In silico detection of binding mode of J-superfamily conotoxin pl14a with Kv1.6 channel

A novel conotoxin pl14a containing 25 amino acid residues with an amidated C-terminus from vermivorous cone snail, Conus planorbis belongs to J-conotoxin superfamily and this is the first conotoxin, which inhibits both nicotinic acetylcholine receptor subtypes and Kv1.6 channel. We have attempted through bioinformatics approaches to elucidate the extent of specificity of pl14a towards Kv1 channel subtypes (Kv1.1-Kv1.6). Our work provides rationale for the relatively high specificity and binding mode of pl14a to Kv1.6 channel. The pl14a peptide contains two types of structural elements, namely the putative dyad (Lys18 and Tyr19) and basic residue ring constituted of arginine residues. We have carried out in silico docking studies so as to assess the contribution of one or combination of both structural elements of pl14a in blocking of Kv1.6 channel. For this purpose, we have built by homology modelling, the theoretical 3D structure of Kv1.6 channel based on the available crystal structure of mammalian shaker Kv1.2 channel. Docking studies suggest that positively charged residues ring may be involved in the blocking mechanism of Kv1.6 channel. The models suggest that the peptide interacts with negatively charged extracellular loops and pore-mouth of the potassium channel and blocks the channel by covering the pore as a lid, akin to previously proposed blocking mechanism of kappaM-conotoxin RIIIK from Conus radiatus to Tsha1 potassium channel. The newly detected pharmacophore for pl14a interacting with Kv1.6 channel provides a pointer to experimental work to validate the observations made here. Based on differences in the number and distribution of the positively-charged residues in other conopeptides from the J-superfamily, we hypothesize different selectivity profiles against subtypes of the potassium channels for these conopeptides.     

东亚钳蝎氯毒素BmKCT的表达和功能鉴定(英文)

从中国东亚钳蝎ButhusmartensiiKarsch的毒腺cDNA文库中分离得到了一个编码毒素蛋白多肽 (命名为BmKCT)前体的全长cDNA序列 .该毒素多肽与已报道的氯毒素 (chlorotoxin)高度同源 ,其蛋白质一级序列有 6 8%的同源性 .为了鉴定BmKCT的生物学功能 ,通过pGEX系统成功地表达了BmKCT ,并用GST亲和层析和凝胶过滤的方法获得了纯化的重组BmKCT毒素蛋白 (rBmKCT) .通过膜片钳实验 ,记录了rBmKCT对人脑星型胶质瘤细胞 (gliomascell)表面的氯离子通道电流的作用 .结果显示 ,BmKCT可以显著抑制人脑星型胶质瘤细胞表面的氯离子通道电流 ,并且这种抑制作用在一定程度上是可逆的 .实验证明 ,在细胞水平上 ,BmKCT是一种新的短链氯离子通道抑制剂 .     

Structural basis of a potent peptide inhibitor designed for Kv1.3 channel, a therapeutic target of autoimmune disease

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.     

Identification of a new specific Kv1.3 channel blocker, Ctri9577, from the scorpion Chaerilus tricostatus

Scorpion toxins are valuable resources for discovering new ion channel modulators and drug candidates. Potassium channel Kv1.3 is an important pharmacological target of T cell-mediated autoimmune diseases, which are encouraging the screening and design of the specific peptide blockers for Kv1.3 channel. Ctri9577, the first neurotoxin gene of Chaerilidae family was cloned from the venom of the scorpion Chaerilus tricostatus through the constructing its cDNA library. The sequence analysis showed that the mature peptide of Ctri9577 contained 39 amino acid residues including six conserved cysteines, whose low sequence similarity indicated that it was a new member of α-KTx15 subfamily. By using expression and purification technology, the recombinant peptide was obtained. Subsequently, the electrophysiological experiments indicated that the Ctri9577 peptide selectively inhibited Kv1.3 channel current with an IC(50) of 0.49±0.45 nM without effectively blocking potassium channels Kv1.1, Kv1.2, hERG and SK3. All these findings not only enrich the knowledge of toxins from the Chaerilidae family, but also present a novel potential drug candidate targeting Kv1.3 channels for the therapy of autoimmune diseases.     

