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BmKK4, a novel toxin from the venom of Asian scorpion Buthus martensi Karsch, inhibits potassium currents in rat hippocampal neurons in vitro
Toxicon 42 (2003) 199–205 www.elsevier.com/locate/toxicon
BmKK4, a novel toxin from the venom of Asian scorpion Buthus martensi Karsch, inhibits potassium currents in rat hippocampal neurons in vitro Ming-Hua Lia, Nai-Xia Zhangb, Xue-Qin Chena, Gong Wub, Hou-Ming Wub, Guo-Yuan Hua,* a
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 555 Zu-Chong-Zhi Road, Shanghai 201203, People’s Republic of China b State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Feng-Lin Road, Shanghai 200032, People’s Republic of China Received 7 March 2003; accepted 27 May 2003
Abstract A novel short-chain peptide BmKK4 was isolated from the venom of Asian scorpion Buthus martensi Karsch. It is composed of 30 amino acids including six cysteine residues, and shares less than 25% sequence identity with the known a-KTx toxins. The action of BmKK4 on voltage-dependent potassium currents was examined in acutely dissociated hippocampal neurons of rat. BmKK4 (10 – 100 mM) inhibited both the delayed rectifier and fast transient potassium current in concentration-dependent manners. The inhibition was reversible and voltage-independent. BmKK4 caused a depolarizing shift (about 10 mV) of the steady-state activation curve of the currents, without changing their steady-state inactivation behavior. The unique amino acid sequence and electrophysiological effects suggest that BmKK4 represent a new subfamily of potassium channel toxins. q 2003 Elsevier Ltd. All rights reserved. Keywords: BmKK4; Buthus martensi Karsch; Hippocampus; Neurotoxin; Potassium channel; Scorpion
1. Introduction Potassium (Kþ) channels play crucial roles in regulating a variety of cellular processes in both excitable and nonexcitable cells, such as setting the resting membrane potential, action potential duration, the delay between a stimulus and the first action potential, discharge patterns, etc. (Storm, 1990). Molecular cloning studies in the recent decades have revealed enormous molecular diversity of Kþ channels. Thus far more than 100 pore-forming subunits of Kþ channels have been cloned. Heteromultimeric assembly of different subunits provides a base for further diversity, and leads to a huge number of functionally diverse Kþ * Corresponding author. Tel.: þ86-21-5080-6778; fax: þ 86-215080-7088. E-mail address: [email protected] (G.-Y. Hu).
channels with distinct biophysical, pharmacological and regulation properties (Coetzee et al., 1999). Investigation of the structure and function of ion channels highly depends on the availability of specific reagents, particularly neurotoxins isolated from terrestrial and marine organisms. In contrast to the enormous molecular and functional diversity of Kþ channels, however, there is in fact a paucity of high-affinity ligands binding to the channels. Such a situation has hindered our understanding how the Kþ channels function in the nervous, cardiovascular and other systems. Short-chain peptides (30 – 40 amino acid residues) isolated from the venoms of different species of scorpions were found to specifically block distinct Kþ channels and named a-KTx toxins (Tytgat et al., 1999; Pongs and Legros, 2000). Among them, charybdotoxin (ChTX, from Israeli scorpion Leiurus quinquestriatus hebraeus), noxiustoxin (NTX, from American scorpion Centruroides noxius
0041-0101/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0041-0101(03)00136-3
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Fig. 1. Amino acid sequence of BmKK4 was aligned according to the cysteine residues (boxed), with other short-chain Kþ channel toxins. The percentage identity with BmKK4 is given at the right. Asterisk indicates amidation at the C terminus. Gaps are presented as dashes. BmP01, BmP03 from Buthus martensi Karsch; ChTX (charybdotoxin) from Leiurus quinquestriatus hebraeus; KTX (kaliotoxin) from Androctonus mauretanicus mauretanicus; NTX (noxiustoxin) from Centruroides noxius hoffmann; MTX (maurotoxin) from Scorpio maurus; ScTX (scyllatoxin) from Leiurus quinquestriatus hebraeus; P01 from Androctonus mauretanicus mauretanicus; TSK(Tskapa) from Buthidae Tityus serrulatus; CoTX1 (cobatoxin1) from Centruroides noxius.
hoffmann) and kaliotoxin (KTX, from North Africa scorpion Androctonus mauretanicus mauretanicus) have been intensively studied. The venom of Asian scorpion Buthus martensi Karsch is also a rich source of a-KTx toxins with 18 peptides already identified, including BmP01, BmP02, BmP03, BmP05, BmKTX, BmTX1 and BmTX2 (Goudet et al., 2002). Pharmacological studies showed that BmP05 affected the small-conductance Ca2þ-activated Kþ channels (Romi-Lebrun et al., 1997b), whereas BmTX1 and BmTX2 acted on both the large-conductance Ca2þ-activated Kþ channels and voltage-gated Kv1.3 channels (Romi-Lebrun et al., 1997a). BmKK4 is a novel short-chain peptide deduced from a cDNA (AJ277729, EMBL) found in a venom gland cDNA library of B. martensi Karsch (Zeng et al., 2001). The peptide is composed of 30 amino acids including six cysteine residues, and shares less than 25% sequence identity with the known a-KTx toxins (Fig. 1). Up to data, however, the biological activity of BmKK4 has not been examined. In the present study, we purified and characterized the peptide from the venom of B. martensi Karsch, and investigated its action on voltage-dependent Kþ currents in rat dissociated hippocampal neurons.
