Toxicon 43 (2004) 961–971 www.elsevier.com/locate/toxicon
A novel scorpion toxin blocking small conductance Ca2þ activated Kþ channelq Chen-Qi Xua,b, Lin-Lin Hec, Bert Broˆned, Marie-France Martin-Eauclairee, Emmy Van Kerkhoved, Zhuan Zhouc, Cheng-Wu Chia,b,* a
Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China b The Institute of Protein Research, Tong Ji University, 1239 Si-Ping Road, Shanghai 200092, China c Institute of Neuroscience, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China d Laboratory of Physiology, Biomed, Limburgs Universitair Centrum, B-3590 Diepenbeek, Belgium e Laboratoire de Biochimie, CNRS UMR 6560, Faculte de Me´decine Nord, IFR Jean Roche, Universite´ de la Me´diterrane´e, Marseille, France Received 19 August 2003; accepted 8 January 2004 Available online 10 May 2004
Abstract Small conductance calcium activated potassium channels (SK) are crucial in the regulation of cell firing frequency in the nervous system and other tissues. In the present work, a novel SK channel blocker, designated BmSKTx1, was purified from the scorpion Buthus martensi Karsh venom. The sequence of the N-terminal 22 amino acid residues was determined by Edman degradation. Using this sequence information, the full-length cDNA and genomic gene of BmSKTx1 were cloned and sequenced. By these analyses, BmSKTx1 was found to be a peptide composed of 31 amino acid residues with three disulfide bonds. It shared little sequence homology with other known scorpion a-KTxs but showed close relationship with SK channel blockers in the phylogenetic tree. According to the previous nomenclature, BmSKTx1 was classified as a-KTx14.1. We examined the effects of BmSKTx1 on different ion channels of rat adrenal chromaffin cells (RACC) and locust dorsal unpaired median (DUM) neurons. BmSKTx1 selectively inhibited apamin-sensitive SK currents in RACC with Kd of 0.72 mM and Hill coefficient of 2.2. And it had no effect on Naþ, Ca2þ, Kv, and BK currents in DUM neuron, indicating that BmSKTx1 was a selective SK toxin. q 2004 Elsevier Ltd. All rights reserved. Keywords: SK channel; Amino acid sequence; Gene cloning; Phylogenetic tree
1. Introduction Small conductance calcium-activated potassium channels (SK channel) play a fundamental role in various tissues, including the nervous system (Rimini et al., 2000), heart q The genomic gene data of BmSKTx1 are available in the GenBank database under the accession number AF295594. * Corresponding author. Address: Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China. Tel.: þ 86-21-5492-1165; fax: þ86-21-5492-1011. E-mail address:
[email protected] (C.-W. Chi).
0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2004.01.018
(Schetz and Anderson, 1995) and others (Park, 1994; Pribnow et al., 1999). After being activated by the increased intracellular Ca2þ concentration, SK channels mediate the slow afterhyperpolarization (sAHP), which may persist for several seconds (Hirschberg et al., 1998). Though the duration of sAHP varies considerably in different cell types, it contributes to spike-frequency adaptation, which protects cells from deleterious effects of continuous firing and is essential for normal neurotransmission (Bond et al., 1999). To date, three SK subtypes, designated SK1-3, have been cloned from rat and human brains (Kohler et al., 1996). The changes of their expression levels trigger many physiological responses. For instance, an over-expression of SK3 can
962
C.-Q. Xu et al. / Toxicon 43 (2004) 961–971
induce an abnormal respiratory response to hypoxia and compromised parturition. This suggests that SK3 channel could be a potential therapeutic target for disorders such as sleep apnea and sudden infant death syndrome and for regulating uterine contractions during labor (Bond et al., 2000). To elucidate the precise function of each SK channel, it is important to search for their selective ligands. Natural peptide blockers have been used as efficient tools for the structure and function studies of SK channels. Apamin, an 18 amino acid neurotoxin from honeybee venom, was the first discovered SK blocker (Habermann, 1984) and it contributed a lot to study the molecular pharmacology of SK channels. By using the 125I-apamin competition assay, six SK blockers were found in different scorpion venoms, e.g. Leiurotoxin I (scyllatoxin or Lei I), (Sabatier et al., 1994), P05 (Sabatier et al., 1993), BmP05 (Romi-Lebrun et al., 1997), Tityus serrulatus k (Tsk) (Legros et al., 1996), Pandinus imperator 1 (Pi1) (Fajloun et al., 2000) and Maurotoxin (MTX) (Kharrat et al., 1997). All these scorpion SK blockers compete with apamin for the binding to the rat brain synapotosomal membrane, indicating that they share the same binding site(s) on SK channel. Because of their functional similarity with apamin, these scorpion toxins do not appear to provide additional benefits to the study on SK channel. Here we report the primary structure, gene cloning and function characterization of a novel SK blocker, BmSKTx1, purified from the scorpion Buthus martensi Karsh (BmK) venom. Like other scorpion toxins, it is a short peptide with three disulfide bonds but shares little sequence homology with other known scorpion SK blockers. Using rat adrenal chromaffin cells (RACC), we found that BmSKTx1 has a limited effect on the apamin-sensitive SK currents in RACC by maximum 65% blockade.