Different residues in channel turret determining the selectivity of ADWX-1 inhibitor peptide between Kv1.1 and Kv1.3 channels

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.     

Blockage of human T lymphocyte Kv1.3 channels by Pi1, a novel class of scorpion toxin

Using the patch-clamp technique we determined that Pandinus imperator toxin Pi1, a recently described peptide toxin having four disulfide bridges instead of the usual three in scorpion toxins, blocked Kv1.3 channels of human T lymphocytes from the extracellular side with a 1:1 stoichiometry. Kv1.3 block was instantaneous and removable with toxin-free extracellular solution. The toxin did not influence activation or inactivation of the channels. We found that Pi1 blocked Kv1.3 with less affinity (K(d) = 11.4 nM) than the structurally related three disulfide bridge containing toxins Pi2 (50 pM) and Pi3 (0.5 nM). The fourth disulfide bridge in Pi1 had no influence on the channel binding ability of the toxin; the less effective block was due to differences in amino acid side chain properties at positions 11 and 35.     

The antibody targeting the E314 peptide of human Kv1.3 pore region serves as a novel, potent and specific channel blocker

Selective blockade of Kv1.3 channels in effector memory T (T(EM)) cells was validated to ameliorate autoimmune or autoimmune-associated diseases. We generated the antibody directed against one peptide of human Kv1.3 (hKv1.3) extracellular loop as a novel and possible Kv1.3 blocker. One peptide of hKv1.3 extracellular loop E3 containing 14 amino acids (E314) was chosen as an antigenic determinant to generate the E314 antibody. The E314 antibody specifically recognized 63.8KD protein stably expressed in hKv1.3-HEK 293 cell lines, whereas it did not recognize or cross-react to human Kv1.1(hKv1.1), Kv1.2(hKv1.2), Kv1.4(hKv1.4), Kv1.5(hKv1.5), KCa3.1(hKCa3.1), HERG, hKCNQ1/hKCNE1, Nav1.5 and Cav1.2 proteins stably expressed in HEK 293 cell lines or in human atrial or ventricular myocytes by Western blotting analysis and immunostaining detection. By the technique of whole-cell patch clamp, the E314 antibody was shown to have a directly inhibitory effect on hKv1.3 currents expressed in HEK 293 or Jurkat T cells and the inhibition showed a concentration-dependence. However, it exerted no significant difference on hKv1.1, hKv1.2, hKv1.4, hKv1.5, hKCa3.1, HERG, hKCNQ1/hKCNE1, L-type Ca(2+) or voltage-gated Na(+) currents. The present study demonstrates that the antibody targeting the E314 peptide of hKv1.3 pore region could be a novel, potent and specific hKv1.3 blocker without affecting a variety of closely related K(v)1 channels, KCa3.1 channels and functional cardiac ion channels underlying central nervous system (CNS) disorders or drug-acquired arrhythmias, which is required as a safe clinic-promising channel blocker.     

Mapping the functional anatomy of BgK on Kv1.1, Kv1.2, and Kv1.3. Clues to design analogs with enhanced selectivity

BgK is a peptide from the sea anemone Bunodosoma granulifera, which blocks Kv1.1, Kv1.2, and Kv1.3 potassium channels. Using 25 analogs substituted at a single position by an alanine residue, we performed the complete mapping of the BgK binding sites for the three Kv1 channels. These binding sites included three common residues (Ser-23, Lys-25, and Tyr-26) and a variable set of additional residues depending on the particular channel. Shortening the side chain of Lys-25 by taking out the four methylene groups dramatically decreased the BgK affinity to all Kv1 channels tested. However, the analog K25Orn displayed increased potency on Kv1.2, which makes this peptide a selective blocker for Kv1.2 (K(D) 50- and 300-fold lower than for Kv1.1 and Kv1.3, respectively). BgK analogs with enhanced selectivity could also be made by substituting residues that are differentially involved in the binding to some of the three Kv1 channels. For example, the analog F6A was found to be >500-fold more potent for Kv1.1 than for Kv1.2 and Kv1.3. These results provide new information about the mechanisms by which a channel blocker distinguishes individual channels among closely related isoforms and give clues for designing analogs with enhanced selectivity.     

