Li MH et al / Acta Pharmacol Sin 2003 Oct; 24 (10): 1016-1020

BmTx3B, a novel scorpion toxin from Buthus martensi Karsch, inhibits delayed rectifier potassium current in rat hippocampal neurons

LI Ming-Hua¹, WANG Yue-Feng², CHEN Xue-Qin¹, ZHANG Nai-Xia², WU Hou-Ming², HU Guo-Yuan^1,3

¹State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203; ²State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China

³ Correspondence to Prof HU Guo-Yuan. Phn 86-21-5080-6778. Fax 86-21-5080-7088. E-mail gyhu@mail.shcnc.ac.cn

Received 2003-04-10 Accepted 2003-08-01

KEY WORDS BmTx3B; Buthus martensi Karsch; hippocampus; potassium channels; scorpion venom

ABSTRACT

AIM: To examine the effect of BmTx3B, a novel short-chain peptide isolated from the venom of Asian scorpion Buthus martensi Karsch, on voltage-gated potassium channels. METHODS: Two types of voltage-dependent potassium currents were recorded from dissociated hippocampal neurons of neonatal rat in whole-cell voltage-clamp mode, and separated based upon their kinetic properties. RESULTS: BmTx3B (10-100 µmol/L) selectively inhibited the delayed rectifier potassium current (I_K), without affecting the fast transient potassium current (I_A). The inhibition of the peptide on I_K was reversible, concentration-dependent and voltage-independent. BmTx3B did not affect the steady-state activation and inactivation kinetics of the current. CONCLUSION: The short-chain scorpion peptide BmTx3B selectively blocked the delayed rectifier potassium channel.

INTRODUCTION

Potassium (K⁺) channels play important roles in regulating various physiological functions in both excitable and non-excitable cells, such as control of action potential and excitability in nerve and muscle cells, regulation of hormone secretion, cell volume, and T lymph cell activation^[1]. Molecular cloning studies have revealed enormous molecular diversity of K⁺ channels: so far more than 100 pore-forming subunits of K⁺ channels have been cloned. Moreover, heteromultimeric assembly of the subunits, co-assembly with auxiliary subunits, post-transcriptional processes and post-translational modifications provide a base for further diversity, thus leading to a huge number of functionally diverse K⁺ channels with different biophysical, pharmacological and regulation properties^[2]. Study of the structure and function of K⁺ channels is dependent on the availability of specific reagents, in particular, the neurotoxins isolated from terrestrial and marine organisms. Short-chain peptides (30-40 amino acid residues) isolated from the venoms of different species of scorpions were found to specifically block distinct K⁺ channels and named -KTx toxins^[3,4]. Among them, charybdotoxin (ChTX, from Israeli scorpion Leiurus quinquestriatus hebraeus), noxiustoxin (NTX, from American scorpion 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 a rich source of -KTx toxins with 18 peptides already identified, including BmP01, BmP02, BmP03, BmP05, BmKTx, BmTx1, BmTx2, etc^[5]. We recently isolated and purified a novel short-chain peptide BmK622 from the venom of Buthus martensi Karsch, and the amino acid sequence of the peptide was determined by the hybrid application of tandem CID MS/MS, Edman degradation and database search^[6]. The peptide is composed of 37 amino acids including 6 cysteine residues (Fig 1), and identical with BmTx3B, a ChTX homologue found in the NCBI database (accession number AAF87224)^[5]. However, thus far nothing is known about its physiological activity. In order to determine whether the peptide is a K⁺ channel-blocking peptide, we investigated the effect of BmTx3B (or BmK622) on voltage-dependent K⁺ currents in rat dissociated hippocampal neurons.

Fig 1. Amino acid sequence alignment of short-chain scorpion peptides from Buthus martensi Karsch in the charybdotoxin subfamily: The sequences are aligned according to their cysteine residues. Gaps are presented as dashes. The percentage sequences identity with charybdotoxin (ChTX) is given at the right. BmTx1, BmTx2, BmTx3A and BmTx3B (or BmK622) from Buthus martensi Karsch; ChTX from Leiurus quinquestriatus hebraeus.

MATERIALS AND METHODS

BmTx3B (purity >95 %) was prepared as described elsewhere^[6]. Other chemicals and reagents were purchased from Sigma, USA.

