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Hg1, Novel Peptide Inhibitor Specific for Kv1.3 Channels fromFirst Scorpion Kunitz-type Potassium Channel Toxin Family*□S

Received for publication, January 19, 2012, and in revised form, February 14, 2012 Published, JBC Papers in Press, February 21, 2012, DOI 10.1074/jbc.M112.343996

Zong-Yun Chen‡1, You-Tian Hu‡1, Wei-Shan Yang‡, Ya-Wen He‡, Jing Feng‡, Bin Wang‡, Rui-Ming Zhao‡,Jiu-Ping Ding§, Zhi-Jian Cao‡, Wen-Xin Li‡2, and Ying-Liang Wu‡3

From the ‡State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China and the §KeyLaboratory of Molecular Biophysics, Ministry of Education, College of Life Science and Technology, Huazhong University of Scienceand Technology, Wuhan 430074, China

Background: The potassium channel inhibitory activity of scorpion Kunitz-type toxins has not yet been determined.Results:We identified the first scorpion Kunitz-type potassium channel toxin family with three groups and seven members.Conclusion: A novel peptide, Hg1, specific for Kv1.3 channel, was found.Significance: Kunitz-type toxins are a new source to screen and design potential peptides for diagnosing and treatingKv1.3-mediated autoimmune diseases.

The potassium channel Kv1.3 is an attractive pharmacologi-cal target for autoimmune diseases. Specific peptide inhibitorsare key prospects for diagnosing and treating these diseases.Here, we identified the first scorpion Kunitz-type potassiumchannel toxin family with three groups and seven members. Inaddition to their function as trypsin inhibitors with dissociationconstants of 140 nM for recombinant LmKTT-1a, 160 nM forLmKTT-1b, 124 nM for LmKTT-1c, 136 nM for BmKTT-1, 420nM for BmKTT-2, 760 nM for BmKTT-3, and 107 nM for Hg1, allseven recombinant scorpion Kunitz-type toxins could block theKv1.3 channel. Electrophysiological experiments showed thatsix of seven scorpion toxins inhibited �50–80% of Kv1.3 chan-nel currents at a concentration of 1�M. The exceptionwas rBm-KTT-3, which had weak activity. The IC50 values of rBmKTT-1,rBmKTT-2, and rHg1 for Kv1.3 channels were �129.7, 371.3,and 6.2 nM, respectively. Further pharmacological experimentsindicated that rHg1 was a highly selective Kv1.3 channel inhib-itor with weak affinity for other potassium channels. DifferentfromclassicalKunitz-typepotassiumchannel toxinswithN-ter-minal regions as the channel-interacting interfaces, the chan-nel-interacting interface ofHg1was in theC-terminal region. Inconclusion, these findings describe the first scorpion Kunitz-type potassium channel toxin family, of which a novel inhibitor,Hg1, is specific for Kv1.3 channels. Their structural and func-tional diversity strongly suggest that Kunitz-type toxins are anew source to screen and design potential peptides for diagnos-ing and treating Kv1.3-mediated autoimmune diseases.

In recent years, Kv1.3 channels have been found to be a phar-macological target for autoimmune diseases (1–3). PotentKv1.3 peptide inhibitors have proven effective on different ani-mal models of autoimmune diseases such as multiple sclerosis,type-1 diabetes, rheumatoid arthritis, and psoriasis (2, 4–6). Inpast years, many peptides isolated from various animal venomscould potently inhibit Kv1.3 channels. These toxins usually had30–40 residues cross-linked by three disulfide bridges, such asOdk2,KTX,OSK1,AOSK1,HsTx1, and ShK (7–10).Until now,the screening and design of novel potent and selective Kv1.3inhibitors derived from animal toxins remain an attractiveprospect for disease diagnosis and treatment (11–13).Kunitz-type toxins are an ancient and multifunctional toxin

