toxins Article Centipede KCNQ Inhibitor SsTx Also Targets KV1.3 Canwei Du 1, Jiameng Li 1, Zicheng Shao 1, James Mwangi 2,3, Runjia Xu 1, Huiwen Tian 1, Guoxiang Mo 1, Ren Lai 1,2,4,* and Shilong Yang 2,4,* 1 College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China; [email protected] (C.D.); [email protected] (J.L.); [email protected] (Z.S.); [email protected] (R.X.); [email protected] (H.T.); [email protected] (G.M.) 2 Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Yunnan Province, Kunming Institute of Zoology, Kunming 650223, Yunnan, China; [email protected] 3 University of Chinese Academy of Sciences, Beijing 100009, China 4 Sino-African Joint Research Center, Chinese Academy of Science, Wuhan 430074, Hubei, China * Correspondence: [email protected] (R.L.); [email protected] (S.Y.) Received: 27 December 2018; Accepted: 27 January 2019; Published: 1 February 2019 Abstract: It was recently discovered that Ssm Spooky Toxin (SsTx) with 53 residues serves as a key killer factor in red-headed centipede’s venom arsenal, due to its potent blockage of the widely expressed KCNQ channels to simultaneously and efficiently disrupt cardiovascular, respiratory, muscular, and nervous systems, suggesting that SsTx is a basic compound for centipedes’ defense and predation. Here, we show that SsTx also inhibits KV1.3 channel, which would amplify the broad-spectrum disruptive effect of blocking KV7 channels. Interestingly, residue R12 in SsTx extends into the selectivity filter to block KV7.4, however, residue K11 in SsTx replaces this ploy when toxin binds on KV1.3. Both SsTx and its mutant SsTx_R12A inhibit cytokines production in T cells without affecting the level of KV1.3 expression. The results further suggest that SsTx is a key molecule for defense and predation in the centipedes’ venoms and it evolves efficient strategy to disturb multiple physiological targets. Keywords: Centipede; SsTx; KV7; KV1.3; toxin Key Contribution: The study identifies the key amino acids for SsTx inhibition on KV1.3 channel, which is different from that on KV7.4 and shows the inhibition on KV1.3 is important in suppressing cytokines secretion in T cells. 1. Introduction With over 3000 species, centipedes, arthropods of the class Chilopoda are widely distributed in both tropical and subtropical regions. Their first pair of legs have been modified into strong mandibles and venomous fangs called forcipules that play a critical role in defense and predation [1]. Centipede stings occur frequently in areas, such as Hawaii, Brazil, Australia, Taiwan, and Japan [2], evoking an issue of social health care. Centipede toxins are composite mixtures, which are responsible for both local and systemic reactions. Recently, we have identified and characterized several peptide toxins from centipede venom and established they act on ion channels that are associated with pain and inflammation [3–7]. For example, a centipede toxin (RhTx) causes intense local pain by targeting the heat activation machinery of nociceptor TRPV1 (transient receptor potential cation channel subfamily V member 1) channel [4]. We also revealed the molecular basis of cardiovascular, respiratory, muscular, and nervous system’s dysfunction due to centipede bites by the identification of Ssm Spooky Toxin (SsTx). It targets KCNQ channels (voltage-gated potassium channel family 7) to exhibit potent lethal Toxins 2019, 11, 76; doi:10.3390/toxins11020076 www.mdpi.com/journal/toxins Toxins 2019, 11, 76 2 of 9 Toxins 2019, 11, x FOR PEER REVIEW 2 of 9 toxicityMechanically, by causing SsTx severe binds vesselsto KV7 channels and respiratory tightly, disordersdue to the orfact seizures. that residue Mechanically, K13 in SsTx SsTx anchors binds the to KtoxinV7 channels to the outer tightly, pore due region to the factof K thatV7 channels residue K13and in residue SsTx anchors R12 in theSsTx toxin extends to the into outer the pore selectivity region offilter KV7 to channels block the and channels residue [8]. R12 The inrefore, SsTx extends SsTx shows into thephysiological selectivity and filter pathological to block the importance channels [8 in]. Therefore,understanding SsTx showsthe efficiency physiological in prey and capture pathological and clinical importance reports in after understanding centipede envenomation. the efficiency inIn preythe present capture study, and clinical we established reports after that centipede SsTx also envenomation. inhibited KV1.3 In thethat present have been study, reported we established to be an thatimportant SsTx also target inhibited for autoimmune KV1.3 that diseases have been [9– reported11]. Through to be the an importantconstruction target of toxin for autoimmune mutants, we diseasesfound that [9– SsTx11]. Through used different the construction side chains ofto toxintarget mutants, KV1.3, which we found allowed that us SsTx to modify used differentthem towards side chainsspecifically to target targeting KV1.3, K whichV1.3 channel allowed in us human to modify T cells. them Furthermore, towards specifically SsTx and targeting its mutant KV 1.3suppressed channel inTem human-effector T cells. cells Furthermore, proliferation SsTxand cytokine and its mutant production suppressed in T cells. Tem-effector cells proliferation and cytokine production in T cells. 