Molecular basis of the inhibition of the fast inactivation of voltage-gated sodium channel Nav1.5 by tarantula toxin Jingzhaotoxin-II

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Molecular basis of the inhibition of the fast inactivation of voltage-gated sodium channel Nav1.5 by tarantula toxin Jingzhaotoxin-II

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Ying Huang a , Xi Zhou a , Cheng Tan a , Yunxiao Zhang a , Huai Tao b , Ping Cheng a , Zhonghua Liu a,∗ a

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College of Life Sciences, Hunan Normal University, Changsha 410081, Hunan, China Department of Biochemistry and Molecular Biology, Hunan University of Chinese Medicine, Changsha 410208, Hunan, China

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a r t i c l e

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Article history: Received 26 January 2015 Received in revised form 13 March 2015 Accepted 16 March 2015 Available online xxx

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Keywords: Jingzhaotoxin-II Nav1.5 Action mechanism

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Introduction

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Jingzhaotoxin-II (JZTX-II) is a 32-residue peptide from the Chinese tarantula Chilobrachys jingzhao venom, and preferentially inhibits the fast inactivation of the voltage-gated sodium channels (VGSCs) in rat cardiac myocytes. In the present study, we elucidated the action mechanism of JZTX-II inhibiting hNav1.5, a VGSC subtype mainly distributed in human cardiac myocytes. Among the four VGSC subtypes tested, hNav1.5 was the most sensitive to JZTX-II (EC50 = 125 ± 4 nM). Although JZTX-II had little or no effect on steady-state inactivation of the residual currents conducted by hNav1.5, it caused a 10 mV hyperpolarized shift of activation. Moreover, JZTX-II increased the recovery rate of hNav1.5 channels, which should lead to a shorter transition from the inactivation to closed state. JZTX-II dissociated from toxin–channel complex via extreme depolarization and subsequently rebound to the channel upon repolarization. Mutagenesis analyses showed that the domain IV (DIV) voltage-sensor domain (VSD) was critical for JZTX-II binding to hNav1.5 and some mutations located in S1–S2 and S3–S4 extracellular loops of hNav1.5 DIV additively reduced the toxin sensitivity of hNav1.5. Our data identified the mechanism underlying JZTX-II inhibiting hNav1.5, similar to scorpion ␣-toxins, involving binding to neurotoxin receptor site 3. © 2015 Published by Elsevier Inc.

Voltage-gated sodium channels (VGSCs) are responsible for the generation and propagation of action potentials in excitable cells [6]. All VGSCs are composed of a pore-forming functional ␣-subunit (260 kDa) and up to four auxiliary ␤ subunits [5,8]. Nine VGSC ␣ subunits (Nav1.1–1.9) and four ␤ subunits (␤1–␤4) have been identified and functionally characterized from mammals [5]. In terms of tetrodotoxin (TTX), the nine isoforms of VGSCs are classified as TTX-sensitive (TTX-S) (Nav1.1–1.4, Nav1.6, and Nav1.7) or TTX-resistant (TTX-R) (Nav1.5, Nav1.8, and Nav1.9) [3,5,34]. The ␣-subunit consists of four homologous domains (DI–DIV), each of which has six transmembrane segments (S1–S6) and a reentrant pore loop between S5 and S6. The central pore is separated by the SS1 and SS2 segments, which form the ion selectivity filter at its extracellular end. The voltage sensor domain

∗ Corresponding author. Tel.: +86 731 88872556. E-mail address: [email protected] (Z. Liu).