family, which have been found in various animal venoms, suchas snake, lizard, cattle tick, cone snail, spider, sea anemone, andscorpion (14–18). Members of this family usually have 50–70residues cross-linked by two or three disulfide bridges. Struc-turally, almost all Kunitz-type toxins adopt the conservedstructural fold with two antiparallel �-sheets and one or twohelical regions (19–21). Functionally, many Kunitz-type toxinshave protease and/or potassium channel inhibiting properties.For example, Kunitz-type toxin bungaruskunin, isolated fromsnake venom, is a serine protease inhibitor (22), but �-dentro-toxin, �-dentrotoxin, dentrotoxin K, and dentrotoxin I, alsofrom snake venom, are potent Kv1.1 channel inhibitors (21).Kunitz-type toxins HWTX-XI from spider and APEKTx1,AKC1, AKC2, and AKC3 from sea anemone are bifunctionaltoxin peptides with both protease and potassium channel-in-hibiting properties (20, 23, 24). Conkunitzin-S1, a 60-residuecone snail Kunitz-type toxin cross-linked by two disulfidebridges, interacts with the shaker potassium channel (19, 25).From scorpion venoms, three Kunitz-type toxins, Hg1, SdPI,and SdPI-2 have been isolated, but only SdPI was found toinhibit trypsin (26, 27). Until now, the potential potassiumchannel inhibitory activity of scorpion Kunitz-type toxin hasnot been determined.To identify novel peptide inhibitors specific for Kv1.3 chan-

nels, we screened scorpion Kunitz-type toxins and evaluatedtheir pharmacological activities for potassium channels. By

* This work was supported by grants from the National Basic Research Pro-gram of China (2010CB529800), the National Natural Sciences Foundationof China (30530140, 31071942, and 30973636), and the Hubei ProvinceNatural Sciences Foundation of China (2009CDA076).

□S This article contains supplemental Figs. S1–S4.1 Both authors contributed equally to this work.2 To whom correspondence may be addressed: State Key Laboratory of Virol-

ogy, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072,China.Tel.:86-0-27-68752831;Fax:86-0-27-68752146;E-mail:[email protected].

3 To whom correspondence may be addressed: State Key Laboratory of Virol-ogy, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072,China. Tel.: 86-0-27-68752831; Fax: 86-0-27-68752146; E-mail: [email protected].

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 17, pp. 13813–13821, April 20, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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cDNA cloning, bioinformatic analyses, and functional evalua-tions, we identified the first scorpion Kunitz-type potassiumchannel toxin family composed of four newmembers (LmKTT-1c, BmKTT-1, BmKTT-2, and BmKTT-3) and three knownmembers (LmKTT-1a, LmKTT-1b, and Hg1) (26, 27). In addi-tion to their functions as trypsin inhibitors, six of the recombi-nant scorpion Kunitz-type toxins also block 50–80% of Kv1.3currents at a concentration of 1 �M. The exception was rBm-KTT-3, which had weak activity. Among these peptides, a spe-cific Kv1.3 inhibitor Hg1 was discovered with an IC50 value of�6.2 � 1.2 nM. Significantly different from classical Kunitz-type potassium channel toxins with the N-terminal region asthe channel-interacting interface, Hg1 adopted the C-terminalregion as the main channel-interacting interface. Our resultsdescribe the first scorpion Kunitz-type potassium channeltoxin family, and the identification of the specific Kv1.3 inhib-itor Hg1. Kunitz-type toxins are a new group of toxins that canbe used to screen and design potential peptides for diagnosingand treating Kv1.3-mediated autoimmune diseases.

MATERIALS AND METHODS

cDNA Library Construction and Screening—Venom glandcDNA libraries of scorpion Buthus martensii, Isometrus macu-lates, Lychas mucronatus, Heterometrus spinifer, Scorpiopstibetanus, and Scorpiops jendeki were constructed as describedin our previous work (26). Some new randomly selected colo-nies were sequenced using the ABI 3730 automated sequencer(Applied Biosystems, Foster City, CA). Sequences were identi-fied for open reading frames using the ORF finder program(http://www.ncbi.nlm.nih.gov/projects/gorf/). After excludingthe signal peptides, the similarity was annotated by searchingagainst the GenBankTMNCBI database (http://www.ncbi.nlm-.nih.gov/blast) using BLAST algorithms. Three known genesencoding Kunitz-type toxins Hg1, SdPI, and SdPI-2, and fournew genes encoding Kunitz-type toxins, BmKTT-1, BmKTT-2,BmKTT-3, and LmKTT-1c were chosen. According to thenomenclature proposed recently for all peptide toxins (41),LmKTT-1a, LmKTT-1b, LmKTT-1c, BmKTT-1, BmKTT-2,and BmKTT-3 would be named �-BUTX-Lm3a, �-BUTX-Lm3b, �-BUTX-Lm3c, �-BUTX-Bm4a, �-BUTX-Bm4b,�-BUTX-Bm4c, respectively, but the simple names will be usedthroughout this paper.Expression and Purification of Scorpion Kunitz-type Toxins—