T1-T1 interactions occur in ER membranes while nascent Kv peptides are still attached to ribosomes

For voltage-gated K+ channels (Kv), it is not clear at which stage during biosynthesis in the endoplasmic reticulum (ER) oligomerization occurs, specifically whether it can begin while nascent peptide chains of individual subunits are still attached to ribosomes. Kv channels possess a T1-recognition domain in the NH2-terminus, which confers subfamily specificity for intersubunit assembly and forms a tetramer. Using pairs of cysteines engineered into the T1-T1 interface and cross-linking methods, we show that specific residues in the T1-T1 interface of different Kv1.3 subunits come into close proximity in the ER, both in microsomal membranes and in Xenopus oocytes. Furthermore, using translocation intermediates containing pairs of engineered cysteines in the T1 interface, we demonstrate that specific residues in the folded T1 domain interface can approach within 2 A of each other and form tetramers while the nascent Kv1.3 peptides are still attached to ribosomes and have translocated across the membrane. ER membranes are required for this interaction, and T1-T1 interactions occur inter-polysomally. Thus, folding of the T1 domain and intersubunit interaction may represent the first assembly event in channel formation.     

Chemical synthesis and characterization of Pi1, a scorpion toxin from Pandinus imperator active on K+ channels.

Pi1 is a 35-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the chactidae scorpion Pandinus imperator. Due to its very low abundance in the venom, we have chemically synthesized this toxin in order to study its biological activity. Enzyme-based proteolytic cleavage of the synthetic Pi1 (sPi1) demonstrates half-cystine pairings between Cys4-Cys25, Cys10-Cys30, Cys14-Cys32 and Cys20-Cys35, which is in agreement with the disulfide bridge organization initially reported on the natural toxin. In vivo, intracerebroventricular injection of sPi1 in mice produces lethal effects with an LD50 of 0.2 microgram per mouse. In vitro, the application of sPi1 induces drastic inhibition of Shaker B (IC50 of 23 nM) and rat Kv1.2 channels (IC50 of 0.44 nM) heterologously expressed in Xenopus laevis oocytes. No effect was observed on rat Kv1.1 and Kv1.3 currents upon synthetic peptide application. Also, sPi1 is able to compete with 125I-labeled apamin for binding onto rat brain synaptosomes with an IC50 of 55 pM. Overall, these results demonstrate that sPi1 displays a large spectrum of activities by blocking both SK- and Kv1-types of K+ channels; a selectivity reminiscent of that of maurotoxin, another structurally related four disulfide-bridged scorpion toxin that exhibits a different half-cystine pairing pattern.     

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.     

Clustering of neuronal potassium channels is independent of their interaction with PSD-95

Voltage-dependent potassium channels regulate membrane excitability and cell-cell communication in the mammalian nervous system, and are found highly localized at distinct neuronal subcellular sites. Kv1 (mammalian Shaker family) potassium channels and the neurexin Caspr2, both of which contain COOH-terminal PDZ domain binding peptide motifs, are found colocalized at high density at juxtaparanodes flanking nodes of Ranvier of myelinated axons. The PDZ domain-containing protein PSD-95, which clusters Kv1 potassium channels in heterologous cells, has been proposed to play a major role in potassium channel clustering in mammalian neurons. Here, we show that PSD-95 colocalizes precisely with Kv1 potassium channels and Caspr2 at juxtaparanodes, and that a macromolecular complex of Kv1 channels and PSD-95 can be immunopurified from mammalian brain and spinal cord. Surprisingly, we find that the high density clustering of Kv1 channels and Caspr2 at juxtaparanodes is normal in a mutant mouse lacking juxtaparanodal PSD-95, and that the indirect interaction between Kv1 channels and Caspr2 is maintained in these mutant mice. These data suggest that the primary function of PSD-95 at juxtaparanodes lies outside of its accepted role in mediating the high density clustering of Kv1 potassium channels at these sites.     