(VSD) is made up of the S1 to S4 segments [9,19,24]. The VSDs are essentially independent structures that directly respond to changes in membrane potential, initiating conformational changes that open or close the pore [3,4,31]. The intracellular loop between DIII and DIV forms the fast inactivation gate, folding in and blocking the pore to cause fast inactivation [11,27]. Neurotoxins such as TTX, ␮-conotoxins, ␣- and ␤-scorpion toxins, sea anemone toxins, and spider toxins interact with VGSCs, blocking sodium currents or modulating the gating properties of these ion channels. At least six different neurotoxin receptor sites (site 1–6) have been identified [1,2,14,29]. A number of animal toxins have been found to modulate the activities of the voltage sensors in DII and DIV which are the two most common toxin-binding sites in VGSCs [25]. Scorpion ␣-toxins bind to the extracellular DIVS3–S4 linker and slow fast inactivation by trapping the DIVS4 voltage sensor in the closed state [20,25]. Scorpion ␤-toxins bind to the extracellular DIIS3–S4 linker to enhance voltage-dependent activation by trapping the DIIS4 voltage sensor in the activated state [7,40]. In contrast, two tarantula toxins, HWTX-IV and ProTx-II can both inhibit channel activation by trapping the DIIS4 voltage sensor

http://dx.doi.org/10.1016/j.peptides.2015.03.012 0196-9781/© 2015 Published by Elsevier Inc.

Please cite this article in press as: Huang Y, et al. Molecular basis of the inhibition of the fast inactivation of voltage-gated sodium channel Nav1.5 by tarantula toxin Jingzhaotoxin-II. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.03.012

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Fig. 1. Inhibitory effect of JZTX-II on four VGSC subtypes expressed in HEK293 cells. rNav1.3 (A), rNav1.4 (B), hNav1.5 (C) and hNav1.7 (D) channels were exposed to JZTX-II. All inward current traces were elicited by a 50-ms depolarization potential to −10 mV from a holding potential of −80 mV. Current traces before and after the application of toxin are shown in black and red, respectively. (E) The inhibition of JZTX-II on rNav1.3 or hNav1.5 was dose-dependent. Every point (mean ± S.E.) was derived from 3–5 separated experimental cells. These data points were fitted according to a sigmoidal equation. Apparent EC50 values were 1.65 ± 0.29 ␮M for rNav1.3 and 125 ± 4 nM for hNav1.5. Because of the low inhibition of the toxin on rNav1.4 and hNav1.7, the apparent EC50 values were not calculated.

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in the resting state and impair channel inactivation by interacting with DIV [28,35,36]. Thus, animal toxins may provide important information about structural mechanisms of channel gating and powerful foundations for the structure–function relationship of VGSCs. Spider venoms contain abundant neurotoxins that target various voltage-gated ion channels, many of which are invaluable receptors or targets for elucidating important physiological functions [2]. Jingzhaotoxin-II (JZTX-II) is a 32-residue peptide isolated from the tarantula Chilobrachys jingzhao [33]. In our previous study, we showed that JZTX-II preferentially inhibits rapid inactivation of TTX-R VGSC on cardiac myocytes, has weak effect on TTX-S VGSCs in rat DRG neurons, TTX-S VGSCs in rat hippocampal neurons and cockroach DUM neurons, and has no effect on TTX-R VGSCs in rat DRG neurons [33]. However, little is known about the molecular basis of its interaction with VGSCs. In this study, we investigated the toxin sensitivity for rNav1.3, rNav1.4, hNav1.5, and hNav1.7, as well as the mechanism of its inhibition on hNav1.5. Of the four examined VGSCs, hNav1.5 was the most sensitive to JZTX-II. The hNav1.5 mutant analyses showed that JZTX-II might inhibit the rapid inactivation by docking to receptor site 3.

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Materials and methods

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Toxin purification

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JZTX-II was purified from the crude venom which was collected from the spider Chilobrachys jingzhao as described by Wang et al. [33]. The purity of the toxin was determined to be >98% by reversed-phase high performance liquid chromatography (RPHPLC), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis, and N-terminal Edman degradation. JZTX-II was lyophilized and stored at −20 ◦ C.