We used the cDNA sequences of LmKTT-1a, LmKTT-1b, andLmKTT-1c from Lychas mucronatus venom gland cDNAlibraries and BmKTT-1, BmKTT-2, and BmKTT-3 from scor-pion B. martensii cDNA libraries as the templates for PCR togenerate respective fragments (27). The PCR product of Hg1was generated by overlapping PCR. All PCR products weredigested with NdeI and XhoI and inserted into expression vec-tor pET-28a. After confirmation by sequencing, the plasmidswere transformed into competent Escherichia coli BL21(DE3)cells for expression.QuikChange site-directedmutagenesis kits(Stratagene, Santa Clara, CA) were used for generating themutants based on the wild-type plasmid pET-28a-Hg1. Allmutant plasmids were verified by DNA sequencing beforeexpression. Kunitz-type toxins and mutants were expressedaccording to our previous protocol (26). For example, the

recombinant LmKTT-1a was found to accumulate exclusivelyin inclusion bodies andwas refolded in vitro. Renatured proteinwas finally purified by HPLC on a C18 column (10 mm � 250mm, 5 �m Dalian Elite). Peaks were detected at 230 nm. Thefraction containing recombinant LmKTT-1a was eluted at20–21 min and further analyzed by MALDI-TOF-MS (Voyag-er-DESTR, Applied Biosystems).Determination and Modeling of Scorpion Kunitz-type Toxin

Structures—The secondary structures of scorpion Kunitz-type toxins and mutants with a control peptide BPTI wereanalyzed by circular dichroism (CD) spectroscopy. All sam-ples were dissolved in water at a concentration of 0.2 mg/ml.Spectra were recorded at 25 °C from 250 to 190 nm with ascan rate of 50 nm/min, on a Jasco-810 spectropolarimeter(Jasco Analytical Instruments, Easton, MD). The CD spectrawere collected from averaging three scans after subtractingthe blank spectrum of water. The three-dimensional struc-ture of Hg1 was modeled using BPTI (PDB4 code 6PTI) as atemplate through the SWISS-MODEL server as we havedescribed previously (28).Molecular Docking—Molecular docking of Hg1 interacting

with the Kv1.3 channel was carried our as previous compu-tational approaches (13, 29). First, the structure of the Kv1.3channel was modeled using KcsA (PDB code 1K4C) as a tem-plate. Second, molecular docking was performed on themodeled Hg1 peptide and Kv1.3 channels using the ZDOCKprogram (30), and the docking results were then filtered byscoring combined with detailed mutagenesis information;Finally, a reasonable Hg1-Kv1.3 complex that was consistentwith the experimental alanine-scanning mutagenesis wasscreened out.Serine Protease Inhibitory Activity Assay—The inhibitory

activities of seven Kunitz-type toxins were tested in the pres-ence of serine proteases as described previously (26). Trypsin(bovine pancreatic trypsin; EC 3.4.21.4), �-chymotrypsin(bovine pancreatic �-chymotrypsin; EC 3.4.21.1), elastase (por-cine pancreatic elastase; EC 3.4.21.36), and their respectivechromogenic substrates, Na-benzoyl-L-arginine, 4-nitroanilidehydrochloride,N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide,N-succinyl-Ala-Ala-Ala-p-nitroanilide, were purchased fromSigma (Sigma-Aldrich). Trypsin was incubated with variousamounts of rHg1 (100 to 400 nM) for 30 min at a final concen-tration of 400 nM. The reactions were initiated by adding vary-ing concentrations of substrate, ranging from 0.1 to 0.8 mM.The initial rate of p-nitroanilide formation wasmonitored con-tinuously at 405 nm for 5min at 25 °C. The inhibitory activity ofrHg1 was estimated by setting the initial velocity obtained withonly protease as 100%. The inhibitory constant (Ki) of thetrypsin-inhibitor complexwas determined by Lineweaver-Burkplots and further slope replotting. Themethods for chymotryp-sin and elastase assay were the same, except the final concen-tration of chymotrypsin was 100 nM.Electrophysiological Recordings—The cDNAs encoding