2. Materials and methods All chemicals and reagents, unless otherwise stated, were purchased from Sigma, USA.
(Fig. 2B). Solution A contained NaAC (20 mM), whereas solution B contained NaAC (20 mM) and NaCl (1 M). Then it was followed by the same separation on Sephadex G-50 column and a similar separation on Mono S cation exchange column eluted with a step gradient of solution A to B, but at pH 4.0 (Fig. 2D). The final purification was achieved on a reverse-phase HPLC column (C18 column, 4.6 £ 250 mm, 5 mm bead size, Alltech) using a linear gradient from 20 to 45% buffer B in buffer A. Buffer A contained TFA (0.1%) and CH3CN (10%) in H2O, and buffer B contained TFA (0.1%) and H2O (20%) in CH3CN. 2.2. Sequence determination of BmKK4 After DTT reduction, the peptide (100 pM) was processed with iodoacetic acid derivatization, and digested with sequencing grade trypsin or endoproteinase Lys-C (2 pM). Digested sample was desalted into a solution consisted of CH3CN: H2O (4:1) and formic acid (0.1%) using C18 ZipTips (Millipore, USA). ES-MS and ESMS/MS spectra were performed using a tandem quadrupoleTOF instrument (Micromass, UK). The sample (1– 2 ml) was loaded into a nanospray needle. For recording ESMS/MS spectra, the quadrupole was operated as a mass filter and used to select the desired precursor ion isotope cluster. During product ion analysis the resolving quadrupole was set to transmit a ^ 2 m=z window round the precursor ion. Precursor ions were directed into a hexapole collision cell. The collision gas was argon and the collision energy was optimized for each analysis.
2.1. Purification of BmKK4 2.3. Preparation of rat dissociated hippocampal neurons Crude venom was collected by electrical stimulation of the telson of scorpions B. martensi Karsch bred in captivity in Henan province, China. Lyophilized venom (9.6 g) was dissolved in NH4HCO3 buffer (50 mM, pH 8.5) and centrifuged at 4000g for 15 min. The supernatant was loaded onto a Sephadex G-50 column (2.5 £ 150 cm) preequilibrated and eluted with the same buffer. A fraction containing the peptide was loaded onto a Mono S cation exchange column (HR 5/5, Pharmacia LKB Biotech, Inc.) eluted with a step gradient of solution A to B at pH 5.0
Hippocampal neurons were prepared as described previously (Sodickson and Bean, 1998; Li and Hu, 2002). Briefly, mini-slices (500 mm) of the hippocampal CA1 region from 5 to 9-day-old Sprague-Dawley rats were cut in oxygenated ice-cold dissociation solution containing (in mM): Na2SO4 (82), K2SO4 (30), MgCl2 (5), N-(2-hydroxyethyl)piperazine-N 0 -(2-ethanesulfonic acid) (HEPES) (10) and glucose (10) at pH 7.3 with NaOH. The slices were incubated in dissociation solution containing protease
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Fig. 2. Isolation and purification of BmKK4. (A) Sephadex G-50 column chromatography of the crude venom from Buthus martensi Karsch. (B) FPLC fractionation of the fraction IV obtained in (A) on a Mono S column. (C) Sephadex G-50 column chromatography of the fraction 1 in (B). (D) FPLC fractionation of the fraction 1–2 in (C) on a Mono S column. (E) RP-HPLC fractionation of the fraction 1 –22 in (D) on a C18 column.
XXIII (3 mg/ml) at 32 8C for 8 min. The solution was then replaced with dissociation solution containing trypsin inhibitor type II-S (1 mg/ml) and bovine serum albumin (1 mg/ml). The slices were allowed to cool to room temperature under an oxygen atmosphere. Before recording, the slices were triturated using a series of fire-polished Pasteur pipettes with decreasing tip diameters. Dissociated neurons were placed in a recording dish and perfused with external solution. The solution contained (in mM): NaCl (135), KCl (5), MgCl2 (2), CaCl2 (1), HEPES (10), glucose (10) and tetrodotoxin (0.001) at pH 7.3 with NaOH.
The delayed rectifier Kþ currents ðIK Þ were elicited by a similar protocol in which a 50 ms interval at 2 50 mV was inserted after the prepulse. Subtraction of IK from Itotal revealed the fast transient Kþ current ðIA Þ (Numann et al., 1987; Klee et al., 1995). Current records were filtered at 2– 10 kHz and sampled at frequencies of 10– 40 kHz. Series resistance was compensated by 75 – 85%. Linear leak and residual capacitance currents were subtracted on-line. BmKK4 was dissolved in the external solution. The toxincontaining solution was applied to the recorded neuron using RSC-100 Rapid Solution Changer with an 18-tube head (BioLogic Co., France).
2.4. Whole-cell voltage-clamp recording 2.5. Data analysis Recording was made from large pyramidal-shaped neurons using an Axopatch 200A amplifier (Axon Instruments, USA) at 21 – 23 8C. The electrodes (tip resistance 2 , 4 MV) were pulled from borosilicate glass pipettes (Hilgenberg, Germany). The pipette solution contained (in mM): potassium gluconate (125), KCl (20), MgCl2 (2), CaCl2 (1), HEPES (10) and EGTA (10) at pH 7.3 with KOH. Voltage protocols were provided by pClamp 6.0.2 software via a DigiData-1200A interface (Axon Instruments, USA). The holding potential was 2 50 mV. As shown in Fig. 3, the total Kþ currents ðItotal Þ were elicited by depolarizing command pulses to þ60 mV in 10 mV steps following a hyperpolarizing prepulse of 400 ms to 2 110 mV.
For analysis, the peak amplitude of IA was measured, whereas the amplitude of IK was determined at 300 ms latency. The data are presented as mean ^ SEM. Student’s two-tailed t-test was used for statistical analysis.
