiple peripheral effects of hyperleptinemia and leptin resistance, the efficacy of leptin transgene expression in the hypothalamic paraventricular nucleus (PVN) to reinstate the central energy homeostasis in obesity was examined. Research Methods and Procedures: A recombinant adeno‐associated viral vector encoding either leptin (rAAV‐lep) or green fluorescent protein (rAAV‐GFP) was microinjected into the PVN of obesity‐prone rats consuming a high‐fat diet (HFD). Results: rAAV‐lep, and not rAAV‐GFP, microinjection significantly reduced energy intake and enhanced energy expenditure, thereby resulting in normalization of weight and blood levels of leptin, insulin, free fatty acids, and glucose concomitant with enhanced ghrelin secretion during the extended period of observation. Discussion: Thus, we show, for the first time, that amelioration of leptin insufficiency with enhanced localized leptin availability in the PVN alone can reverse dietary obesity and the attendant hyperinsulinemia and concurrently block the central stimulatory effects of elevated endogenous ghrelin on food intake and adiposity.     

TGF‐β gene transfer and overexpression via rAAV vectors stimulates chondrogenic events in human bone marrow aspirates

Genetic modification of marrow concentrates may provide convenient approaches to enhance the chondrogenic differentiation processes and improve the repair capacities in sites of cartilage defects following administration in the lesions. Here, we provided clinically adapted recombinant adeno‐associated virus (rAAV) vectors to human bone marrow aspirates to promote the expression of the potent transforming growth factor beta (TGF‐β) as a means to regulate the biological and chondrogenic activities in the samples in vitro. Successful TGF‐β gene transfer and expression via rAAV was reached relative to control (lacZ) treatment (from 511.1 to 16.1 pg rhTGF‐β/mg total proteins after 21 days), allowing to durably enhance the levels of cell proliferation, matrix synthesis, and chondrogenic differentiation. Strikingly, in the conditions applied here, application of the candidate TGF‐β vector was also capable of reducing the hypertrophic and osteogenic differentiation processes in the aspirates, showing the potential benefits of using this particular vector to directly modify marrow concentrates to generate single‐step, effective approaches that aim at improving articular cartilage repair in vivo.     

ImKTx1, a new Kv1.3 channel blocker with a unique primary structure

Toxins from the venoms of scorpion, snake, and spider are valuable tools to probe the structure-function relationship of ion channels. In this investigation, a new toxin gene encoding the peptide ImKTx1 was isolated from the venom gland of the scorpion Isometrus maculates by constructing cDNA library method, and the recombinant ImKTx1 peptide was characterized physiologically. The mature peptide of ImKTx1 has 39 amino acid residues including six cross-linked cysteines. The electrophysiological experiments showed that the recombinant ImKTx1 peptide had a pharmacological profile where it inhibited Kv1.3 channel currents with IC(50) of 1.70 n± 1.35 μM, whereas 10 μM rImKTx1 peptide inhibited about 40% Kv1.1 and 42% Kv1.2 channel currents, respectively. In addition, 10 μM rImKTx1 had no effect on the Nav1.2 and Nav1.4 channel currents. Multiple sequence alignments showed that ImKTx1 had no homologous toxin peptide, but it was similar with Ca(2+) channel toxins from scorpion and spider in the arrangement of cysteine residues. These results indicate that ImKTx1 is a new Kv1.3 channel blocker with a unique primary structure. Our results indicate the diversity of K(+) channel toxins from scorpion venoms and also provide a new molecular template targeting Kv1.3 channel.