mKv1.1, hKv1.2, mKv1.3, and hSKCa3 were provided gener-ously by professor Stephan Grissmer (University of Ulm, Ulm,

4 The abbreviation used is: PDB, Protein Data Bank.

Scorpion Kunitz-type Inhibitor Specific for Kv1.3 Channels

13814 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 17 • APRIL 20, 2012

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Germany) and professorGeorge Chandy (University of Califor-nia, Irvine, CA).We saved themBKCa channel. All of the chan-nels were subcloned into the pIRES2-EGFP vector (Clontech,Mountain View, CA) and transformed into HEK293 cells. Thewhole-cell patch clamp was used to measure and record thechannel currents according to a previously described procedure(13, 31). Peptides were dissolved in stock solutions containing1% BSA and diluted into solutions containing 0.01% BSA fortoxin application in electrophysiological experiments.Results are shown asmean� S.E., withn being the number of

experiments. The significance between two means was calcu-latedwith the Student’s t test usingOrigin software (version 6.0,Microcal Software, Northampton, MA). Differences in themean values were considered significant at probability �0.05.Using IGOR software (WaveMetrics, Lake Oswego, OR), con-centration versus response relationships were fitted accordingto the modified Hill equation: Itoxin/Icontrol � 1/1 � ([toxin]/IC50), where I is the peak current, and [toxin] is the concentra-tion of toxin. The parameters to be fitted were concentration athalf-maximal effect (IC50).

RESULTS

Primary Structures of Scorpion Kunitz-type Toxins—On thebasis of our cDNA libraries and further random sequencing,four new genes encoding Kunitz-type toxins were obtained.Three were isolated from scorpion B. martensii, which werenamed BmKTT-1, BmKTT-2, and BmKTT-3, and one was iso-lated from scorpion L. mucronatus and named LmKTT-1c.Combined with the three known Kunitz-type toxins, Hg1,LmKTT-1a, and LmKTT-1b (26, 27), seven scorpion Kunitz-type toxins can be classified into three groups according to theirdisulfide bridge patterns (Fig. 1). Hg1 and BmKTT-3 belong tothe first group, which adopted classical disulfide pairings simi-lar to HWTX-XI toxin from spider (20), dendrotoxin K fromsnake (32), and APEKTx1 from sea anemone (23). LmKTT-1a,LmKTT-1b, LmKTT-1c, and BmKTT-1 belong to the secondgroup, which adopted a unique cystine framework that wedescribed in our previous work (26). These toxins lack the nor-mal CysII–CysIV disulfide bonds present in BmKTT-3 fromgroup I but contain two new cysteine residues near the C ter-minus of the mature peptide. Different from all known Kunitz-type toxins from various animals, BmKTT-2 in the third grouphas eight cysteine residues, which may adopt novel disulfidebonds (17). Our findings demonstrate the molecular diversityof scorpion Kunitz-type toxins.

Preparation and Structural Analysis of Scorpion Kunitz-typeToxins—To evaluate the function of scorpion Kunitz-type tox-ins, we obtained seven recombinant toxins as described previ-ously (26, 27). Expression and purification of LmKTT-1a toxinwere as follows. A His6 tag and a thrombin cleavage site werefused to LmKTT-1a at the N terminus. Inclusion bodies ofLmKTT-1a fusion peptide were suspended in LB medium andrefolded successfully in vitro. The soluble protein was furtherseparated by RP-HPLC and SDS-PAGE (supplemental Fig. S1,A and B). By MALDI-TOF MS, the molecular weight was8657.8 Da, which is in good agreement with the calculatedmolecular mass of 8658.6 Da (supplemental Fig. S1C).BPTI is a classical Kunitz-type peptide (33). By circular

dichroism spectroscopy, all seven recombinant scorpionKunitz-type toxins were found to have similar secondary struc-tures to BPTI (supplemental Fig. S2), which suggests the con-served structures of Kunitz-type peptides.Trypsin Inhibitory Activities of Scorpion Kunitz-type Toxins—