Plasmids and construction of hNav1.5 mutants All mutations of hNav1.5 were constructed using the Gene Tailor Site-Directed Mutagenesis System (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The forward and reverse primers with a length of 30 base nucleotides were designed with only one nucleotide change in the codons. Sitedirected mutations were produced by PCR with corresponding primers. All mutations were sequenced to confirm that the appropriate mutations were made. Transient transfection A VGSC plasmid, such as wild type VGSC plasmid (rNav1.3, rNav1.4, hNav1.5, or hNav1.7) or mutant hNav1.5 channel plasmid, and a plasmid for green fluorescent protein were transiently transfected into human embryonic kidney 293 (HEK293) cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. HEK293 cells were grown under standard tissue culture conditions (5% CO2 ; 37 ◦ C) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The hNav1.7 channel was cotransfected with the ␤1 subunit [17] to increase the current density, whereas the rNav1.3, rNav1.4 and hNav1.5 channels were transfected without any ␤ subunit. Cells with green fluorescent protein fluorescence were selected for whole-cell patch-clamp recordings at 36–72 h after transfection. Whole-cell patch-clamp recording Sodium current recording was performed using whole-cell patch-clamp technique at room temperature (20–25 ◦ C) through an EPC-9 patch clamp amplifier (HEKA Electronics, Germany). Suction pipettes with access resistance of 2.0–3.0 M were made from borosilicate glass capillary tubes (VWR micropipettes; VWR

Please cite this article in press as: Huang Y, et al. Molecular basis of the inhibition of the fast inactivation of voltage-gated sodium channel Nav1.5 by tarantula toxin Jingzhaotoxin-II. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.03.012

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Fig. 2. The effect of JZTX-II on the kinetics of the hNav1.5 current expressed in HEK293 cells. (A) Current–voltage (I–V) curves for hNav1.5 before (solid circles) and after (open circles) 0.5 ␮M JZTX-II treatment. Currents were induced by 50 ms depolarizing steps to various test potentials from a holding potential of −80 mV. Test pulses ranged from −80 to +80 mV with a 10 mV increment. (B) JZTX-II (0.5 ␮M; open circles) shifted the conductance–voltage relationships of hNav1.5 to more negative potentials as compared with control (solid circles). (C) The voltage dependence of steady-state inactivation was estimated using a standard two-pulse protocol, in which sodium currents were induced by a 50-ms depolarizing potential of −10 mV following a 500-ms prepulse at potentials that ranged from −130 to −10 mV with a 10-mV increment. JZTX-II (0.5 ␮M) had no obvious effect on steady-state inactivation. (D) The cells were held at −80 mV and prepulsed to −10 mV for 50 ms to inactivate hNav1.5 currents, then returned to the recovery potential (−80 mV) for increasing recovery durations prior with a test pulse to −10 mV. Typical current traces were recorded from cells expressing hNav1.5 before (top) and after (below) the application of 0.5 ␮M JZTX-II, indicating the rate of recovery from inactivation (repriming) at −80 mV. (E) Data points represent the ratios of the peak currents induced by test pulse (Itest ) to those induced by conditioning pulse (Icond ). Plotted against the duration of the interval between two pulses, they have been fitted according to a single exponential equation. The open and solid circle symbols present for control and 0.5 ␮M JZTX-II, respectively. The kinetics of current recovery from fast inactivation before and after the application of JZTX-II were 8.7 ± 0.7 and 3.5 ± 0.4 ms (n = 5), respectively.

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Co., West Chester, PA, USA) using a two-step vertical microelectrode puller (PC-10; Narishige, Tokyo, Japan). The pipette solution contained 140 mM CsF, 1 mM EGTA, 10 mM NaCl, and 10 mM HEPES (pH 7.3). The external solution contained 140 mM NaCl, 3 mM KCl, 1 mM MgCl2 , 1 mM CaCl2 , and 10 mM HEPES (pH 7.3). Experimental data were collected and analyzed by

using the Pulse/Pulsefit program (version 8.0; HEKA Electronics). Macroscopic sodium currents were usually filtered at 5 kHz and sampled at 20 kHz. Series resistance was kept at 3–5 M and compensated to 75–85% for all cells; linear capacitative and leakage currents were digitally subtracted by using the P/4 protocol.