3. Results 3.1. Purification and identification of BmKK4 The soluble venom was initially separated into four fractions (I – IV) by gel-filtration chromatography on
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Fig. 3. BmKK4 inhibits the Kþ currents in hippocampal pyramidal neurons. Upper, middle and lower traces are the current families for the total Kþ currents ðItotal Þ; delayed rectifier Kþ currents ðIK Þ and fast transient Kþ currents ðIA Þ; respectively, in control and in presence of BmKK4 (10 mM). The upper and middle traces were evoked with command pulse protocols shown in the insets. The lower traces are the subtractions of the middle from the upper ones.
a Sephadex G-50 (Fig. 2A). Fraction IV was further separated into 13 fractions on a Mono S cation exchange column (Fig. 2B). After another separation of fraction 1 on a Sephadex G-50 column, four fractions (1 – 1, 1 – 2, 1 – 3 and 1 – 4) were obtained (Fig. 2C). A further separation of the fraction 1 – 2 on a Mono S column gave two fractions (1 – 21, 1 – 22) (Fig. 2D). A pure peptide (6.8 mg) was obtained after the separation of fraction 1 – 22 on a reverse-phase HPLC column (Fig. 2E). The amino acid sequence of the pure peptide was determined via tandem CID MS/MS analysis of its enzymatic fragments. The intact sequence obtained is listed as follows: ZTQCQ SVRDC QQYCL/I TPDRC SYGTC YCKTT(NH2). Following a database search, the peptide was identified with BmKK4 that was deduced from a cDNA
(AJ277729, EMBL) found in a venom gland cDNA library of B. martensi Karsch (Zeng et al., 2001). 3.2. Effects of BmKK4 on voltage-dependent potassium currents in hippocampal neurons With the voltage protocols and subtraction procedure used, two types of outward current could be simultaneously recorded from the same neuron (Fig. 3, left panel). The slowly inactivating outward current was reversibly blocked by TEA (IC50 ¼ 3.1 ^ 0.6 mM, n ¼ 5Þ; thus corresponded to the delayed rectifier Kþ current ðIK Þ: The rapidly inactivating outward current was reversibly blocked by 4-AP (IC50 ¼ 4.9 ^ 0.7 mM, n ¼ 5), and represented the fast transient Kþ current ðIA Þ:
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Fig. 4. The inhibitory effects of BmKK4 on IK and IA : (A) Time course of inhibition caused by BmKK4 (10 mM) in another neuron. (B) Concentration-response curves of BmKK4 in inhibition of IK and IA ðn ¼ 6 – 9Þ: (C) Current– voltage ðI=VÞ relationship of IK before and during application of BmKK4 (10 mM). (D) Current –voltage ðI=VÞ relationship of IA before and during application of the toxin. The data in (C) and (D) were from the same neuron shown in Fig. 3.
BmKK4 (10 – 100 mM) inhibited both IK and IA in concentration-dependent manners (Fig. 3, right panel). The inhibitory actions had a rapid onset and were reversible upon washout (Fig. 4A). When the concentration– response relationships of BmKK4 in inhibition of the Kþ currents were analyzed, the two curves are nearly superimposed. At the maximum concentration (100 mM) tested, the amplitude of IK and IA reduced to 65.7 ^ 3.2 and 68.2 ^ 0.8 of the control levels, respectively (Fig. 4B). Due to the limited amount of toxin obtained, the IC50 values were not determined. The actions of BmKK4 on the current – voltage ðI=VÞ relations of IK and IA are shown in Fig. 4C and D, respectively. The inhibition of BmKK4 on the Kþ currents was voltage-independent. In the presence of BmKK4 (10 mM), the amplitude of IK in steps to 0, þ20, þ 40 and þ60 mV reduced to 81 ^ 6, 77 ^ 7, 73 ^ 9 and 73 ^ 6% of the control levels ðn ¼ 6; P . 0:05 vs. 0 mV), whereas the amplitude of IA in response to the same steps was 86 ^ 6, 78 ^ 8, 77 ^ 9 and 83 ^ 7% of the control levels ðn ¼ 6; P . 0:05 vs. 0 mV). 3.3. Effects of BmKK4 on kinetic behaviors of voltagedependent potassium currents in hippocampal neurons Application of BmKK4 resulted in a depolarizing shift of the steady-state activation curve for both the Kþ currents
(Fig. 5A and C), without significant effect on their steadystate inactivation behavior (Fig. 5B and D). The voltages for half-maximal activation ðVH Þ of IK and IA were 2 1.2 ^ 3.3 and 224.7 ^ 3.9 mV, respectively, in control solution. Application of BmKK4 (10 mM) changed the VH values to þ9.3 ^ 2.0 mV ðn ¼ 5; P ¼ 0:008Þ and 2 13.9 ^ 4.6 mV ðn ¼ 7; P ¼ 0:003Þ; respectively. The effects were reversible: the VH values returned to 2 2.8 ^ 1.2 and 224.8 ^ 1.8 mV, respectively, upon washout.
4. Discussion The present study demonstrates that BmKK4, a novel short-chain peptide isolated from the venom of B. martensi Karsch, inhibits voltage-dependent Kþ currents in rat hippocampal neurons. BmKK4 shares less than 25% sequence identity with the known a-KTx toxins (Fig. 1), thus has been proposed to represent a new subfamily of Kþ channel toxins (a-KTX 17.1) (Goudet et al., 2002). Several known a-KTx toxins in the venom of B. martensi Karsch have been reported to selectively depress the fast transient Kþ current, without effect on the delayed rectifier Kþ current in different preparations. For example, BmP01 and BmP02 (previously coded BmP-3) inhibited the peak component of voltage-dependent Kþ current (corresponding to IA Þ in rat hippocampal neurons (Wu et al., 1998, 2000).