Based on the conserved structures of scorpion Kunitz-type tox-ins, they were assayed for inhibitory activity against trypsin,

FIGURE 1. Primary structures of scorpion Kunitz-type toxins. Seven Kunitz-type toxins from scorpion, including four new Kunitz-type toxins; BmKTT-1,BmKTT-2, and BmKTT-3 from scorpion B. martensii, LmKTT-1c from scorpion Lychas mucronatus, and three known Kunitz toxins, LmKKT-1a, LmKTT-1b, and Hg1.Identical and similar residues are noted. The cysteine residues are marked with Roman numerals.

FIGURE 2. Inhibitory effects of scorpion Kunitz-type toxins on trypsin.A, inhibitory effects of rHg1 peptide on trypsin with BPTI and BSA as controls.B, inhibitory effects of seven scorpion Kunitz-type toxins at different concen-trations on trypsin using the same conditions. Data represent the mean � S.E.of at least three experiments.



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chymotrypsin, and elastase. All seven recombinant toxinsexhibited apparent inhibitory effects on trypsin (Fig. 2), butno inhibitory effect on chymotrypsin or elastase, even athigher concentrations. Recombinant LmKTT-1a, LmKTT-1b,LmKTT-1c, BmKTT-1, and Hg1 could completely inhibit thetrypsin activity at the ratio of 1:1 with a dissociation constant of140, 160, 124, 136, and 107 nM, respectively (supplemental Fig.S3). The rBmKTT-2 could completely inhibit the trypsin activ-ity at a ratio of �1.5:1, with a dissociation constant of 420 nM,and rBmKTT-3 could inhibit �85% of the trypsin activity at aratio of �4:1, with a dissociation constant of 760 nM (Table 1).

Inhibitory Activities of Scorpion Kunitz-type Toxins on Kv1.3Potassium Channel—The pharmacological activities of thescorpion Kunitz-type toxins on Kv1.3 channels were evaluated.Fig. 3,A andC–H, show the inhibitory effects onKv1.3 currentsat a concentration of 1 �M. rLmKTT-1a, rLmKTT-1b, andrLmKTT-1c could inhibit �50% of Kv1.3 channels currents,whereas rHg1, rBmKTT-1, and rBmKTT-2 could inhibit�60–80% of Kv1.3 channels currents. The rBmKTT-3 had a weakeffect on Kv1.3 channel currents. However, rHg1 at a decreasedconcentration of 100 nM still had a significant inhibitory effecton Kv1.3 channels (Fig. 3B). Concentration-dependent experi-ments further showed that rBmKTT-1, rBmKTT-2, and rHg1inhibited Kv1.3 channel currents with IC50 values of 129.7 �31.3 nM, 371.3 � 82.1, and 6.2 � 1.2 nM, respectively (Fig. 3,I–K). These results provide the first characterization of a scor-pion Kunitz-type potassium channel toxin family, which con-tains seven newmembers and can inhibit both potassium chan-nels and trypsin.Hg1, Selective Inhibitor for Kv1.3 Channels—Based on the

pharmacological properties of rHg1, the first potent Kv1.3channel inhibitor with a Kunitz-type fold, we further investi-gated its effects on different types of potassium channels. Asshown in Fig. 4, rHg1 could inhibit �50% of the Kv1.1 andKv1.2 channel currents at a concentration of 1�M (Fig. 4,A andB), and had little effect on SKCa3 and BKCa channel currents atidentical concentrations (Fig. 4, C and D). These data indicatethat Hg1 is a selective peptide inhibitor specific for Kv1.3channels.