2. Materials and methods 2.1. Materials The crude venom of scorpion BmK was obtained from a scorpion farm (Henan province, China). Tetrodotoxin was purchased from Alomone Labs (Jerusalem, Israel). Apamin and bovine serum albumin were from Sigma (St Louis, MO, USA). Na25I, Sephadex G-50 and DEAE Sephacel were purchased from Amersham Pharmacia (Uppsala, Sweden). HPLC-grade acetonitrile and Trifluoroacetic acid (TFA) were from Merck (Darmstadat, Germany). The 30 RACE and 50 RACE kit and TRIzol Reagent were from Life Technology (Gainthersburg, MD, USA). PCR preps DNA purification system, Minipreps DNA purification system and pGEM-T Easy vector system were from Promega (Madison, WI, USA). E. Coli strain DH5a was used for transformation of pGEM-T Easy vector. Other solvents and reagents were of analytical grade.
2.2. Purification of BmSKTx1 The venom of scorpion Buthus martensi Karsh (300 mg) was first fractionated by a Sephadex G-50 column as previously described (Wang et al., 2001). Peak 2 containing various neurotoxins was applied to a DEAE Sephacel column (3 £ 6.5 cm2) previously equilibrated with 20 mM Na2CO3 – NaHCO3 buffer, pH 10.5. The breakthrough fraction was pooled and loaded to a semi-preparative RP-HPLC C18 column (1 £ 25 cm2, Beckman) equilibrated with buffer A (0.1% TFA in water) at a flow rate of 2 ml/ min. Proteins were eluted by a two-step gradient system: 0– 36% buffer B (70% acetonitrile in buffer A) for 36 min and 37 –46% buffer B for 15 min. Fraction 2 from the C18 column was applied to a Mono-S column (0.5 £ 5 cm2, Pharmacia) equilibrated with 50 mM HAc-NaAc buffer, pH 4.3 at a flow rate of 1 ml/min. Elution was carried out by a linear gradient of 0 – 1 M NaCl in the acetate buffer for 40 min. The component of the first peak (BmSKTx1) was used for the following studies. 2.3. 3 0 and 5 0 RACE 30 and 50 RACE were performed as previously described (Wu et al., 1999) A gene specific primer 1 [50 -TT(TC) GC(GATC) AT(ACT) AA(GA) TG(TC) GC-30 ] corresponding to the N-terminal sequence (Phe-Ala-Ile-LysCys-Ala) of BmSKTx1 was synthesized. Using the gene specific primer 1 and an abridged amplification primer provided in the kit, the 30 -end partial cDNA of BmSKTx1 was amplified by PCR. The PCR product containing polyA tail was directly cloned into pGEM-T Easy vector for sequencing. On the basis of the 30 -end partial cDNA sequence of BmSKTx1, the antisense primer 2 [50 -TGT ACA AGC GCA AAA TCC-30 ] corresponding to the residues 31-26, and its nested antisense primer 3 [50 -A TCC ATT TCT GCA AGG AG-30 ] corresponding to the 26-21 residues, were designed and synthesized. With the same strategy as described previously (Wu et al., 1999), the 50 -end cDNA of BmSKTx1was then amplified and sequenced. 2.4. Amplification of genomic DNA In accordance with the determined cDNA sequence of BmSKTx1, the gene specific primer 4 [50 -AGA TTT TAA AAT TGT ATA CTT TTC C-30 ], corresponding to the 30-6 base upstream of the initial codon ATG, and its antisense primer 5 [50 -CAA TGA TTA ATT TTA CAC ATA CTG G30 ], corresponding to the 48-24 base downstream of the second stop codon TAG, were designed. Using the genomic DNA extracted from the telsons of the scorpion BmK as a template, the genomic gene of BmSKTx1 was then amplified by PCR. The PCR product was directly cloned into pGEM-T Easy vector for sequencing.
C.-Q. Xu et al. / Toxicon 43 (2004) 961–971
2.5. Patch clamp recording on RACC Rat adrenal medulla chromaffin cells were prepared as described previously (Zhou and Misler, 1995). Cells were studied under whole cell voltage clamp using the nystatin perforated patch-clamp technique (Horn and Marty, 1988). The holding potential was 270 mV. For the measurement of the SK current, we used an internal pipette solution containing (in mM) 145 KCl, 8 NaCl, 1 MgCl2, 10 HEPES and 250 mg/ml nystatin, pH 7.2, and an standard external bath solution containing (in mM) 125 NaCl, 2.8 KCl, 10 CaCl2, 1 MgCl2 and 10 HEPES, pH 7.4. Experiments were performed with Axon 200B (Axon Instruments, CA, USA) or the PC-2B (INBIO Inc., Wuhan, China) patch clamp amplifiers using the pClamp 8 data acquisition system. BmSKTx1 and the control/wash solution were puffed locally to the cell via RCP-2B multi-channel micro perfusion system (INBIO Inc., Wuhan, China). The puffing pipette of 100 mm tip diameter was located about 120 mm away from the cell. Data were analyzed with Igor software (AveMatrix, OR, USA). Dose-dependent curves were fit to the equation: g ¼ 100={1 þ ð½toxin=Kd Þn }: g is the relative response (%); n is the Hill coefficient; [toxin] is the toxin concentration; and Kd is the dissociation constant.