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Fig. 3. The kinetics of dissociation and reassociation of JZTX-II-Nav1.5 complex upon strong depolarization and repolarization, respectively. (A) Traces demonstrating time dependent dissociation of JZTX-II at +120 mV. Cells expressing hNav1.5 were incubated in 0.5 ␮M JZTX-II for 5 min at a holding potential of −100 mV to allow binding. The current traces showed that sustained current amplitudes were decreased more when the depolarization lasted for longer, indicating that the dissociation of JZTX-II from hNav1.5 could be accelerated with longer depolarization. The rate of toxin dissociation was determined with the illustrated pulse paradigm by stepping to a depolarizing pulse of +120, +100, or +80 mV for 1–1024 ms, returning to −100 mV for 20 ms to allow recovery from fast inactivation and then assessing the effect of the depolarizing pulse with a 50-ms test pulse to −10 mV. (B) Time course of dissociation of 0.5 ␮M JZTX-II from cells expressing hNav1.5 at +120 mV ( = 28 ± 2 ms), +100 mV ( = 54 ± 4 ms), and +80 mV ( = 185 ± 37 ms) (n = 5; P < 0.001). (C) Current traces demonstrating time dependent reassociation of JZTX-II at −100 mV. Cells expressing hNav1.5 were incubated in 0.5 ␮M JZTX-II. Toxin was dissociated from the channels using a 300-ms depolarization to +100 mV. The rate of toxin association was measured by stepping to a hyperpolarizing pulse of −120, −100, or −80 mV for increasing times. The effect of repolarization on toxin action was assessed with a 50-ms test pulse to −10 mV. (D) The time course of association of 0.5 ␮M JZTX-II from cells expressing hNav1.5 was determined at −120 mV ( = 18.1 ± 1.9 s), −100 mV ( = 18.2 ± 1.1 s), and −80 mV ( = 14.0 ± 2.3 s) (n = 5; P > 0.05).

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Data analysis

Results

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Data analysis was performed using the Pulse/Pulsefit (HEKA, Germany) and Sigmaplot 10.0 (Sigma, St. Louis, MO, USA) software programs. All data points are presented as mean ± standard error and n represents the number of separate experimental cells. The degree of fast inactivation was assayed by measuring the I5 ms /Ipeak ratio where I5 ms is the current measured at the depolarization of 5 ms. Concentration–response curves were fitted by the sigma plot Sigmoidal equation as follows: y = a/(1 + exp(−(x−EC50 )/b)), in which EC50 is the concentration of toxin at half-maximal efficacy, a and b are the constants. Steady-state activation and inactivation curves were fitted using the Boltzmann equation: y = 1/(1 + exp((V−V1/2 )/k), in which V is the test potential, V1/2 is the midpoint voltage of kinetics, and k is the slope factor. The current recovery from inactivation, the toxin dissociation and reassociation curves were fitted using a single exponential equation f(t) = A e−t/ +C, where A represents the amplitude of the current, t is the time,  is the time course, and C is the constant.

Selectivity of JZTX-II for VGSC subtypes

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The amino acid sequence of JZTX-II was determined to be GCGTMWSPCSTEKPCCDNFSCQPAIKWCIWSP by Edman degradation in our previous study [33]. Previous work also demonstrated that JZTX-II preferentially inhibits the rapid inactivation of TTX-R VGSC in cardiac myocytes with the EC50 value of 0.26 ± 0.09 ␮M [33]. However, the effects of JZTX-II on specific VGSC subtypes remain unclear. Therefore, the inhibitory activity of JZTX-II on rNav1.3, rNav1.4, hNav1.5 or hNav1.7 channels transiently expressed on HEK293 cells was examined in this study. Using whole-cell voltage clamp recording techniques, the current of each subtype was triggered by 50-ms depolarization potentials to −10 mV from a holding potential of −80 mV for every 5 s. JZTX-II displayed different inhibitory potency on the fast inactivation of the four VGSC subtype currents as revealed by I5 ms /Ipeak ratio, which accounts for 79.2% for hNav1.5, 7.3% for rNav1.3, 20.4% for rNav1.4