2. Results 2. Results 2.1. SsTx Inhibits KV1.3 2.1. SsTx Inhibits KV1.3 As described in our previous work [8], centipede prey on animals via SsTx inhibiting the KV7.4. SsTxAs consists described of in53 our amino previous acids work refolded [8], centipede by two prey intracellular on animals disulfide via SsTx inhibitingbonds (Figure the K V1A).7.4. SsTx consists of 53 amino acids refolded by two intracellular disulfide bonds (Figure1A). Surprisingly, Surprisingly, we found that SsTx toxin also inhibited KV1.3. As illustrated in Figure1A, SsTx potently we found that SsTx toxin also inhibited K 1.3. As illustrated in Figure1A, SsTx potently inhibited inhibited KV1.3 currents, with an IC50 valueV of 5.26 μM (Figure 1B). As a pore blocker, SsTx inhibited K 1.3 currents, with an IC value of 5.26 µM (Figure1B). As a pore blocker, SsTx inhibited K 1.3 KVV1.3 channel in a voltage50-dependent manner (Figure 1C), similar to the inhibition of KV7.4. TakenV channeltogether, in our a voltage-dependent results, which are manner consistent (Figure with1C), a similarprevious to report the inhibition [12], suggest of K V 7.4.that Taken the βαββ together,-type our results, which are consistent with a previous report [12], suggest that the βαββ-type toxins have a toxins have a higher affinity for KV7 and KV1.3 channels. The fact that SsTx shows no effect on Slo1 higherchannel affinity implies for KthatV7 and the K V1.3binding channels. surface The factof thatthe SsTxscorpion shows toxin no effect charybdotoxin on Slo1 channel may implies have thatsophisticatedly the binding surfaceevolved of to the use scorpion a different toxin mechanism charybdotoxin to inhibit may Slo1 have channel sophisticatedly [13–15]. evolved to use a different mechanism to inhibit Slo1 channel [13–15]. Figure 1. The effect of Ssm Spooky Toxin (SsTx) on KV1.3. (A)KV1.3 currents were inhibited by 10 µM Figure 1. The effect of Ssm Spooky Toxin (SsTx) on KV1.3. (A) KV1.3 currents were inhibited by 10 μM SsTx. The black and red traces represent bath solution and solution containing 10 µM SsTx, respectively. SsTx. The black and red traces represent bath solution and solution containing 10 μM SsTx, The upper insert shows the sequence of SsTx and the cysteine residues are presented in red color. respectively. The upper insert shows the sequence of SsTx and the cysteine residues are presented in (B) Concentration-response curves displaying the inhibition of SsTx on KV7.4 and KV1.3. Data were red color. (B) Concentration-response curves displaying the inhibition of SsTx on KV7.4 and KV1.3. fitted with a Hill equation. The IC50 values are 2.80 ± 0.23 µM for KV7.4 (n = 5 cells) and 5.26 ± 0.56 µM Data were fitted with a Hill equation. The IC50 values are 2.80 ± 0.23 μM for KV7.4 (n = 5 cells) and 5.26 for KV1.3 (n = 10 cells). (C) The relationship between the inhibitory percentage of 10 µM SsTx on KV1.3 ± 0.56 μM for KV1.3 (n = 10 cells). (C) The relationship between the inhibitory percentage of 10 μM and the test pulses. The cells were held at −80 mV (n = 4–6 cells). SsTx on KV1.3 and the test pulses. The cells were held at −80 mV (n = 4–6 cells). 2.2. K11 and K13 in SsTx Are Crucial for Inhibiting KV1.3 2.2. K11 and K13 in SsTx Are Crucial for Inhibiting KV1.3 Toxins with multiple functions have been widely utilized to probe the structure–function relationshipToxins ofwith ion channelsmultiple [16functions,17]. Given have that been SsTx widely targets bothutilized KV1.3 to andprobe KV 7the channels, structure we– studiedfunction therelationship key residues of ion for channels their bio-activity [16,17]. Given on KV that1.3 andSsTx K targetsV7 channels. both K OurV1.3 previousand KV7 channels, results demonstrate we studied thatthe therekey residues are two for direct their interactions bio-activity betweenon KV1.3 SsTxand K andV7 channels KV7.4: The. Our side previous chainof results K13 ondemonstrate the toxin anchorsthat there it toare the two outer direct pore interactions region of KbetweenV7.4, and SsTx the and side K chainV7.4: T ofhe R12 side extends chain of into K13 the on selectivity the toxin filteranchors (Figure it to2 theA). outer Because pore blockage region of of K KV7.4,V7 channels and the side is considered chain of R12 to beextends toxic, into such the information selectivity mayfilter direct(Figure our 2A).