963
the samples were centrifuged at 11,000g for 1 min. The supernatants were aspirated and the pellets were washed twice with 1 ml cold buffer. The radioactivity associated with the pellets was measured using a g Packard spectrometer (RIASTAR systeme) and non-specific binding (, 10% of the total binding) was subtracted. 2.8. Sequence analysis and construction of a phylogenetic tree The amino acid sequences of BmSKTx1, other scorpion SK toxins and 26 toxins belonging to other a-KTx subfamilies were selected to do analysis (Dudina et al., 2001; Strong et al., 2001; Tytgat et al., 1999; Vacher et al., 2001). Multiple alignment of these sequences was performed by the means of CLUSTALX programme (Thompson et al., 1997). This alignment was input into MEGA2 (Molecular Evolutionary Genetics Analysis Software Version 2) (Kumar et al., 2001) to obtain an unrooted Neighbor-Joining (NJ) tree. Evolutionary distances were computed by Poisson Correction and the bootstrap was set 1000 replications.
3. Results
2.6. Patch clamp recording on locust DUM neurons The cell bodies of the dorsal unpaired median (DUM) neurons of Locusta migratoria were isolated as previously described (Brone et al., 2003). The patch clamp technique was used in the whole cell voltage clamp configuration (Hamill et al., 1981). Whole cell currents were measured in voltage clamp experiments with a PC controlled EPC9 patch clamp amplifier (HEKA Elektronik, Lambrecht, Germany). The holding potential was 2 100 mV. The extracellular solution contained (in mM) 172.5 NaCl, 6.5 KCl, 7.7 MgCl2, 2 CaCl2, 10 HEPES and 13 glucose, pH 6.80. Tetrodotoxin (TTX, 100 nM) was added to block Naþ currents when necessary. The internal pipette solution contained (in mM) 160 K gluconate, 6.5 NaCl, 1 CaCl2, 10 EGTA, 10 HEPES, 2 MgATP, and 45 glucose, pH 6.65. Data were analyzed with Origin 6.0 (Micoral Software, Northampton, USA). 2.7.
125
I-apamin competition assay
The preparations of synaptosomal membranes (P2) from the rat brain and 125I-apamin (2 Ci/mol) were achieved as previously described (Seagar et al., 1985). A buffer, 25 mM Tris – HCl, 10 mM KCl and 0.1% bovine serum albumin, pH 7.4, was used for both incubation and washing. BmSKTx1 or other competitors (50 ml) at a final concentration from 0.1 pM to 1 mM were incubated with 125I-apamin (50 ml) at 10 pM and 0.4 mg of P2 (400 ml). The non-specific binding was determined in the presence of a large excess of unlabeled apamin (100 nM). After 60 min incubation on ice,
3.1. Purification, amino acid sequencing and molecular mass determination BmSKTx1 was purified from the scorpion Buthus martensi Karsh venom by conventional column chromatography as detailed in Methods. Several peaks were separated by semi-preparative HPLC and BmSKTx1 was found in Fraction 2 (Fig. 1A). Fraction 2 was then subjected to the Mono S column, and BmSKTx1 was eluted in a sharp discrete peak (Fig. 1B). The overall yield of BmSKTx1 was 500 mg, which accounts for less than 1% of the total protein in the crude venom. The mass spectrometric analysis of the purified BmSKx1 gave a single signal with a molecular mass of 3242.0 Da, indicating the material was pure. BmSKTx1 was directly subjected to the sequence analysis and a single sequence of the N-terminal 22 residues, TPFAIKCATNAXCSXKCPGNXP-, was unequivocally determined except for a few unidentified residues represented by X. During the purification, BmSKTx1 flowed through the DEAE Sephacel column at pH 10.5 and tightly bound to the Mono S (SP Sepharose) column, indicating BmSKTx1 is a basic peptide. 3.2. cDNA and genomic DNA sequences On the basis of the determined amino acid sequence, the cDNA coding for BmSKTx1 was cloned and sequenced (Fig. 2). It encodes a single peptide of 54 amino acids residues, in which the determined sequence of BmSKTx1 locates in the middle of the sequence. These results show
964
C.-Q. Xu et al. / Toxicon 43 (2004) 961–971
Fig. 1. Purification of BmSKTx1. (A) RP-HPLC profile. The breakthrough fraction from the DEAE-Sephacel column was applied to a semipreparative C18 column equilibrated with buffer A, 0.1% TFA in water. The column was eluted with a linear gradient by two steps: 0–36% buffer B (70% acetonitrile in buffer A) for 36 min and 36– 46% buffer B for 15 min. (B) Ion-exchange chromatographic profile of Mono-S column. Fraction 2 from the HPLC C18 column was applied to Mono-S column equilibrated with HAc-NaAc buffer (pH 4.3). Proteins or peptides were eluted with a linear salt gradient (0–1 M NaCl) in the acetate buffer. The fraction marked by asterisk was BmSKTx1. Inset, mass spectrometric pattern obtained for BmSKTx1.
BmSKTx1 is synthesized as a precursor and the mature peptide generates after a signal peptide is removed. The deduced amino acid sequence of mature BmSKTx1 was consistent with its determined sequence. The determined molecular mass of BmSKTx1 (3242.0 Da) matches to the calculated value, 3241.8 Da, which is obtained from the composition from Thr at position 1 to the end of the coding sequence. This shows that the mature peptide is composed of 31 residues and all six cysteine residues are engaged in internal disulfide bonds. Distinctive from other reported cDNAs of scorpion toxins (Wu et al., 1999; Xiong et al., 1999), the cDNA of BmSKTx1 was devoid of the polyadenylation signal
(AATAAA) in its 30 UTR, but it had two stop codons (TGA and TAG) instead. Using the determined cDNA sequence of BmSKTx1, its genomic gene was amplified from the total genomic DNA extracted from telsons of the scorpion BmK (Fig. 2). The gene consists of two exons disrupted by an intron of 79 bp. The intron was inserted into the alanine codon at 27 position upstream of the mature peptide. In accordance with the feature of introns in eukaryotic genes, it was flanked by gt/ag donor – receptor pair (Breathnach and Chambon, 1981). The AT content was up to 78%. This high AT content was suggested to be prerequisite for the intron splicing (Goodall and Filipowicz, 1989).