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Fig. 4. JZTX-II interacts with the voltage sensor region in hNav1.5 DIV. The inhibitory effect of 0.5 ␮M JZTX-II on wild type (wt)-hNav1.5 (A), Nav1.5/1.3 D4D1pore chimera (B), Nav1.5/1.3 D1pore chimera (C) and Nav1.5/1.3 D4VSD chimera (D). All inward current traces were elicited by a 50-ms depolarization potential to −10 mV from a holding potential of −80 mV. (E) The inhibitory effect of the toxin on the four channels was dose-dependent. (F) The EC50 values were 125 ± 4 nM for wt-hNav1.5, 5.60 ± 0.77 ␮M for Nav1.5/1.3 D4D1pore chimera, 300 ± 11 nM for Nav1.5/1.3 D1pore chimera and 6.52 ± 0.60 ␮M for Nav1.5/1.3 D4VSD chimera (n = 3–5).

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and 23.0% for hNav1.7 (Fig. 1A–D). It should be noted that the former two subtypes were treated with 0.5 ␮M JZTX-II, while the last two subtypes were treated with 5 ␮M JZTX-II. JZTX-II also exerted a weak concentration-dependent reduction of hNav1.7 currents accompanied by a slowing of sodium current inactivation similar to delta-ACTXs [22]. The fast inactivation inhibition of hNav1.5 and rNav1.3 currents by the toxin was dose-dependent, indicating the apparent EC50 value was 125 ± 4 nM for hNav1.5 or 1.65 ± 0.29 ␮M for rNav1.3 (Fig. 1E; n = 3–5). Because of the very low inhibition by the toxin, the EC50 values for rNav1.4 and hNav1.7 could not be accurately calculated. These data indicate that hNav1.5, a VGSC subtype preferentially expressed in cardiac myocytes, is the most sensitive to JZTX-II. Effects of JZTX-II on the activation and inactivation kinetics of hNav1.5 Certain VGSC toxins, such as CssIV, ProTx-II, scorpion ␣-toxins, and the sea anemone toxins, shift the threshold of VGSC activation to more positive or negative potentials [7,21,36,41] The current–voltage (I–V) curves of hNav1.5 were examined before and after the application of 0.5 ␮M JZTX-II (Fig. 2A), indicating that the toxin treatment resulted in a 10 mV hyperpolarization shift of the maximum activation voltage (from −20 mV in control to −30 mV in the presence of toxin) without evidently altering the initial activation and reversal potentials. Accordingly, a hyperpolarization shift of the steady-state activation curve was observed after 0.5 ␮M JZTX-II treatment (Fig. 2B; n = 5). The half-activation voltage (V1/2 ) of hNav1.5 was −36.5 ± 0.3 and −46.6 ± 0.9 mV in absence and presence of 0.5 ␮M JZTX-II, respectively. These data indicate that it might be easier to activate hNav1.5 current in the presence of JZTX-II by a weak depolarization. Steady-state inactivation is another important property of VGSCs, which modulate the excitability of neurons [13] and it can be modified by scorpion and spider toxins [2,28,29]. Therefore, we investigated the effect of JZTX-II on steady-state inactivation of hNav1.5 using a standard two-pulse protocol. As shown in