Preparation of rat dissociated hippocampal neurons Hippocampal neurons were prepared as described previously^{[7, 8]}. Neonatal (5_9-day-old) Sprague-Dawley rats were supplied by Shanghai Laboratory Animal Center, CAS [SPF, SCXK (Shanghai) 2002-0010]. Mini-slices (500 µm) of the hippocampal CA1 region were cut in oxygenated ice-cold dissociation solution containing (in mmol/L): Na₂SO₄ 82, K₂SO₄ 30, MgCl₂ 5, N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid] (HEPES) 10 and glucose 10 at pH 7.3 with NaOH. The slices were incubated in dissociation solution containing protease XXIII 3 g/L at 32 ºC for 8 min. The solution was then replaced with dissociation solution containing trypsin inhibitor type II-S 1 g/L and bovine serum albumin 1 g/L. 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 containing (in mmol/L): NaCl 135, KCl 5, MgCl₂ 2, CaCl₂ 1, HEPES 10, glucose 10 and tetrodotoxin 0.001 at pH 7.3 with NaOH.

Whole-cell voltage-clamp recording The recording was made from large pyramidal-shaped neurons (10-20 µm in diameter) using an Axopatch 200A amplifier (Axon Instruments, USA) at 21-23 ºC. The electrodes (tip resistance 2~4 M) were pulled from borosilicate glass pipettes (Hilgenberg, Germany). The pipette solution contained (in mmol/L): potassium gluconate 125, KCl 20, MgCl₂ 2, CaCl₂ 1, HEPES 10 and egtazic acid 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 -50 mV. The total K⁺ currents were elicited by depolarizing command pulses from -70 mV to +60 mV in 10 mV steps following a hyperpolarizing prepulse of 400 ms to -110 mV. The delayed rectifier K⁺ currents (I_K) were elicited by a similar protocol in which a 50 ms interval at -50 mV was inserted after the prepulse. Subtraction of I_K from the total K⁺ current revealed the fast transient K⁺ current (I_A) ^[9,10]. 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. BmTx3B was dissolved in the external solution. The toxin-containing solution was applied to the recorded neuron using RSC-100 Rapid Solution Changer with an 18-tube head (BioLogic Co France).

Activation and inactivation kinetics analysis The same pulse protocol to elicit I_K was used to investigate its steady-state activation kinetics. Steady-state inactivation of I_K was studied by using a protocol with a series of hyperpolarizing prepulses of 600 ms from -110 to -10 mV (10 mV steps) followed by a step to a fixed voltage (0 mV). The normalized conductance is plotted against the membrane potential, and fitted with a Boltzmann equation: G/G_max=1/{1+exp[-(V-V _H)/k]}, where G/G_max is the normalized conductance, V is the membrane potential, V_H (or V_h) is the voltage at half-maximal activation (or inactivation), and k is the slope factor.

Data analysis The amplitude of I_K was measured at a latency of 300 ms. The peak amplitude of I_A was also determined. The data are presented as mean±SD. Student's two-tailed t-test was used for statistical analysis.

RESULTS

With the voltage protocols and subtraction procedure used, the delayed rectifier (I_K) and fast transient K⁺ current (I_A) could be simultaneously recorded from the same neuron. BmTx3B (10-100 µmol/L) selectively inhibited I_K. On contrary, the same concentration of BmTx3B did not cause a significant reduction in the amplitude of I_A (Fig 2A). The inhibition of BmTx3B on I_K was voltage-independent (Fig 2B). In the presence of 10 µmol/L BmTx3B, the amplitude of I_K in steps to 0, +20, +40, and +60 mV was reduced to 80 %±7 %, 79 %±7 %, 80 %±5 %, and 81 %±5 % of the control levels (n=6, P>0.05 vs 0 mV).

Fig 2. BmTx3B selectively inhibits the delayed rectifier K⁺ current in hippocampal pyramidal neurons. (A) The left and right records are the delayed rectifier (I_K) and fast transient K⁺ current (I_A), respectively, in control and in the presence of BmTx3B 100 µmol/L. (B) Current-voltage (I/V) relationship of I_K before and during application of BmTx3B 100 µmol/L. (C) Current-voltage (I/V) relationship of I_A before and during application of BmTx3B 100 µmol/L.

The inhibitory effect of BmTx3B on I_K had a rapid onset, and was reversible upon washout (Fig 3A). Furthermore, the effect was concentration-dependent. In the presence of BmTx3B 100 µmol/L, the amplitude of I_K reduced by 50 % (Fig 3B). However, the IC₅₀ value was not determined due to the limitation of purified peptide obtained. The activation and inactivation kinetics of I_K were studied when BmTx3B 100 µmol/L was applied. The peptide did not result in any change in the steady-state activation and inactivation kinetics of the current (Fig 3C, 3D).