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Fig. 5. Steady-state activation curves (A, C) and inactivation curves (B, D) of IK and IA constructed in the absence and presence of BmKK4 (10 mM). Pulse protocols and subtraction procedure similar to those in Fig. 3 were used to study the steady-state activation. Steady-state inactivation was studied using pulse protocols with hyperpolarizing prepulses from 2110 to 210 mV (10 mV steps) followed by a step to a fixed voltage (0 mV). In each panel the mean of normalized conductance (n ¼ 5–7) is plotted against the membrane potential, and fitted with a Boltzmann equation: G=Gmax ¼ 1={1 þ exp½2ðV 2 VH Þ=k}; where G=Gmax is the mean of normalized conductance, V is the membrane potential, VH (or Vh Þ is the voltage for half-maximal activation (or inactivation), and k is the slope factor.
BmP02 and BmP03 were also found to depress the transient outward Kþ current ðIto Þ in rat ventricular myocytes (Tong et al., 2000). On the contrary, BmKK4 exerted inhibitory effect on both IA and IK in hippocampal neurons with similar potencies. The mechanisms underlying the blockade of voltagegated K þ channels by a-KTx toxins have been intensively explored in the recent decade. It was found that five residues (Lys27, Met29, Asn30, Arg34 and Tyr36) in two C-terminal b-strands and b-turn played important roles in the binding of ChTX, BmTX1 or BmTX2 to voltage-gated Kþ channel (Goldstein et al., 1994; Blanc et al., 1998). Among the five residues, Lys27 was identified to be crucial for toxin affinity, and proposed to plug directly into the pore, coming in the vicinity of the selectivity filter of Kþ channel (Aiyar et al., 1996). In BmKK4, a different residue Arg19 replaces the important residue Lys27 (Fig. 1). The substitution seems to be sufficient to explain the low potency of BmKK4 found in the present study. In fact, a similar phenomenon has been seen in the two short-chain peptides Pi4 and Pi7 isolated from the venom of the scorpion Pandinus
imperator: peptide Pi4 with a critical residue Lys26 is a potent blocker of Shaker B Kþ channels (Kv1.1 channel); whereas peptide Pi7, in which Arg26 replaces the residue Lys26, is totally inactive towards the channels (Olamendi-Portugal et al., 1998). Alternatively, it is likely that BmKK4 mainly affects Ca2þ-activated Kþ currents, rather than voltage-dependent Kþ currents. Because Ca2þ-activated Kþ current accounts for less than 2% of the total sustained Kþ current recorded in our preparation (Santos et al., 1998; Li and Hu, 2002), this question was not further addressed. If the substitution Arg19 for Lys26 in BmKK4 markedly attenuates its affinity for the Kþ channels, and prevents it occluding the channel pore, how does the toxin exert its inhibitory action on the voltage-dependent Kþ currents? We noticed that BmKK4-induced inhibition was accompanied by a reversible depolarizing shift (nearly 10 mV) of the steady-state activation curve of the Kþ currents. Similar shift of the voltage-dependent activation has been used to explain the Pb2þ-induced inhibition of the delayed rectifier Kþ current in hippocampal neurons (Medeja et al., 1997). It is feasible that BmKK4 may depress the Kþ currents via
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a novel mechanism distinct from the classical model proposed for a-KTx toxins in blockade of Kþ channels. The exact mechanism underlying BmKK4-induced inhibition of the Kþ currents need to be further investigated.
References Aiyar, J., Rizzi, J.P., Gutman, G.A., Chandy, K.G., 1996. The signature sequence of voltage-gated potassium channels projects into the external vestibule. J. Biol. Chem. 271, 31013– 31016. Blanc, E., Romi-Lebrun, R., Bornet, O., Nakajima, T., Darbon, H., 1998. Solution structure of two new toxins from the venom of the Chinese scorpion Buthus martensi Karsch blockers of potassium channels. Biochemistry 37, 12412–12418. Coetzee, W.A., Amarillo, Y., Chiu, J., Chow, A., Lau, D., McCormack, T., Moreno, H., Nadal, M.S., Ozaita, A., Pountney, D., Saganich, M., Vega-Saenz De Miera, E., Rudy, B., 1999. Molecular diversity of Kþ channels. Ann. NY Acad. Sci. 868, 233 –285. Goldstein, S.A.N., Pheasant, D.J., Miller, C., 1994. The charybdotoxin receptor of a Shaker Kþ channel: peptide and channel residues mediating molecular recognition. Neuron 12, 1377–1388. 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. Klee, R., Ficker, E., Heinemann, U., 1995. Comparison of voltagedependent potassium currents in rat pyramidal neurons acutely isolated from hippocampal regions CA1 and CA3. J. Neurophysiol. 74, 1982–1995. Li, Y., Hu, G.-Y., 2002. Huperzine A, a nootropic agent, inhibits fast transient potassium current in rat dissociated hippocampal neurons. Neurosci. Lett. 324, 25 –28. Madeja, M., Mußhoff, U., Binding, N., Witting, U., Speckmann, E.-J., 1997. Effects of Pb2þ on delayed-rectifier potassium channels in acutely isolated hippocampal neurons. J. Neurophysiol. 78, 2649–2654. Numann, R.E., Wadman, W.J., Wong, R.K.S., 1987. Outward currents of single hippocampal cells obtained from the adult guinea-pig. J. Physiol. 393, 331 –353. Olamendi-Portugal, T., Gomez-Lagunas, F., Gurrola, G.B., Possani, L.D., 1998. Two similar peptides from the venom of the scorpion