Fig. 2C, the estimated midpoint of steady-state inactivation was −66.8 ± 0.7 mV for control and −64.6 ± 1.1 mV for 0.5 ␮M JZTX-II treatment (n = 5). These data indicate that JZTX-II had little or no effect on the steady-state inactivation of the channel, despite its capacity to inhibit the rapid inactivation of hNav1.5. Some scorpion ␣-toxins also have no effect on steady-state fast inactivation, whereas they remarkably inhibit the fast inactivation of VGSCs [18]. Effects of JZTX-II on the repriming kinetics of hNav1.5 The effect of JZTX-II on recovery rate from fast inactivation was examined by using a standard two-pulse protocol in which the cell clamped at −80 mV was applied with a 50 ms conditioning pulse of −10 mV, then a 50 ms second test pulse of −10 mV was triggered after a repolarizing interpulse to −80 mV during an interval between 0.5 ms and 1 s. Under control condition, almost no current was induced by the second test pulse at the interpulse (Itest ) duration of 0.5 ms. In the 30–35-ms interpulse time, currents induced by Itest were as large as that induced by first conditioning pulse (Icond ), indicating full recovery from inactivation (Fig. 2D). Repriming (recovery from inactivation) was estimated by calculating the ratio of Itest to Icond (Fig. 2E; n = 5). It was seen that 0.5 ␮M JZTX-II significantly accelerated channel recovery from inactivation, and in the presence of JZTX-II for the interpulse times above 16 ms, fast inactivation inhibition was found in all induced current traces by both test and conditioning pulses. A significant decrease in the time required for current recovery from fast inactivation before and after the application of 0.5 ␮M JZTX-II was observed from 8.7 ± 0.7 to 3.5 ± 0.4 ms (n = 5). This result shows that the toxin bound to hNav1.5 might decrease the amount of inactivation and accelerate the dissociation of the inactivation particle of hNav1.5, causing a shorter transition from the inactivated to closed state. Kinetics of dissociation and reassociation of JZTX-II from hNav1.5 Binding of toxins were reversed by prolonged strong depolarizations, such as occurs for HWTX-IV, HNTX-III and ␣-scorpions

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Fig. 5. Effects of single-site mutations located in hNav1.5 DIVS1–S2 and DIVS3–S4 linkers on JZTX-II inhibitory potency. (A and B) Concentration dependence for the inhibition of JZTX-II on two hNav1.5 mutants (T1548A, D1549A) in D4 S1–S2 linker and four hNav1.5 mutants (D1609A, Y1614A, F1615A and S1617A) in D4 S3–S4 linker. The apparent EC50 values were 400 ±12 nM, 445 ± 21 nM, 1.54 ± 0.02 ␮M, 512 ± 40 nM, 392 ± 47 nM, and 341 ± 19 nM for T1548A, D1549A, D1609A, Y1614A, F1615A and S1617A, respectively (n = 3–5). (C and D) Changes in apparent toxin affinity (Mut EC50 /WT EC50 ) were plotted for individual mutants in the DIV S1–S2 region (C) and the DIV S3–S4 region (D). Six crucial determinants of JZTX-II binding to hNav1.5 are shade in gray and the most crucial D1609 is marked with an asterisk (*).

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[16,32,35]. Therefore, we examined whether the inhibitory effect of JZTX-II on hNav1.5 could be reversed by prolonged strong depolarizations using the protocol described by Wang et al. [32] with some modifications. Clamped at −100 mV, experimental cells expressing hNav1.5 were stepped to a depolarizing potential of +120 mV for 1–1024 ms to induce dissociation, hyperpolarized to −100 mV for 300 ms to reverse inactivation, and followed by a pulse of −10 mV for measurement of sodium currents (Fig. 3A, inset). As shown in Fig. 3A, in the presence of 0.5 ␮M JZTX-II, gradually longer strong depolarizations led to faster and more complete hNav1.5 channel inactivation, indicating JZTX-II dissociation. Depolarization to +100 mV lasting 256 ms resulted in practically complete dissociation of toxin and loss of toxin effect (Fig. 3B). It should be noted that the toxin dissociation rate was determined by the ratios of I5 ms to Ipeak , which were elicited during the test pulse and plotted as a function of conditioning pulse duration. In the presence of 0.5 ␮M JZTX-II, the dissociation time constant after strong depolarizations was 28 ± 2 ms at +120 mV, 54 ± 4 ms at +100 mV, and 185 ± 37 ms at +80 mV (Fig. 3B; n = 5; P < 0.001). These results showed that the rate of JZTX-II dissociation was voltage-dependent, with more rapid dissociation at stronger depolarized potentials. The rate of toxin reassociation of the unbound JZTX-II to hNav1.5 was also assessed using another two-pulse protocol, comprising a 300-ms depolarization to +100 mV to cause toxin dissociation, followed by gradually longer repolarizations to allow toxin rebinding,