Engineering-specific pharmacological binding sites for peptidyl inhibitors of potassium channels into KcsA

The bacterial potassium channel, KcsA, can be modified to express a high-affinity receptor site for the scorpion toxin kaliotoxin (KTX) by substituting subregion I in the P region of KcsA with the one present in the human voltage-gated potassium channel Kv1.3 [Legros, C., Pollmann, V., Knaus, H. G., Farrell, A. M., Darbon, H., Bougis, P. E., Martin-Eauclaire, M. F., and Pongs, O. (2000) J. Biol. Chem. 275, 16918-16924]. This approach opened the way to investigate whether sequence differences in subregion I of Kv1 channels correlate with the distinct pharmacological profiles of peptide inhibitors. A panel of six chimeras between KcsA and human Kv1.1-6 were constructed, expressed in Escherichia coli, purified to homogeneity, and assessed in filter binding assays using either monoiodo-tyrosine-KTX ([(125)I]KTX) or monoiodo-tyrosine-hongotoxin(1)(A19Y/Y37F) ([(125)I]HgTX(1)(A19Y/Y37F)). The KcsA-Kv1.X chimeras were found to have lower affinities for these ligands than the corresponding mammalian Kv1.X channels, indicating that other parts of the channels may contribute to binding or that subtle structural differences exist between these channels. The properties of the KcsA-Kv1.X chimeras were also characterized in surface plasmon resonance experiments. KcsA-Kv1.3 chimeras were immobilized on the surface of a sensor chip for determining, in real time, binding of the peptides. KTX binding properties to immobilized KcsA-Kv1.3 chimera were similar to those determined by filtration techniques. Taken together, our results demonstrate that the pharmacological profile of peptide toxins can be incorporated into KcsA-Kv1.X chimeras containing the subregion I of the corresponding mammalian Kv1.X channels. This innovative approach may facilitate the high-throughput screening of ligand libraries aimed at the discovery of novel potassium channel modulators.     

Synaptosome-associated protein of 25 kilodaltons modulates Kv2.1 voltage-dependent K(+) channels in neuroendocrine islet beta-cells through an interaction with the channel N terminus

Insulin secretion is initiated by ionic events involving membrane depolarization and Ca(2+) entry, whereas exocytic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate exocytosis itself. In the present study, we characterize the interaction of the SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) with the beta-cell voltage-dependent K(+) channel Kv2.1. Expression of Kv2.1, SNAP-25, and syntaxin 1A was detected in human islet lysates by Western blot, and coimmunoprecipitation studies showed that heterologously expressed SNAP-25 and syntaxin 1A associate with Kv2.1. SNAP-25 reduced currents from recombinant Kv2.1 channels by approximately 70% without affecting channel localization. This inhibitory effect could be partially alleviated by codialysis of a Kv2.1N-terminal peptide that can bind in vitro SNAP-25, but not the Kv2.1C-terminal peptide. Similarly, SNAP-25 blocked voltage-dependent outward K(+) currents from rat beta-cells by approximately 40%, an effect that was completely reversed by codialysis of the Kv2.1N fragment. Finally, SNAP-25 had no effect on outward K(+) currents in beta-cells where Kv2.1 channels had been functionally knocked out using a dominant-negative approach, indicating that the interaction is specific to Kv2.1 channels as compared with other beta-cell Kv channels. This study demonstrates that SNAP-25 can regulate Kv2.1 through an interaction at the channel N terminus and supports the hypothesis that SNARE proteins modulate secretion through their involvement in regulation of membrane ion channels in addition to exocytic membrane fusion.     