C.-Q. Xu et al. / Toxicon 43 (2004) 961–971
965
Fig. 2. Genomic DNA sequence and deduced amino acid sequence of BmSKTx1. The two exons are written in capital letters, while the intron and untranslated regions in small letters. The deduced mature toxin sequence is shown in bold. The primers for 30 RACE, 50 RACE and genomic gene PCR are indicated with arrows. Numbers at the top of the nucleotide sequence correspond to the positions of amino acid residues.
3.3. Classification of BmSKTx1 Scorpion Kþ channel toxins have been classified into three families based on their primary sequences and function: a-KTx, b-KTx and g-KTx (Tytgat et al., 1999). a-KTx is the biggest family containing more than 80 members active on Kv, BK or SK channels. They are shortchain toxins with 23-41 amino acids containing three or four disulfide bonds. b-KTxs are long-chain toxins composed of 60-64 amino acids but contain only three disulfide bonds. g-KTxs are HERG channel blockers with 36-47 amino acids containing three or four disulfide bonds. According to its length and the conserved cysteine pattern shared by all a-KTxs, BmSKTx1 should be classified into a-KTx family. Since BmSKTx1 was found to be a SK channel toxin, we compared it with other reported scorpion SK toxins and 26 toxins belonging to other a-KTx subfamilies. BmSKTx1 shows nearly no homology with other a-KTxs except for the cysteine pattern (Fig. 3A). So that BmSKTx1 should be classified into a new subfamily of a-KTx. With the BmSKTx1 data provided by us, Goudet C et al. classified this toxin into Subfamily 14 of a-KTx in his review of toxins from scorpion BmK (Goudet et al., 2002). Since BmSKTx1 is the first purified toxin in subfamily 14 and has definite biological function described as following, we classified it as a-KTx 14.1. To study the evolutionary relationship of BmSKTx1 with other a-KTxs, an unrooted NJ tree was constructed by MEGA2 programme (Fig. 3B). Toxins from different subfamilies had reasonable positions in the tree, which is consistent with the previous nomenclature (Tytgat et al., 1999). BmSKTx1 clustered in the same branch with BmP02
and BmP03 whose biological targets have not been definitely determined. But BmSKTx1 also showed very close relationship with BmP05, P05 and Lei I, which implicated that they might have similar biological function. 3.4. Selectivity of BmSKTx1 To determine the bio-target of BmSKTx1, we first examined BmSKTx1 on whole cell currents in RACC. The result indicated that this toxin only influenced the hyperpolarization activated tail currents. It is well demonstrated by different investigators that the main part of hyperpolarization activated slow tail currents are SK currents and these currents can be completely blocked by apamin or scyllatoxin-analoge BmP05 (Neely and Lingle, 1992; Wu et al., 2002; Dunn, 1999; Park, 1994). In this study, we used their standard protocol to induce the hyperpolarization activated tail currents for studying the effect of BmSKTx1. These currents disappeared almost completely in the 0 mM Ca2þ bath solution (Fig. 4A) and could be totally blocked by apamin (data not shown), indicating they were nearly pure SK currents. We found that BmSKTx1 can block these SK currents and the blockade was completely reversed by washing (Fig. 4B). As shown in Fig. 4A, the outward currents during 0 mV step decreased obviously when no Ca2þ entered RACC in the case of 0 mM Ca2þ bath solution. Therefore, one can image that the outward currents would decrease obviously too if Ca2þ channels were blocked. Since no decrease of outward currents was observed after applying BmSKTx1 (data not shown), it can be sure that BmSKTx1 did not affect the Ca2þ
966
C.-Q. Xu et al. / Toxicon 43 (2004) 961–971
Fig. 3. Multiple sequence alignment and phylogenetic tree of scorpion a-KTxs. (A) Amino acid sequence alignment of BmSKTx1, other scorpion SK toxins and 26 toxins belonging to other a-KTx subfamilies. Gaps are inserted to make better alignments. SK channel blockers are underlined. The conserved residues are shown in shadow. (B) Unrooted phylogenetic tree. An unrooted Neighbor-Joining (NJ) tree was constructed by the sequence alignment in A. BmSKTx1 was shown in bold and underlined. This toxin clustered in the same branch with BmP02 and BmP03, and showed very close relationship with BmP05, P05 and Lei I.
entry into RACC. Thus, the inhibition of SK currents observed upon BmSKTx1 application must be due to the direct blockade of SK channel. Although 2 mM BmSKTx1 inhibited a greater portion of SK currents than 200 nM apamin in Fig. 4B, BmSKTx1 had weaker effect on SK currents than apamin in the same concentration. The maximum blockade given by BmSKTx1 was only 65% of the total SK currents (Fig. 4C), while recombinant BmP05OH (Fig. 4D) or apamin could almost completely block the total SK currents. Furthermore, the Hill coefficient of BmSKTx1 was determined to be 2.2, about two times of that observed with rBmP05-OH, suggesting that there might be at least two binding regions for BmSKTx1 on the channel.