and a final test pulse of −10 mV to assess JZTX-II effect (Fig. 3C, inset). The toxin progressively rebound to hNav1.5 after longer repolarizations with complete reassociation at 131 s (Fig. 3C). In the presence of 0.5 ␮M JZTX-II, the association time constant was 18.1 ± 1.9 s at −120 mV, 18.2 ± 1.1 s at −100 mV, and 14.0 ± 2.3 s at −80 mV (Fig. 3D; n = 5; P > 0.05), indicating that toxin reassociation was voltage-independent. JZTX-II interacts with the voltage sensor region in the DIV of hNav1.5 Our data indicate that JZTX-II might share a similar effect with some scorpion ␣-toxins, and therefore it would structurally interact with the receptor site 3 of hNav1.5, which is composed of the extracellular loops connecting S5–S6 of DI, and S1–S2 and S3–S4 of DIV [12,20,25]. Thus, our initial attempts focused on finding whether the inhibition resulted from interaction with a specific domain of this channel. Chimeras of hNav1.5 domains combined with varying homologous domains of rNav1.3 were constructed and expressed in HEK293 cells. The chimeric channels were named as follows: for example, Nav1.5/1.3 D4D1pore chimera represents the replacement of DI pore region and the whole DIV from hNav1.5 with the counterparts from rNav1.3. As shown in Fig. 4A–C, Nav1.5/1.3 D4D1pore chimera but not Nav1.5/1.3 D1pore chimera greatly reduced the inhibition of JZTX-II. This data

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suggested that the interaction sites might lie somewhere within DIV. Nav1.5/1.3 D4VSD chimera resulted in a >50-fold loss of potency for the inhibition by JZTX-II (Fig. 4D and F). Apparent EC50 values were 125 ± 4 nM for Nav1.5, 5.60 ± 0.77 ␮M for Nav1.5/1.3 D4D1pore chimera, 300 ± 11 nM for Nav1.5/1.3 D1pore chimera and 6.52 ± 0.60 ␮M for Nav1.5/1.3 D4VSD chimera (Fig. 4E; n = 3–5). These data show that the lost potency for inhibition by JZTX-II on Nav1.5/1.3 D4VSD chimera was similar with that on Nav1.5/1.3 D4D1pore chimera (Fig. 4F), indicating that the main interaction site for JZTX-II probably might lie within the voltage sensor region of DIV. Effects of single-site mutations located in hNav1.5 DIVS1–S2 and DIVS3–S4 linkers on JZTX-II inhibitory potency

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To better understand the molecular mechanism of JZTX-II inhibition and identify specific residues that determined the sensitivity of hNav1.5 to JZTX-II, we investigated the interaction of each residue at the hNav1.5 DIVS1–S2 and DIVS3–S4 linker with JZTX-II using the alanine-scanning technique. We found that the single mutation of six amino acid residues (T1548, D1549 in the DIVS1–S2 region, and D1609, Y1614, F1615 and S1617 in the DIVS3–S4 region) produced substantial decreases in JZTX-II inhibition. As seen in Fig. 5A and B, the apparent EC50 values were 400 ± 12 nM, 445 ± 21 nM, 1.54 ± 0.02 ␮M, 512 ± 40 nM, 392 ± 47 nM, and 341 ± 19 nM (n = 3–5) for T1548A, D1549A, D1609A, Y1614A, F1615A and S1617A, respectively. Alanine mutation of each of these six residues reduced toxin sensitivity by 3.2-, 3.6-, 12.3-, 4.1-, 3.1-, and 2.7-fold, respectively. However, alanine mutations of other residues altered JZTX-II binding affinity by <2.2fold (Fig. 5C and D). Obviously, among the mutated residues, D1609 is the most important for JZTX-II binding. As indicated in Fig. 6, JZTX-II (0.5 ␮M) inhibited 79.2 ± 2.2% of the wt-Nav1.5 fast inactivation (Fig. 6A), whereas it only inhibited the fast inactivation of mutant D1609A by 4.2 ± 2.5% (Fig. 6B).