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Pandinus imperator, one highly effective blocker and the other inactive on Kþ channels. Toxicon 36, 759 –770. Pongs, O., Legros, C., 2000. Pharmacology of voltage-gated potassium channels. In: Kurachi, E.Y., Mishina, M. (Eds.), Pharmacology of Ionic Channel Function: Activators and Inhibitors, Springer, Berlin, pp. 177–196. Romi-Lebrun, R., Lebrun, B., Martin-Eauclaire, M.-F., Ishiguro, M., Escoubas, P., Wu, F.Q., Hisada, M., Pongs, O., Nakajima, T., 1997a. Purification, characterization, and synthesis of three novel toxins from the Chinese scorpion Buthus martensi, which act on Kþ channels. Biochemistry 36, 13473–13482. Romi-Lebrun, R., Martin-Eauclaire, M.-F., Escoubas, P., Wu, F.Q., Lebrun, B., Hisada, M., Nakajima, T., 1997b. Characterization of four toxins from Buthus martensi scorpion venom, which act on apamine-sensitive Ca2þ-activated Kþ channels. Eur. J. Biochem. 245, 457–464. Santos, A.I., Wadman, W.J., Costa, P.F., 1998. Sustained potassium currents in maturing CA1 hippocampal neurons. Dev. Brain Res. 108, 13 –21. Sodickson, D.L., Bean, B.P., 1998. Neurotransmitter activation of inwardly rectifying potassium current in dissociated hippocampal CA3 neurons: interactions among multiple receptors. J. Neurosci. 18, 8153– 8162. Storm, J.F., 1990. Potassium currents in hippocampal pyramidal cells. Prog. Brain Res. 83, 161– 187. Tong, Q.-C., Zhang, Y., Li, D.-P., Zhou, Z.-N., Ji, Y.-H., 2000. The blocking effect of BmP02, one novel short-chain scorpion peptide on transient outward Kþ channel of adult rat ventricular myocyte. Regul. Peptides 90, 85–92. 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 insolated from scorpion venoms: a-KTx molecular subfamilies. Trends Pharmacol. Sci. 20, 444 –447. Wu, G., Wei, D.-S., He, F.-H., Hu, G.-Y., Wu, H.-M., 1998. A Kþ channel-blocking peptide from venom of Chinese scorpion Buthus martensii Karsch. Acta Pharmacol. Sin. 19, 317 –321. Wu, G., Li, Y., Wei, D., He, F., Jiang, S., Hu, G., Wu, H., 2000. Solution structure of BmP01 from the venom of scorpion Buthus martensii Karsch. Biochem. Biophys. Res. Commun. 276, 1148–1154. Zeng, X.-C., Peng, F., Luo, F., Zhu, S.-Y., Liu, H., Li, W.-X., 2001. Molecular cloning and characterization offour scorpion Kþ-toxinlike peptides: a new subfamily of venom peptides (a-KTx14) and genomic analysis of a member. Biochimie 83, 883–889.
BmKK4, a novel toxin from the venom of Asian scorpion Buthus martensi Karsch, inhibits potassium currents in rat hippocampal neurons in vitro Ming-Hua Lia, Nai-Xia Zhangb, Xue-Qin Chena, Gong Wub, Hou-Ming Wub, Guo-Yuan Hua,* a
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 555 Zu-Chong-Zhi Road, Shanghai 201203, People’s Republic of China b State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Feng-Lin Road, Shanghai 200032, People’s Republic of China Received 7 March 2003; accepted 27 May 2003
Abstract A novel short-chain peptide BmKK4 was isolated from the venom of Asian scorpion Buthus martensi Karsch. It is composed of 30 amino acids including six cysteine residues, and shares less than 25% sequence identity with the known a-KTx toxins. The action of BmKK4 on voltage-dependent potassium currents was examined in acutely dissociated hippocampal neurons of rat. BmKK4 (10 – 100 mM) inhibited both the delayed rectifier and fast transient potassium current in concentration-dependent manners. The inhibition was reversible and voltage-independent. BmKK4 caused a depolarizing shift (about 10 mV) of the steady-state activation curve of the currents, without changing their steady-state inactivation behavior. The unique amino acid sequence and electrophysiological effects suggest that BmKK4 represent a new subfamily of potassium channel toxins. q 2003 Elsevier Ltd. All rights reserved. Keywords: BmKK4; Buthus martensi Karsch; Hippocampus; Neurotoxin; Potassium channel; Scorpion
1. Introduction Potassium (Kþ) channels play crucial roles in regulating a variety of cellular processes in both excitable and nonexcitable cells, such as setting the resting membrane potential, action potential duration, the delay between a stimulus and the first action potential, discharge patterns, etc. (Storm, 1990). Molecular cloning studies in the recent decades have revealed enormous molecular diversity of Kþ channels. Thus far more than 100 pore-forming subunits of Kþ channels have been cloned. Heteromultimeric assembly of different subunits provides a base for further diversity, and leads to a huge number of functionally diverse Kþ * Corresponding author. Tel.: þ86-21-5080-6778; fax: þ 86-215080-7088. E-mail address: [email protected] (G.-Y. Hu).