To study the selectivity of BmSKTx1, we further examined the effect of BmSKTx1 on different ion channels present in the isolated locust DUM neurons (Fig. 5). The locust DUM neurons have Naþ, Kv, BK and Ca2þ channels but do not have SK channels. Thus, these cells could be useful to check the selectivity of BmSKTx1. Voltage gated currents were elicited by depolarizing the locust DUM neurons from a holding potential of 2100 mV to 0 mV for 40 ms. BmSKTx1 had no effect on the inward currents (mainly Naþ) or the outward currents (mainly Kþ, including Kv and BK currents) (Fig. 5A). Since the inward Ca2þ currents was completely masked by the large outward Kþ currents, the effect of BmSKTx1 on Ca2þ currents
C.-Q. Xu et al. / Toxicon 43 (2004) 961–971
967
Fig. 3 (continued )
cannot be observed directly. However, this effect can be indirectly observed by the change of large Ca2þ-activated BK currents. As a positive control, 1 mM Cd2þ was used to directly block Ca2þ currents and indirectly blocked Ca2þactivated Kþ currents. In Fig. 5B, the inward Naþ current was blocked by TTX firstly, and then 1 mM Cd2þ was applied to DUM neuron. A consequent decrease of voltage gated outward currents was observed unequivocally due to the indirect blockade of BK currents. Thus we could safely state that BmSKTx1 had no effect on Ca2þ channel because it did not cause any decrease of the voltage gated outward currents. We subsequently tested BmSKTx1 on hyperpolarization activated tail currents elicited at 2 120 mV following a 1 s command step to 0 mV from the holding potential (Fig. 5C). Since no SK currents have been found in DUM neuron (Wicher et al., 2001), the tail currents might be composed of Kir currents and/or some resting Naþ and Ca2þ currents (Grolleau and Lapied, 2000). BmSKTx1 also had no effect on these currents. All these negative results suggested that BmSKTx1 had no effect on Naþ, Ca2þ, Kv,
BK and probably Kir channels. Therefore, BmSKTx1 was found to be a selective SK channel toxin. 3.5. 125I-apamin competition assay To our knowledge, all previously reported scorpion SK toxins can compete with apamin for the binding on the rat brain synaptosome (Sabatier et al., 1994; Legros et al., 1996; Romi-Lebrun et al., 1997; Sabatier et al., 1993; Fajloun et al., 2000; Kharrat et al., 1997). Thus, it is worthy to check whether BmSKTx1 could compete with apamin also. Fig. 6 illustrated that BmSKTx1 did not affect 125I-apamin (10 pM) binding to the membrane, even at doses over 1 mM, while rBmP05-OH and apamin inhibited the binding in a dose-dependent manner. This result suggested that BmSKTx1 might bind to distinctive sites on SK channel. In these experiments, IC50 values were 10 nM for rBmP05OH and 20 pM for synthetic BmP05-NH2. The large difference in IC50 values between rBmP05-OH and synthetic BmP05-NH2 is probably due to the lack of
968
C.-Q. Xu et al. / Toxicon 43 (2004) 961–971
amidation on the C-terminal residue of rBmPO5-OH (Sabatier et al., 1993).
4. Discussion
Fig. 4. Effect of BmSKTx1 on SK currents in RACC. (A) A protocol of pure SK currents in RACC. The slow tail currents was elicited at 2100 mV following a 1 s command step to 0 mV from a holding potential of 270 mV. In 10 mM Ca2þ bath solution, the slow tail currents were the nearly pure SK currents up to 65 pA which were activated by the Ca2þ influx through Ca2þ channels during the 1 s depolarization pre-pulse. These currents disappeared almost completely in 0 mM Ca2þ bath solution ðn ¼ 20Þ: (B) The inhibitory effect of BmSKTx1 and apamin on SK currents. Two micromolar BmSKTx1 could block SK currents obviously ðn ¼ 20Þ: As a positive control, 0.2 mM apamin (close to its Kd) was also used to block SK currents. Their inhibitory effects could be washed
The known endogenous SK channels are tetramers, composed of homo- or hetero-subunits of SK1, 2 or 3 subtypes. The SK1-3 subtypes share high sequence homology and have similar properties with one another (Kohler et al., 1996). Their distributions, physiological functions and pharmacology were mainly determined by apamin and other SK blockers. According to the block selectivity of apamin, the known SK subtypes are classified into two classes, namely, apamin-sensitive (SK2 and SK3) and apamin-insensitive subtypes (SK1). However, apamin is not specific enough to further distinguish different apaminsensitive subtypes. Unfortunately, Lei I and other scorpion SK blockers show low selectivity too. Even the most selective blocker, Lei I, exhibits only five-fold higher selectivity for SK2 over SK3 (Shakkottai et al., 2001). Furthermore, all above blockers share the same binding site(s) on SK channel with apamin, and cannot provide more information about SK channel. Therefore, a novel ligand with high selectivity and different binding mechanism eagerly need to be discovered. In this study, a selective SK channel blocker BmSKTx1 was purified from the scorpion BmK. This toxin has low sequence homology with other scorpion a-KTxs but it shows close relationship with SK blockers in the phylogenetic tree. According to the previous nomenclature, BmSKTx1 was classified as a-KTx14.1. To determine its bio-target and selectivity, we used RACC and DUM neuron to test BmSKTx1 on different ion channels. In RACC, BmSKTx1 was found to only block SK currents. The locust DUM neurons were chosen to study the selectivity of BmSKTx1 because these cells have many ion channels but no SK channels. Fig. 5 showed that BmSKTx1 did not influence voltage gated currents and hyperpolarization activated tail currents in DUM neurons, including Naþ, Ca2þ, Kv, and BK currents. These results indicate that BmSKTx1 is a specific blocker for the SK channel. The total tail SK currents in RACC could be almost fully blocked by apamin and BmP05, indicating that these SK currents are apamin-sensitive. Given the fact that SK2 and SK3 currents are sensitive to apamin but SK1 current is R out by the control buffer. (C and D) The dose-dependent curves for inhibition of SK currents by BmSKTx1 and rBmP05-OH. Each point shown is the mean percentage of the blocked currents of 5–27 cells at a given dose ^ SE The curve of BmSKTx1 was fitted well with Hill ¼ 2.2 and Kd ¼ 0:72 mM, and its maximum block was only 65% of the total SK currents. While the curve of rBmP05-OH was fitted well with Hill ¼ 0.95 and Kd ¼ 0:92 mM, and the maximum block was nearly 100%.