FIGURE 3. Effects of seven scorpion Kunitz-type toxins on mKv1.3 channel currents. A and B, blocking effects of rHg1 on mKv1.3 K� currents. C, blockingeffects of rBmKKT-3 on mKv1.3 currents. D, blocking effects of rLmKTT-1a on mKv1.3 currents. E, blocking effects of rLmKTT-1b on mKv1.3 currents. F, blockingeffects of rLmKTT-1c on mKv1.3 currents. G, blocking effects of rBmKTT-1 on mKv1.3 currents. H, blocking effects of rBmKTT-2 on mKv1.3 currents. I, concen-tration-dependent inhibition of mKv1.3 channels by rBmKTT-1. J, concentration-dependent inhibition of mKv1.3 channels by rBmKTT-2. K, concentration-de-pendent inhibition of mKv1.3 channels by rHg1. Data represent the mean � S.D. of at least three experiments.

TABLE 1Kunitz-type potassium channel toxins from animal venoms

Name SourceTrypsinactivity

Channelactivity Ref.

nM nMHg 1 Scorpion 107 6.2 (Kv1.3) Ref. 26 and this workaLmKTT-1a Scorpion 140 �1000 (Kv1.3) Ref. 25 and this workaLmKTT-1b Scorpion 160 �1000 (Kv1.3) Ref. 25 and this workbLmKTT-1c Scorpion 124 �1000 (Kv1.3) This workBmKTT-1 Scorpion 136 129.7 (Kv1.3) This workBmKTT-2 Scorpion 420 371.3 (Kv1.3) This workBmKTT-3 Scorpion 760 �1000 (Kv1.3) This work�-DTX Snake �c 0.004 (Kv1.1) Ref. 20�-DTX Snake � �0.01 (Kv1.1)HWTX-XI Spider 23 2570 (Kv1.1) Ref. 19Conk-S1 Conus � 502 (Shaker) Ref. 24APEKTx1 Sea anemone 124 0.9 (Kv1.1) Ref. 22AKC1 Sea anemone �30 2800 (Kv1.2) Ref. 23AKC2 Sea anemone �30 1100 (Kv1.2)AKC3 Sea anemone �30 1300 (Kv1.2)a First function characterization in this work.b First characterization of potassium channel inhibitory function in this work.c �, lack of activity.



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Unique Molecular Mechanism of Hg1 Blocking Kv1.3 Chan-nels—By using the alanine-scanning strategy, we investigatedthe molecular mechanism of Hg1 toxin blocking Kv1.3 chan-nels. As shown in Fig. 5,A–H, there were no apparent effects of

His-2, His-3, Asn-4, Arg-5, Leu-9, Leu-10, and Lys-13 residueson the toxin pharmacological activities, whereas there were lessconformational changes for toxin mutants (Fig. 5, I and J).These data indicate that Hg1 toxin did not use N-terminal res-

FIGURE 4. Effects of rHg1 on other potassium channel currents. A, blocking effects of rHg1 on mKv1.1 currents. B, blocking effects of rHg1 on hKv1.2 currents.C, blocking effects of rHg1 on hSKCa3 currents. D, blocking effects of rHg1 on mBKCa currents.

FIGURE 5. Effects of the Hg1 mutants on mKv1.3 channel currents. A–H, representative current traces of mKv1.3 channel showing the blockage of currentsby Hg1 and its mutants. A, 100 nM Hg1; B, 100 nM Hg1-H2A; C, 100 nM Hg1-H3A; D, 100 nM Hg1-N4A; E, 100 nM Hg1-R5A; F, 100 nM Hg1-L9A; G, 100 nM Hg1-L10A;H, 100 nM Hg1-K13A. I, circular dichroism spectra of Hg1, Hg1-H2A, Hg1-H3A, and Hg1-N4A peptides. J, circular dichroism spectra of Hg1, Hg1-R5A, Hg1-L9A,Hg1-L10A, and Hg1-K13A peptides. deg, degrees.