channels with distinct biophysical, pharmacological and regulation properties (Coetzee et al., 1999). Investigation of the structure and function of ion channels highly depends on the availability of specific reagents, particularly neurotoxins isolated from terrestrial and marine organisms. In contrast to the enormous molecular and functional diversity of Kþ channels, however, there is in fact a paucity of high-affinity ligands binding to the channels. Such a situation has hindered our understanding how the Kþ channels function in the nervous, cardiovascular and other systems. Short-chain peptides (30 – 40 amino acid residues) isolated from the venoms of different species of scorpions were found to specifically block distinct Kþ channels and named a-KTx toxins (Tytgat et al., 1999; Pongs and Legros, 2000). Among them, charybdotoxin (ChTX, from Israeli scorpion Leiurus quinquestriatus hebraeus), noxiustoxin (NTX, from American scorpion Centruroides noxius
0041-0101/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0041-0101(03)00136-3
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Fig. 1. Amino acid sequence of BmKK4 was aligned according to the cysteine residues (boxed), with other short-chain Kþ channel toxins. The percentage identity with BmKK4 is given at the right. Asterisk indicates amidation at the C terminus. Gaps are presented as dashes. BmP01, BmP03 from Buthus martensi Karsch; ChTX (charybdotoxin) from Leiurus quinquestriatus hebraeus; KTX (kaliotoxin) from Androctonus mauretanicus mauretanicus; NTX (noxiustoxin) from Centruroides noxius hoffmann; MTX (maurotoxin) from Scorpio maurus; ScTX (scyllatoxin) from Leiurus quinquestriatus hebraeus; P01 from Androctonus mauretanicus mauretanicus; TSK(Tskapa) from Buthidae Tityus serrulatus; CoTX1 (cobatoxin1) from Centruroides noxius.
hoffmann) and kaliotoxin (KTX, from North Africa scorpion Androctonus mauretanicus mauretanicus) have been intensively studied. The venom of Asian scorpion Buthus martensi Karsch is also a rich source of a-KTx toxins with 18 peptides already identified, including BmP01, BmP02, BmP03, BmP05, BmKTX, BmTX1 and BmTX2 (Goudet et al., 2002). Pharmacological studies showed that BmP05 affected the small-conductance Ca2þ-activated Kþ channels (Romi-Lebrun et al., 1997b), whereas BmTX1 and BmTX2 acted on both the large-conductance Ca2þ-activated Kþ channels and voltage-gated Kv1.3 channels (Romi-Lebrun et al., 1997a). BmKK4 is a novel short-chain peptide deduced from a cDNA (AJ277729, EMBL) found in a venom gland cDNA library of B. martensi Karsch (Zeng et al., 2001). The peptide is composed of 30 amino acids including six cysteine residues, and shares less than 25% sequence identity with the known a-KTx toxins (Fig. 1). Up to data, however, the biological activity of BmKK4 has not been examined. In the present study, we purified and characterized the peptide from the venom of B. martensi Karsch, and investigated its action on voltage-dependent Kþ currents in rat dissociated hippocampal neurons.
2. Materials and methods All chemicals and reagents, unless otherwise stated, were purchased from Sigma, USA.
(Fig. 2B). Solution A contained NaAC (20 mM), whereas solution B contained NaAC (20 mM) and NaCl (1 M). Then it was followed by the same separation on Sephadex G-50 column and a similar separation on Mono S cation exchange column eluted with a step gradient of solution A to B, but at pH 4.0 (Fig. 2D). The final purification was achieved on a reverse-phase HPLC column (C18 column, 4.6 £ 250 mm, 5 mm bead size, Alltech) using a linear gradient from 20 to 45% buffer B in buffer A. Buffer A contained TFA (0.1%) and CH3CN (10%) in H2O, and buffer B contained TFA (0.1%) and H2O (20%) in CH3CN. 2.2. Sequence determination of BmKK4 After DTT reduction, the peptide (100 pM) was processed with iodoacetic acid derivatization, and digested with sequencing grade trypsin or endoproteinase Lys-C (2 pM). Digested sample was desalted into a solution consisted of CH3CN: H2O (4:1) and formic acid (0.1%) using C18 ZipTips (Millipore, USA). ES-MS and ESMS/MS spectra were performed using a tandem quadrupoleTOF instrument (Micromass, UK). The sample (1– 2 ml) was loaded into a nanospray needle. For recording ESMS/MS spectra, the quadrupole was operated as a mass filter and used to select the desired precursor ion isotope cluster. During product ion analysis the resolving quadrupole was set to transmit a ^ 2 m=z window round the precursor ion. Precursor ions were directed into a hexapole collision cell. The collision gas was argon and the collision energy was optimized for each analysis.
2.1. Purification of BmKK4 2.3. Preparation of rat dissociated hippocampal neurons Crude venom was collected by electrical stimulation of the telson of scorpions B. martensi Karsch bred in captivity in Henan province, China. Lyophilized venom (9.6 g) was dissolved in NH4HCO3 buffer (50 mM, pH 8.5) and centrifuged at 4000g for 15 min. The supernatant was loaded onto a Sephadex G-50 column (2.5 £ 150 cm) preequilibrated and eluted with the same buffer. A fraction containing the peptide was loaded onto a Mono S cation exchange column (HR 5/5, Pharmacia LKB Biotech, Inc.) eluted with a step gradient of solution A to B at pH 5.0
Hippocampal neurons were prepared as described previously (Sodickson and Bean, 1998; Li and Hu, 2002). Briefly, mini-slices (500 mm) of the hippocampal CA1 region from 5 to 9-day-old Sprague-Dawley rats were cut in oxygenated ice-cold dissociation solution containing (in mM): Na2SO4 (82), K2SO4 (30), MgCl2 (5), N-(2-hydroxyethyl)piperazine-N 0 -(2-ethanesulfonic acid) (HEPES) (10) and glucose (10) at pH 7.3 with NaOH. The slices were incubated in dissociation solution containing protease
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Fig. 2. Isolation and purification of BmKK4. (A) Sephadex G-50 column chromatography of the crude venom from Buthus martensi Karsch. (B) FPLC fractionation of the fraction IV obtained in (A) on a Mono S column. (C) Sephadex G-50 column chromatography of the fraction 1 in (B). (D) FPLC fractionation of the fraction 1–2 in (C) on a Mono S column. (E) RP-HPLC fractionation of the fraction 1 –22 in (D) on a C18 column.