C.-Q. Xu et al. / Toxicon 43 (2004) 961–971
969
BmSKTx1 is 2.2 instead of 1 for BmP05 suggesting the presence of at least two binding sites for BmSKTx1. To our knowledge, no other SK blockers were reported to have a Hill coefficient of 2 and they all have one binding face locating on the intermediate region of SK channels (Rodriguez de la Vega et al., 2003). Therefore, BmSKTx1 might present a unique interaction face with the SK channel. In previous and present studies, BmP05 and other scorpion SK toxins have been found to share the same binding site(s) with apamin by the 125I-apamin competition assay (Sabatier et al., 1994; Legros et al., 1996; Romi-Lebrun et al., 1997; Sabatier et al., 1993; Fajloun et al., 2000; Kharrat et al., 1997). In contrast, BmSKTx1 does not compete with apamin for the binding on rat brain synaptosomal membrane, which is consistent with the proposal that BmSKTx1 might have unique binding regions on SK channels (Fig. 6). It is not the first case that some toxins were reported to block the same channel but bind to different sites. For instance, Hanatoxin and AgTx2 could simultaneously bind to an individual Kþ channel because Hanatoxin bound the S3 segment but AgTx2 directly blocked the channel pore (Li-Smerin and Swartz, 2001; Swartz and MacKinnon, 1997). In the case of Naþ channel blockers, a and b scorpion long-chain toxins were also found to be able to target the same Naþ channel by binding to site 3 and site 4, respectively (Cestele and Catterall, 2000). However, the possibility could not be fully occluded that the rat brain synaptosome might have a different SK expression pattern as compared to RACC. This question will be addressed in the following-up works. On the other hand, we believe that it will be worth unraveling the binding mechanism of BmSKTx1 to SK channel and these data will enrich our understanding about toxin and channel interaction. Fig. 5. Effect of BmSKTx1 on different ion channels in isolated Locust DUM neurons. The currents in control condition and in the presence of 2 mM BmSKTx1 or 1 mM Cd2þ were overlaid. (A) Voltage gated currents were elicited by the pulse protocol as shown in the top panel. BmSKTx1 had no effect on these currents. (B) 1 mM Cd2þ was applied to directly block inward voltage gated Ca2þ currents and indirectly block outward Ca2þ activated currents. The inward Naþ current is blocked by tetrodotoxin (TTX) in both current traces. (C) Hyperpolarization activated tail currents were elicited at -120 mV following a 1 s command step to 0 mV from the holding potential. The tail currents were enlarged in the lower panel. BmSKTx1 also had no effect on these currents.
apamin-insensitive, the total tail SK currents in RACC might be composed of SK2 and SK3 currents. BmSKTx1 maximally blocked 65% of the total apamin-sensitive SK currents, suggesting that this toxin might be selective only on SK2 or SK3. This idea will be verified by testing the toxin on exogenously expressed SK1, SK2 and SK3 channels in the future. Anyway, the selectivity of BmSKTx1 to different apamin-sensitive SK currents is higher than apamin and BmP05. Compared with BmP05, BmSKTx1 might have different binding sites on SK channel because the Hill coefficient for
Fig. 6. 125I-apamin competition assay. Inhibition of the 125I-apamin binding on the rat brain synaptosomal fraction P2 by competitors. B0 is the binding of 125I-apamin (10 pM) in the absence of competitors, and B is the binding in the presence of competitors (0.1 pM – 1 mM). Non-specific binding was subtracted for the calculation of the ratios. Every value was the mean of triplicate assays. The IC50 for apamin and rBmP05-OH were 2 and 10 nM, respectively. However, BmSKTx1 could not compete with 125Iapamin. B, rBmP05-OH; O, BmSKTx1; P, Apamin.