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idues to inhibit the Kv1.3 channel, whichwas different from theknown molecular mechanism of Kunitz-type toxins such as�-dendrotoxin and HWTX-XI. These toxins mainly use N-ter-minal residues to block Kv1.1 channels (20, 34). Among classi-cal animal toxins affecting potassium channels, basic residuesas critical residues are common features (35–37). We thenfocused the second cluster of basic residues, located at the Cterminus al region ofHg1 toxin (Fig. 1) and found the dominanteffects of Lys-56, Arg-57, Phe-61, and Lys-63 residues on thetoxin affinities when using identical 100 nM concentration ofwild-type and mutant Hg1 toxin with less conformationalchanges (Fig. 6, A–E). The IC50 values were 582.0 � 184.5 nMfor Hg1-K56A, 305.0 � 93.7 nM for Hg1-R57A, 360.1 � 116.5nM for Hg1-F61A, and 457.9 � 187.3 nM for Hg1-K63Amutants. Replacement by alanine reduced the ability of thetoxin to inhibit Kv1.3 channels by �94-, 49-, 58-, and 74-fold,respectively (Fig. 6F). These structure and function relation-ships demonstrate that Hg1 toxin mainly uses the C-terminalregion as the channel-interacting interface to inhibit Kv1.3channels.To further reveal the recognitionmechanism of Hg1 peptide

toward Kv1.3 channels, a structural model of the Hg1-Kv1.3complex was obtained through our previous computationalapproaches (13, 34). The importance of four Lys-56, Arg-57,Phe-61, and Lys-63 residues was indicated by the structuralanalysis. Lys-56 is the pore-blocking residue, which is sur-rounded by the pore region residues from Kv1.3 channelswithin a contact distance of 4 Å (Fig. 7A). Phe-61 mainly con-tacts Pro-280, His-307, Val-309, and Lys-314 residues of thechannel A chain and Asp-387 residue of channel D chain byhydrophobic interactions (Fig. 7B). Lys-63mainly contacts Ser-378, Ser-379, Gly-380, and Asn-382 residues of the channel Dchain (Fig. 7C), and Arg-57 mainly contacts Pro-377, Ser-378,

Ser-379, His-404, Pro-405, and Val-406 of the channel D chain(Fig. 7D). The structural model of the Hg1-Kv1.3 complex sup-ported the inference thatHg1mainly used theC-terminal as thechannel-interacting surface.

DISCUSSION

The Kv1.3 potassium channel is an attractive pharmacologi-cal target for autoimmune diseases, and specific peptide inhib-itors are useful tools for diagnosing and treating these diseases(38, 39). To screen and design the potent and selective peptideinhibitors, efforts to improve peptide specificity are continuing(11–13). Kunitz-type toxins are a kind of ancient toxin familythat has been identified in many animal venoms, such as thoseof snake, cone snail, spider, sea anemone, and scorpion (14–18). Several Kunitz-type toxins have been found to inhibitpotassium channels (Table 1).In this work, we adopted a new strategy to screen novel pep-

tide inhibitor specific for Kv1.3 channels from scorpionKunitz-type toxins.We identified the first scorpion Kunitz-type potas-sium channel toxin family with three groups and sevenmembers, from which a novel peptide inhibitor, Hg1, specificfor Kv1.3 channels, was obtained. Overall, our work providedfollowing unique structural and functional features of theKunitz-type potassium channel toxin family.Molecular Diversity of Kunitz-type Potassium Channel

Toxins—Combined with the known Kunitz-type toxinsaffecting potassium channels (Table 1) (21), 17 toxins werefound to block potassium channels. Among these, there weresignificant differences in toxin sequences, sequence lengths,and number and distribution of cysteine residues (20). Mostnotably, BmKTT-2 toxin from scorpion was found to formfour disulfide bridges, which is different from all knownKunitz-type animal toxins (17). The finding of seven addi-

FIGURE 6. Functional importance of residues in the C-terminal region of Hg1. A–D, representative current traces of mKv1.3 channels showing the block ofcurrents by Hg1 mutants: A, 100 nM Hg1-K56A; B, 100 nM Hg1-R57A; C, 100 nM Hg1-F61A; and D, 100 nM Hg1-K63A. E, the circular dichroism spectra analyses ofHg1, Hg1-K56A, Hg1-R57A, Hg1-F61A, and Hg1-K63A peptides. F, concentration-dependent inhibition of Kv1.3 channel currents by Hg1, Hg1-K56A, Hg1-R57A,Hg1-F61A, and Hg1-K63A peptides. Data represent the mean � S.D. of at least three experiments.