XXIII (3 mg/ml) at 32 8C for 8 min. The solution was then replaced with dissociation solution containing trypsin inhibitor type II-S (1 mg/ml) and bovine serum albumin (1 mg/ml). The slices were allowed to cool to room temperature under an oxygen atmosphere. Before recording, the slices were triturated using a series of fire-polished Pasteur pipettes with decreasing tip diameters. Dissociated neurons were placed in a recording dish and perfused with external solution. The solution contained (in mM): NaCl (135), KCl (5), MgCl2 (2), CaCl2 (1), HEPES (10), glucose (10) and tetrodotoxin (0.001) at pH 7.3 with NaOH.
The delayed rectifier Kþ currents ðIK Þ were elicited by a similar protocol in which a 50 ms interval at 2 50 mV was inserted after the prepulse. Subtraction of IK from Itotal revealed the fast transient Kþ current ðIA Þ (Numann et al., 1987; Klee et al., 1995). Current records were filtered at 2– 10 kHz and sampled at frequencies of 10– 40 kHz. Series resistance was compensated by 75 – 85%. Linear leak and residual capacitance currents were subtracted on-line. BmKK4 was dissolved in the external solution. The toxincontaining solution was applied to the recorded neuron using RSC-100 Rapid Solution Changer with an 18-tube head (BioLogic Co., France).
2.4. Whole-cell voltage-clamp recording 2.5. Data analysis Recording was made from large pyramidal-shaped neurons using an Axopatch 200A amplifier (Axon Instruments, USA) at 21 – 23 8C. The electrodes (tip resistance 2 , 4 MV) were pulled from borosilicate glass pipettes (Hilgenberg, Germany). The pipette solution contained (in mM): potassium gluconate (125), KCl (20), MgCl2 (2), CaCl2 (1), HEPES (10) and EGTA (10) at pH 7.3 with KOH. Voltage protocols were provided by pClamp 6.0.2 software via a DigiData-1200A interface (Axon Instruments, USA). The holding potential was 2 50 mV. As shown in Fig. 3, the total Kþ currents ðItotal Þ were elicited by depolarizing command pulses to þ60 mV in 10 mV steps following a hyperpolarizing prepulse of 400 ms to 2 110 mV.
For analysis, the peak amplitude of IA was measured, whereas the amplitude of IK was determined at 300 ms latency. The data are presented as mean ^ SEM. Student’s two-tailed t-test was used for statistical analysis.
3. Results 3.1. Purification and identification of BmKK4 The soluble venom was initially separated into four fractions (I – IV) by gel-filtration chromatography on
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Fig. 3. BmKK4 inhibits the Kþ currents in hippocampal pyramidal neurons. Upper, middle and lower traces are the current families for the total Kþ currents ðItotal Þ; delayed rectifier Kþ currents ðIK Þ and fast transient Kþ currents ðIA Þ; respectively, in control and in presence of BmKK4 (10 mM). The upper and middle traces were evoked with command pulse protocols shown in the insets. The lower traces are the subtractions of the middle from the upper ones.
a Sephadex G-50 (Fig. 2A). Fraction IV was further separated into 13 fractions on a Mono S cation exchange column (Fig. 2B). After another separation of fraction 1 on a Sephadex G-50 column, four fractions (1 – 1, 1 – 2, 1 – 3 and 1 – 4) were obtained (Fig. 2C). A further separation of the fraction 1 – 2 on a Mono S column gave two fractions (1 – 21, 1 – 22) (Fig. 2D). A pure peptide (6.8 mg) was obtained after the separation of fraction 1 – 22 on a reverse-phase HPLC column (Fig. 2E). The amino acid sequence of the pure peptide was determined via tandem CID MS/MS analysis of its enzymatic fragments. The intact sequence obtained is listed as follows: ZTQCQ SVRDC QQYCL/I TPDRC SYGTC YCKTT(NH2). Following a database search, the peptide was identified with BmKK4 that was deduced from a cDNA
(AJ277729, EMBL) found in a venom gland cDNA library of B. martensi Karsch (Zeng et al., 2001). 3.2. Effects of BmKK4 on voltage-dependent potassium currents in hippocampal neurons With the voltage protocols and subtraction procedure used, two types of outward current could be simultaneously recorded from the same neuron (Fig. 3, left panel). The slowly inactivating outward current was reversibly blocked by TEA (IC50 ¼ 3.1 ^ 0.6 mM, n ¼ 5Þ; thus corresponded to the delayed rectifier Kþ current ðIK Þ: The rapidly inactivating outward current was reversibly blocked by 4-AP (IC50 ¼ 4.9 ^ 0.7 mM, n ¼ 5), and represented the fast transient Kþ current ðIA Þ:
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Fig. 4. The inhibitory effects of BmKK4 on IK and IA : (A) Time course of inhibition caused by BmKK4 (10 mM) in another neuron. (B) Concentration-response curves of BmKK4 in inhibition of IK and IA ðn ¼ 6 – 9Þ: (C) Current– voltage ðI=VÞ relationship of IK before and during application of BmKK4 (10 mM). (D) Current –voltage ðI=VÞ relationship of IA before and during application of the toxin. The data in (C) and (D) were from the same neuron shown in Fig. 3.