970
C.-Q. Xu et al. / Toxicon 43 (2004) 961–971
To make a conclusion, BmSKTx1 is a distinctive SK channel blockers with unique primary structure. Although its SK subtype selectivity and binding mechanism remain to be clarified, this toxin could provide a novel tool to further study SK channels.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (30170210 to C.W. Chi, and 39970238, 39970371 to Z. Zhou), China 973 program (G2000077800 to C.W. Chi) and a bilateral grant between Flanders and People’s Republic of China BIL00/06.
References Bond, C.T., Maylie, J., Adelman, J.P., 1999. Small-conductance calcium-activated potassium channels. Ann. N Y Acad. Sci. 868, 370 –378. Bond, C.T., Sprengel, R., Bissonnette, J.M., Kaufmann, W.A., Pribnow, D., Neelands, T., Storck, T., Baetscher, M., Jerecic, J., Maylie, J., Knaus, H.G., Seeburg, P.H., Adelman, J.P., 2000. Respiration and parturition affected by conditional overexpression of the Ca2 þ -activated K þ channel subunit, SK3. Science 289, 1942–1946. Breathnach, R., Chambon, P., 1981. Organization and expression of eucaryotic split genes coding for proteins. Annu. Rev. Biochem. 50, 349 –383. Brone, B., Tytgat, J., Wang, D.C., Van Kerkhove, E., 2003. Characterization of Na(þ ) currents in isolated dorsal unpaired median neurons of Locusta migratoria and effect of the alphalike scorpion toxin BmK M1. J. Insect Physiol. 49, 171 –182. Cestele, S., Catterall, W.A., 2000. Molecular mechanisms of neurotoxin action on voltage-gated sodium channels. Biochimie 82, 883 –892. Dudina, E.E., Korolkova, Y.V., Bocharova, N.E., Koshelev, S.G., Egorov, T.A., Huys, I., Tytgat, J., Grishin, E.V., 2001. OsK2, a new selective inhibitor of Kv1.2 potassium channels purified from the venom of the scorpion Orthochirus scrobiculosus. Biochem. Biophys. Res. Commun. 286, 841–847. Dunn, P.M., 1999. UCL 1684: a potent blocker of Ca2 þ -activated K þ channels in rat adrenal chromaffin cells in culture. Eur. J. Pharmacol. 368, 119 –123. Fajloun, Z., Carlier, E., Lecomte, C., Geib, S., Di Luccio, E., Bichet, D., Mabrouk, K., Rochat, H., De Waard, M., Sabatier, J.M., 2000. Chemical synthesis and characterization of Pi1, a scorpion toxin from Pandinus imperator active on K þ channels. Eur. J. Biochem. 267, 5149– 5155. Goodall, G.J., Filipowicz, W., 1989. The AU-rich sequences present in the introns of plant nuclear pre-mRNAs are required for splicing. Cell 58, 473– 483. Goudet, C., Chi, C.W., Tytgat, J., 2002. An overview of toxins and genes from the venom of the Asian scorpion Buthus martensi Karsch. Toxicon 40, 1239–1258. Grolleau, F., Lapied, B., 2000. Dorsal unpaired median neurones in the insect central nervous system: towards a better
understanding of the ionic mechanisms underlying spontaneous electrical activity. J. Exp. Biol. 203, 1633–1648. Habermann, E., 1984. Apamin. Pharmacol Ther. 25, 255 –270. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391, 85– 100. Hirschberg, B., Maylie, J., Adelman, J.P., Marrion, N.V., 1998. Gating of recombinant small-conductance Ca-activated K þ channels by calcium. J. Gen. Physiol. 111, 565 –581. Horn, R., Marty, A., 1988. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J. Gen. Physiol. 92, 145–159. Kharrat, R., Mansuelle, P., Sampieri, F., Crest, M., Oughideni, R., Van Rietschoten, J., Martin-Eauclaire, M.F., Rochat, H., El Ayeb, M., 1997. Maurotoxin, a four disulfide bridge toxin from Scorpio maurus venom: purification, structure and action on potassium channels. FEBS Lett. 406, 284 –290. Kohler, M., Hirschberg, B., Bond, C.T., Kinzie, J.M., Marrion, N.V., Maylie, J., Adelman, J.P., 1996. Small-conductance, calcium-activated potassium channels from mammalian brain. Science 273, 1709–1714. Kumar, S., Tamura, K., Jakobsen, I.B., Nei, M., 2001. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17, 1244– 1245. Legros, C., Oughuideni, R., Darbon, H., Rochat, H., Bougis, P.E., Martin-Eauclaire, M.F., 1996. Characterization of a new peptide from Tityus serrulatus scorpion venom which is a ligand of the apamin-binding site. FEBS Lett. 390, 81– 84. Li-Smerin, Y., Swartz, K.J., 2001. Helical structure of the COOH terminus of S3 and its contribution to the gating modifier toxin receptor in voltage-gated ion channels. J. Gen. Physiol. 117, 205 –218. Neely, A., Lingle, C.J., 1992. Two components of calcium-activated potassium current in rat adrenal chromaffin cells. J. Physiol. 453, 97–131. Park, Y.B., 1994. Ion selectivity and gating of small conductance Ca(2 þ )-activated K þ channels in cultured rat adrenal chromaffin cells. J. Physiol. 481, 555–570. Pribnow, D., Johnson-Pais, T., Bond, C.T., Keen, J., Johnson, R.A., Janowsky, A., Silvia, C., Thayer, M., Maylie, J., Adelman, J.P., 1999. Skeletal muscle and small-conductance calcium-activated potassium channels. Muscle Nerve 22, 742–750. Rimini, R., Rimland, J.M., Terstappen, G.C., 2000. Quantitative expression analysis of the small conductance calcium-activated potassium channels, SK1, SK2 and SK3, in human brain. Brain Res. Mol. Brain Res. 85, 218 –220. Rodriguez de la Vega, R.C., Merino, E., Becerril, B., Possani, L.D., 2003. Novel interactions between Kþ channels and scorpion toxins. Trends Pharmacol. Sci. 24, 222–227. Romi-Lebrun, R., Martin-Eauclaire, M.F., Escoubas, P., Wu, F.Q., Lebrun, B., Hisada, M., Nakajima, T., 1997. Characterization of four toxins from Buthus martensi scorpion venom, which act on apamin-sensitive Ca2 þ -activated K þ channels. Eur. J. Biochem. 245, 457–464. Sabatier, J.M., Fremont, V., Mabrouk, K., Crest, M., Darbon, H., Rochat, H., Van Rietschoten, J., Martin-Eauclaire, M.F., 1994. Leiurotoxin I, a scorpion toxin specific for Ca(2 þ )activated K þ channels. Structure – activity analysis using synthetic analogs. Int. J. Pept. Protein Res. 43, 486 –495.