Fig 3. The inhibitory effects of BmTx3B on the delayed rectifier K⁺ current. (A) Time course of inhibition caused by BmTx3B 100 mmol/L in a hippocampal neuron. (B) Concentration-response curves of BmTx3B in inhibition of I_K (n=6) and I_A (n=6). ^aP> 0.05, ^cP<0.01 vs the control level. (C) Steady-state activation curves (n=7), and (D) steady-state inactivation curves (n=5) of I_K constructed in control and in the presence of BmTx3B 100 µmol/L. In (C) and (D), the smooth lines show best fits with Boltzmann equations.

DISCUSSION

BmTx3B (or BmK622) shares 40 % sequence identity with ChTX (Fig 1), and has been proposed as a new member in the ChTX subfamily of -KTx toxins (-KTx 1.12)^[5]. In the present study we demonstrated that BmTx3B exerted a selective inhibitory effect on the delayed rectifier K⁺ current, without affecting the fast transient K⁺ current in rat hippocampal neurons. The pharmacological profile of BmTx3B is distinct from that previously reported for other -KTx toxins in the venom of Asian scorpion Buthus martensi Karsch. For example, both BmP01 and BmP02 (previously coded BmP-3) selectively inhibited the peak component of voltage-dependent K⁺ current (corresponding to I_A) in rat hippocampal neurons^[11,12]. BmP02 and BmP03 were also found to inhibit the transient outward K⁺ current (I_to), without affecting the delayed rectifier (I_K) and inward rectifier K⁺ current (I_KI) in rat ventricular myo-cytes^[13]. In addition, BmTx3A (previously named BmTx3) was shown to be a selective inhibitor of the A-type K⁺ current in rat striatal neurons in culture^[14].

In addition to blocking voltage-gated K⁺ channels, K⁺ channel toxins in the venom of Buthus martensi Karsch also affected Ca²⁺-activated K⁺ channels. BmTx1 and BmTx2 were found to block rat brain Kv1.3 channels expressed in Xenopus oocytes, and to inhibit [¹²⁵I]ChTX binding to the large-conductance Ca²⁺-activated K⁺ channels (BK) on bovine aorta sarcolemmal membranes^[15], whereas BmP05 and BmP03 inhibited [¹²⁵I]apamin binding to the small-conductance Ca²⁺-activated K⁺ channels (SK) in synaptosomal membranes of rat brain^[16]. It is likely that BmKK4 mainly affects Ca²⁺-activated K⁺ channels, rather than voltage-gated K⁺ channels. However, Ca²⁺-activated K⁺ current accounted for less than 2 % of the total sustained K⁺ current recorded in dissociated hippocampal neurons of neonatal rat^[8,17], and only a small fraction (16 %-22 %) of the current in dissociated hippocampal neurons of adult and old rat^[18]. This question should be addressed using proper methods, such as [¹²⁵I] ChTX binding assay^[15] and [¹²⁵I] apamin binding assay ^[16].

Compared with BmTx1, BmTx2 and BmKTx, that completely block rat brain Kv1.3 channels with IC₅₀ values of around 1 nmol/L^[15], BmTx3B depressed I_K in hippocampal neurons with a low potency (causing a 50 % inhibition at 100 µmol/L). The deference could be explained by comparing the solution structure of BmTx3B with those of ChTX, BmTx1 and BmTx2. A mutation study has demonstrated that 5 residues (Lys²⁷, Met²⁹, Asn³⁰, Arg³⁴, and Tyr³⁶) in two C-terminal -strands and -turn play important roles for the binding of ChTX to Kv channel^[19]. Both BmTx1 and BmTx2 have the same residues in the relevant positions, thus also exhibit high affinity for Kv channel^[20]. The 4 residues (Lys²⁸, Asn³¹, Arg³⁵, and Tyr³⁷) in BmTx3B are equivalent to the residues Lys²⁷, Asn³⁰, Arg³⁴ and Tyr³⁶ in ChTX. However, a different residue Gln³⁰ in BmTx3B replaces the non-conservative residue Met²⁹ in ChTX, which has been proposed to be crucial for its interaction with Thr⁴⁴⁹ in Shaker Kv channel^[21]. It was demonstrated that mutation of Met²⁹ to Gly²⁹ in ChTX caused a 10⁴ times increase in its K_d value towards Shaker Kv channel^[19].

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