Characterization of a new Kv1.3 channel-specific blocker, J123, from the scorpion Buthus martensii Karsch

The potassium channel Kv1.3 is an attractive pharmacological target for T-cell-mediated autoimmune diseases, and specific and selective peptidic blockers of Kv1.3 channels have served as valuable therapeutic leads for treating these diseases. Here, we found a new peptide toxin, J123, with 43 amino acids including six cysteine residues by screening the venomous cDNA library of scorpion Buthus martensii Karsch, which has been used as traditional medicine in China for more than 2000 years. The sequence analysis suggested that peptide J123 constituted a new member of the alpha-KTx toxins. The electrophysiological experiments further indicated that peptide J123 has a novel pharmacological profiles: it blocked Kv1.3 channel with high potency (IC(50)=0.79nM), and exhibited good selectivity on Kv1.3 over Kv1.1 (>1000-fold) and Kv1.2 (about 30-fold), respectively. Furthermore, peptide J123 had no activity on SKCa2 and SKCa3 channels at micromolar concentration level. Based on the pharmacological activities, the possible channel-interacting surface of peptide J123 was also predicted by molecular modeling and docking. All these data not only enrich the knowledge of the structure-function relationship of the new Kv1.3-speicific peptide but also present a potential drug candidate for selectively targeting Kv1.3 channels.     

Chemical synthesis and 1H-NMR 3D structure determination of AgTx2-MTX chimera, a new potential blocker for Kv1.2 channel, derived from MTX and AgTx2 scorpion toxins

Agitoxin 2 (AgTx2) is a 38-residue scorpion toxin, cross-linked by three disulfide bridges, which acts on voltage-gated K(+) (Kv) channels. Maurotoxin (MTX) is a 34-residue scorpion toxin with an uncommon four-disulfide bridge reticulation, acting on both Ca(2+)-activated and Kv channels. A 39-mer chimeric peptide, named AgTx2-MTX, was designed from the sequence of the two toxins and chemically synthesized. It encompasses residues 1-5 of AgTx2, followed by the complete sequence of MTX. As established by enzyme cleavage, the new AgTx2-MTX molecule displays half-cystine pairings of the type C1-C5, C2-C6, C3-C7, and C4-C8, which is different from that of MTX. The 3D structure of AgTx2-MTX solved by (1)H-NMR, revealed both alpha-helical and beta-sheet structures, consistent with a common alpha/beta scaffold of scorpion toxins. Pharmacological assays of AgTx2-MTX revealed that this new molecule is more potent than both original toxins in blocking rat Kv1.2 channel. Docking simulations, performed with the 3D structure of AgTx2-MTX, confirmed this result and demonstrated the participation of the N-terminal domain of AgTx2 in its increased affinity for Kv1.2 through additional molecular contacts. Altogether, the data indicated that replacement of the N-terminal domain of MTX by the one of AgTx2 in the AgTx2-MTX chimera results in a reorganization of the disulfide bridge arrangement and an increase of affinity to the Kv1.2 channel.     

N-type Inactivation Features of Kv4.2 Channel Gating

We examined whether the N-terminus of Kv4.2 A-type channels (4.2NT) possesses an autoinhibitory N-terminal peptide domain, which, similar to the one of Shaker, mediates inactivation of the open state. We found that chimeric Kv2.1(4.2NT) channels, where the cytoplasmic Kv2.1 N-terminus had been replaced by corresponding Kv4.2 domains, inactivated relatively fast, with a mean time constant of 120 ms as compared to 3.4 s in Kv2.1 wild-type. Notably, Kv2.1(4.2NT) showed features typically observed for Shaker N-type inactivation: fast inactivation of Kv2.1(4.2NT) channels was slowed by intracellular tetraethylammonium and removed by N-terminal truncation (Δ40). Kv2.1(4.2NT) channels reopened during recovery from inactivation, and recovery was accelerated in high external K⁺. Moreover, the application of synthetic N-terminal Kv4.2 and ShB peptides to inside-out patches containing slowly inactivating Kv2.1 channels mimicked N-type inactivation. Kv4.2 channels, after fractional inactivation, mediated tail currents with biphasic decay, indicative of passage through the open state during recovery from inactivation. Biphasic tail current kinetics were less prominent in Kv4.2/KChIP2.1 channel complexes and virtually absent in Kv4.2Δ40 channels. N-type inactivation features of Kv4.2 open-state inactivation, which may be suppressed by KChIP association, were also revealed by the finding that application of Kv4.2 N-terminal peptide accelerated the decay kinetics of both Kv4.2Δ40 and Kv4.2/KChIP2.1 patch currents. However, double mutant cycle analysis of N-terminal inactivating and pore domains indicated differences in the energetics and structural determinants between Kv4.2 and Shaker N-type inactivation.     