C.-Q. Xu et al. / Toxicon 43 (2004) 961–971 Sabatier, J.M., Zerrouk, H., Darbon, H., Mabrouk, K., Benslimane, A., Rochat, H., Martin-Eauclaire, M.F., Van Rietschoten, J., 1993. P05, a new leiurotoxin I-like scorpion toxin: synthesis and structure-activity relationships of the alphaamidated analog, a ligand of Ca(2 þ )-activated K þ channels with increased affinity. Biochemistry 32, 2763–2770. Schetz, J.A., Anderson, P.A., 1995. Pharmacology of the highaffinity apamin receptor in rabbit heart. Cardiovasc. Res. 30, 755–762. Seagar, M.J., Labbe-Jullie, C., Granier, C., Van Rietschoten, J., Couraud, F., 1985. Photoaffinity labeling of components of the apamin-sensitive K þ channel in neuronal membranes. J. Biol. Chem. 260, 3895–3898. Shakkottai, V.G., Regaya, I., Wulff, H., Fajloun, Z., Tomita, H., Fathallah, M., Cahalan, M.D., Gargus, J.J., Sabatier, J.M., Chandy, K.G., 2001. Design and characterization of a highly selective peptide inhibitor of the small conductance calciumactivated K þ channel, SkCa2. J. Biol. Chem 276, 43145– 43151. Strong, P.N., Clark, G.S., Armugam, A., De-Allie, F.A., Joseph, J.S., Yemul, V., Deshpande, J.M., Kamat, R., Gadre, S.V., Gopalakrishnakone, P., Kini, R.M., Owen, D.G., Jeyaseelan, K., 2001. Tamulustoxin: a novel potassium channel blocker from the venom of the Indian red scorpion Mesobuthus tamulus. Arch. Biochem. Biophys. 385, 138–144. Swartz, K.J., MacKinnon, R., 1997. Mapping the receptor site for hanatoxin, a gating modifier of voltage-dependent K þ channels. Neuron 18, 675 –682. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882.
971
Tytgat, J., Chandy, K.G., Garcia, M.L., Gutman, G.A., MartinEauclaire, M.F., van der Walt, J.J., Possani, L.D., 1999. A unified nomenclature for short-chain peptides isolated from scorpion venoms: alpha-KTx molecular subfamilies. Trends Pharmacol. Sci. 20, 444 –447. Vacher, H., Romi-Lebrun, R., Mourre, C., Lebrun, B., Kourrich, S., Masmejean, F., Nakajima, T., Legros, C., Crest, M., Bougis, P.E., Martin-Eauclaire, M.F., 2001. A new class of scorpion toxin binding sites related to an A-type K þ channel: pharmacological characterization and localization in rat brain. FEBS Lett. 501, 31 –36. Wang, C.G., He, X.L., Shao, F., Liu, W., Ling, M.H., Wang, D.C., Chi, C.W., 2001. Molecular characterization of an anti-epilepsy peptide from the scorpion Buthus martensi Karsch. Eur. J. Biochem. 268, 2480–2485. Wicher, D., Walther, C., Wicher, C., 2001. Non-synaptic ion channels in insects—basic properties of currents and their modulation in neurons and skeletal muscles. Prog. Neurobiol. 64, 431–525. Wu, J.J., Dai, L., Lan, Z.D., Chi, C.W., 1999. Genomic organization of three neurotoxins active on small conductance Ca2 þ activated potassium channels from the scorpion Buthus martensi Karsch. FEBS Lett. 452, 360–364. Wu, J.J., He, L.L., Zhou, Z., Chi, C.W., 2002. Gene expression, mutation, and structure-function relationship of scorpion toxin BmP05 active on SK(Ca) channels. Biochemistry 41, 2844–2849. Xiong, Y.M., Ling, M.H., Lan, Z.D., Wang, D.C., Chi, C.W., 1999. The cDNA sequence of an excitatory insect selective neurotoxin from the scorpion Buthus martensi Karsch. Toxicon 37, 335 –341. Zhou, Z., Misler, S., 1995. Action potential-induced quantal secretion of catecholamines from rat adrenal chromaffin cells. J. Biol. Chem. 270, 3498–3505.