BmKK4 (10 – 100 mM) inhibited both IK and IA in concentration-dependent manners (Fig. 3, right panel). The inhibitory actions had a rapid onset and were reversible upon washout (Fig. 4A). When the concentration– response relationships of BmKK4 in inhibition of the Kþ currents were analyzed, the two curves are nearly superimposed. At the maximum concentration (100 mM) tested, the amplitude of IK and IA reduced to 65.7 ^ 3.2 and 68.2 ^ 0.8 of the control levels, respectively (Fig. 4B). Due to the limited amount of toxin obtained, the IC50 values were not determined. The actions of BmKK4 on the current – voltage ðI=VÞ relations of IK and IA are shown in Fig. 4C and D, respectively. The inhibition of BmKK4 on the Kþ currents was voltage-independent. In the presence of BmKK4 (10 mM), the amplitude of IK in steps to 0, þ20, þ 40 and þ60 mV reduced to 81 ^ 6, 77 ^ 7, 73 ^ 9 and 73 ^ 6% of the control levels ðn ¼ 6; P . 0:05 vs. 0 mV), whereas the amplitude of IA in response to the same steps was 86 ^ 6, 78 ^ 8, 77 ^ 9 and 83 ^ 7% of the control levels ðn ¼ 6; P . 0:05 vs. 0 mV). 3.3. Effects of BmKK4 on kinetic behaviors of voltagedependent potassium currents in hippocampal neurons Application of BmKK4 resulted in a depolarizing shift of the steady-state activation curve for both the Kþ currents
(Fig. 5A and C), without significant effect on their steadystate inactivation behavior (Fig. 5B and D). The voltages for half-maximal activation ðVH Þ of IK and IA were 2 1.2 ^ 3.3 and 224.7 ^ 3.9 mV, respectively, in control solution. Application of BmKK4 (10 mM) changed the VH values to þ9.3 ^ 2.0 mV ðn ¼ 5; P ¼ 0:008Þ and 2 13.9 ^ 4.6 mV ðn ¼ 7; P ¼ 0:003Þ; respectively. The effects were reversible: the VH values returned to 2 2.8 ^ 1.2 and 224.8 ^ 1.8 mV, respectively, upon washout.
4. Discussion The present study demonstrates that BmKK4, a novel short-chain peptide isolated from the venom of B. martensi Karsch, inhibits voltage-dependent Kþ currents in rat hippocampal neurons. BmKK4 shares less than 25% sequence identity with the known a-KTx toxins (Fig. 1), thus has been proposed to represent a new subfamily of Kþ channel toxins (a-KTX 17.1) (Goudet et al., 2002). Several known a-KTx toxins in the venom of B. martensi Karsch have been reported to selectively depress the fast transient Kþ current, without effect on the delayed rectifier Kþ current in different preparations. For example, BmP01 and BmP02 (previously coded BmP-3) inhibited the peak component of voltage-dependent Kþ current (corresponding to IA Þ in rat hippocampal neurons (Wu et al., 1998, 2000).
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Fig. 5. Steady-state activation curves (A, C) and inactivation curves (B, D) of IK and IA constructed in the absence and presence of BmKK4 (10 mM). Pulse protocols and subtraction procedure similar to those in Fig. 3 were used to study the steady-state activation. Steady-state inactivation was studied using pulse protocols with hyperpolarizing prepulses from 2110 to 210 mV (10 mV steps) followed by a step to a fixed voltage (0 mV). In each panel the mean of normalized conductance (n ¼ 5–7) is plotted against the membrane potential, and fitted with a Boltzmann equation: G=Gmax ¼ 1={1 þ exp½2ðV 2 VH Þ=k}; where G=Gmax is the mean of normalized conductance, V is the membrane potential, VH (or Vh Þ is the voltage for half-maximal activation (or inactivation), and k is the slope factor.
BmP02 and BmP03 were also found to depress the transient outward Kþ current ðIto Þ in rat ventricular myocytes (Tong et al., 2000). On the contrary, BmKK4 exerted inhibitory effect on both IA and IK in hippocampal neurons with similar potencies. The mechanisms underlying the blockade of voltagegated K þ channels by a-KTx toxins have been intensively explored in the recent decade. It was found that five residues (Lys27, Met29, Asn30, Arg34 and Tyr36) in two C-terminal b-strands and b-turn played important roles in the binding of ChTX, BmTX1 or BmTX2 to voltage-gated Kþ channel (Goldstein et al., 1994; Blanc et al., 1998). Among the five residues, Lys27 was identified to be crucial for toxin affinity, and proposed to plug directly into the pore, coming in the vicinity of the selectivity filter of Kþ channel (Aiyar et al., 1996). In BmKK4, a different residue Arg19 replaces the important residue Lys27 (Fig. 1). The substitution seems to be sufficient to explain the low potency of BmKK4 found in the present study. In fact, a similar phenomenon has been seen in the two short-chain peptides Pi4 and Pi7 isolated from the venom of the scorpion Pandinus
imperator: peptide Pi4 with a critical residue Lys26 is a potent blocker of Shaker B Kþ channels (Kv1.1 channel); whereas peptide Pi7, in which Arg26 replaces the residue Lys26, is totally inactive towards the channels (Olamendi-Portugal et al., 1998). Alternatively, it is likely that BmKK4 mainly affects Ca2þ-activated Kþ currents, rather than voltage-dependent Kþ currents. Because Ca2þ-activated Kþ current accounts for less than 2% of the total sustained Kþ current recorded in our preparation (Santos et al., 1998; Li and Hu, 2002), this question was not further addressed. If the substitution Arg19 for Lys26 in BmKK4 markedly attenuates its affinity for the Kþ channels, and prevents it occluding the channel pore, how does the toxin exert its inhibitory action on the voltage-dependent Kþ currents? We noticed that BmKK4-induced inhibition was accompanied by a reversible depolarizing shift (nearly 10 mV) of the steady-state activation curve of the Kþ currents. Similar shift of the voltage-dependent activation has been used to explain the Pb2þ-induced inhibition of the delayed rectifier Kþ current in hippocampal neurons (Medeja et al., 1997). It is feasible that BmKK4 may depress the Kþ currents via
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a novel mechanism distinct from the classical model proposed for a-KTx toxins in blockade of Kþ channels. The exact mechanism underlying BmKK4-induced inhibition of the Kþ currents need to be further investigated.
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