Electrostatic interaction between inactivation ball and T1–S1 linker region of Kv1.4 channel

Inactivation of potassium channels plays an important role in shaping the electrical signaling properties of nerve and muscle cells. The rapid inactivation of Kv1.4 has been assumed to be controlled by a “ball and chain” inactivation mechanism. Besides hydrophobic interaction between inactivation ball and the channel's inner pore, the electrostatic interaction has also been proved to participate in the “ball and chain” inactivation process of Kv1.4 channel. Based on the crystal structure of Kv1.2 channel, the acidic T1–S1 linker is indicated to be a candidate interacting with the positively charged hydrophilic region of the inactivation domain. In this study, through mutating the charged residues to amino acids of opposite polar, we identified the electrostatic interaction between the inactivation ball and the T1–S1 linker region of Kv1.4 channel. Inserting negatively charged peptide at the amino terminal of Kv1.4 channel further confirmed the electrostatic interaction between the two regions.     

Electrostatic interaction between inactivation ball and T1-S1 linker region of Kv1.4 channel

Inactivation of potassium channels plays an important role in shaping the electrical signaling properties of nerve and muscle cells. The rapid inactivation of Kv1.4 has been assumed to be controlled by a "ball and chain" inactivation mechanism. Besides hydrophobic interaction between inactivation ball and the channel's inner pore, the electrostatic interaction has also been proved to participate in the "ball and chain" inactivation process of Kv1.4 channel. Based on the crystal structure of Kv1.2 channel, the acidic T1-S1 linker is indicated to be a candidate interacting with the positively charged hydrophilic region of the inactivation domain. In this study, through mutating the charged residues to amino acids of opposite polar, we identified the electrostatic interaction between the inactivation ball and the T1-S1 linker region of Kv1.4 channel. Inserting negatively charged peptide at the amino terminal of Kv1.4 channel further confirmed the electrostatic interaction between the two regions.     

Inhibition of Kv2.1 voltage-dependent K+ channels in pancreatic beta-cells enhances glucose-dependent insulin secretion

Voltage-dependent (Kv) outward K(+) currents repolarize beta-cell action potentials during a glucose stimulus to limit Ca(2+) entry and insulin secretion. Dominant-negative "knockout" of Kv2 family channels enhances glucose-stimulated insulin secretion. Here we show that a putative Kv2.1 antagonist (C-1) stimulates insulin secretion from MIN6 insulinoma cells in a glucose- and dose-dependent manner while blocking voltage-dependent outward K(+) currents. C-1-blocked recombinant Kv2.1-mediated currents more specifically than currents mediated by Kv1, -3, and -4 family channels (Kv1.4, 3.1, 4.2). Additionally, C-1 had little effect on currents recorded from MIN6 cells expressing a dominant-negative Kv2.1 alpha-subunit. The insulinotropic effect of acute Kv2.1 inhibition resulted from enhanced membrane depolarization and augmented intracellular Ca(2+) responses to glucose. Immunohistochemical staining of mouse pancreas sections showed that expression of Kv2.1 correlated highly with insulin-containing beta-cells, consistent with the ability of C-1 to block voltage-dependent outward K(+) currents in isolated mouse beta-cells. Antagonism of Kv2.1 in an ex vivo perfused mouse pancreas model enhanced first- and second-phase insulin secretion, whereas glucagon secretion was unaffected. The present study demonstrates that Kv2.1 is an important component of beta-cell stimulus-secretion coupling, and a compound that enhances, but does not initiate, beta-cell electrical activity by acting on Kv2.1 would be a useful antidiabetic agent.