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tional scorpion Kunitz-type toxins strongly enriched themolecular diversity of Kunitz-type toxins inhibiting potas-sium channels.Conserved Structures of Kunitz-type Toxins—Studies have

shown that the backbone of dendrotoxin I, a potassium channelblocker from snake venom, superimposes on BPTI with an root

mean square deviation of�1.7 Å (33). Using circular dichroismspectroscopy analyses, we also found structural similaritybetween BPTI peptide and seven scorpion Kunitz-type toxins,especially with different disulfide bridges (supplemental Fig.S2). The structural similarity ofHg1 andBPTIwas shownby theBPTI structure (PDB code 6PTI) and the Hg1 structural model

FIGURE 7. Interaction of Hg1 with mKv1.3 channels explored by computational simulation. A, the key residue Lys-56 was surrounded mainly by the poreregion residues from Kv1.3 channels within a contact distance of 4 Å. B–D, Phe-61, Lys-63, and Arg-57 contacted the residues of mKv1.3 channels, respectively.

FIGURE 8. Differential binding interfaces of Kunitz-type toxins blocking potassium channels. A, sequence alignments of Kunitz-type potassium channeltoxins Hg1 from scorpion, �-DTX from snake, and HWTX-XI from spider. B, the main functional residues of Kunitz-type toxin �-DTX (structure modeled withDTX-K as template, PDB code 1DTK) interacting with the Kv1.1 channel. C, the main functional residues of Kunitz-type toxin HWTX-XI (PDB code 2JOT)interacting with the Kv1.1 channel. D, the main functional residues of Kunitz-type toxin Hg1 (structure modeled with BPTI as template, PDB code 6PTI)interacting with Kv1.3 channels.



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(supplemental Fig. S4). This indicates that structures were con-served for Kunitz-type toxins.Functional Diversity of Kunitz-type Potassium Channel

Toxins—Previous reports showed several Kunitz-type toxinscould affect Kv1.1, Kv1.2, and Shaker channels (Table 1). In thiswork, we found that scorpion Kunitz-type toxins could inhibitKv1.3 channels. Except for the scorpion Kunitz-type toxinrBmKTT-3, the other six toxins inhibited �50–80% of theKv1.3 currents at a concentration of 1 �M (Fig. 3). The IC50value of rHg1 for Kv1.3 channels was 6.2 � 1.2 nM. The func-tional diversity of Kunitz-type potassium channel toxins willhopefully encourage a functional evaluation of this kind of tox-ins on additional potassium channels.Unique Inhibitory Mechanism of Kunitz-type Potassium

Channel Toxins—Kunitz-type toxins �-dendrotoxin andHWTX-XI mainly use N-terminal residues to block Kv1.1channels (Fig. 8, A–C) (20, 40). In contrast, Hg1 toxin mainlyuses C-terminal residues as a channel-interacting interface toinhibit Kv1.3 channels (Fig. 8D).In conclusion, we have characterized the first scorpion

Kunitz-type potassium channel toxin family with the uniquepharmacological property of blocking Kv1.3 channels. Fromthis toxin family, a potent and selective Kv1.3 channel inhibitor,Hg1, was identified. Hg1 is the first Kunitz-type toxin identifiedthat interacts with potassium channels by its C-terminal regionas the main channel interacting interface. The structural andfunctional diversity of these Kunitz-type potassium channeltoxins may provide a new source of potassium channel inhibi-tors used for the diagnosis and treatment of autoimmunedisorders.

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Rui-Ming Zhao, Jiu-Ping Ding, Zhi-Jian Cao, Wen-Xin Li and Ying-Liang WuZong-Yun Chen, You-Tian Hu, Wei-Shan Yang, Ya-Wen He, Jing Feng, Bin Wang,

Kunitz-type Potassium Channel Toxin FamilyHg1, Novel Peptide Inhibitor Specific for Kv1.3 Channels from First Scorpion

doi: 10.1074/jbc.M112.343996 originally published online February 21, 20122012, 287:13813-13821.J. Biol. Chem.

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hg1,novelpeptideinhibitorspecificforkv1.3channelsfrom ... · constants of 140 nm for recombinant...

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