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In contrast to pore-blocking toxins, JZTX-II is a gating modifier, inhibiting the fast inactivation of hNav1.5, similar to some scorpion ␣-toxins [41]. JZTX-II caused a hyperpolarizing shift in the voltage dependent activation of hNav1.5 and had no effect on steady-state inactivation. Like scorpion ␣-toxin Lqh-II [10], the toxin–channel complex was separated by strong depolarizations. JZTX-II dissociated more rapidly at higher depolarized potentials. Additionally, JZTX-II was able to gradually rebind to hNav1.5 after longer repolarizations. The toxin-bound channel could deactivate or return to a closed configuration more rapidly than the free channel in the absence of JZTX-II. Therefore, JZTX-II should share the same action mechanism as some scorpion ␣-toxins by binding to site 3 of hNav1.5. Neurotoxins acting on site 3, such as scorpion ␣-toxins and sea anemone toxins, slow the rapid inactivation of some VGSCs by binding to the extracellular DIV S1–S2 and S3–S4 linkers [23,25]. In our study, we identified six amino acid residues (T1548, D1549 in the S1–S2 region, and D1609, Y1614, F1615 and S1617 in the S3–S4 region) in the extracellular linkers of DIV VSD that produced substantial decreases in the inhibitory potency of JZTX-II on hNav1.5 rapid inactivation. Of the six residues, D1609 might be the most important residue whose mutation caused the biggest decrease in JZTX-II inhibitory potency on hNav5. As shown in Fig. 6C, the amino acid residue in the position corresponding to D1609 in hNav1.5 is the negatively charged D or E in the JZTX-II-sensitive VGSC (Nav1.3, Nav1.4 or Nav1.7), while it is a neutral or a positively charged amino acid residue in the JZTX-II-resistant VGSC (rNav1.8 or rNav1.9) [33].

Fig. 6. D1609 is the most crucial to the inhibitory effect of JZTX-II on hNav1.5 and conserved in JZTX-II-sensitive subtypes. WT Nav1.5 (A) and mutant D1609A (B) were expressed in HEK-293 cells and exposed to 0.5 ␮M JZTX-II. All inward current traces were elicited by a 50-ms depolarization potentials to −10 mV from a holding potential of −80 mV. (C) Alignment of the sequences of DIV S3–S4 linkers of VGSC subtypes indicated. The residues corresponding to D1609 are shaded in gray.

This hints that the acidic residue (D or E) might be the determinant involved in the selectivity of JZTX-II on the VGSC subtypes. In a comparison with the amino acid residues in some VGSCs important for the interaction with some scorpion ␣-toxins, this acidic residue might constitute the common receptor site for the binding of site 3 toxins [25,32]. The corresponding amino acid residue E1613 is critical to the scorpion ␣-toxin binding to Nav1.2 [25]. On the other hand, because of the conservation of this acidic residue in JZTX-II-sensitive VGSCs, our data do not explain the different inhibitory potency of the toxin on these subtypes. We proposed that some other residues located in DIV or even in other domains of hNav1.5 might contribute to this sensitivity difference. The identification of these residues would be useful in defining a receptor site for designing selective modifiers of hNav1.5. In our previous study, we had isolated and characterized other three toxins which acted on Nav1.5 from the venom of Chilobrachys jingzhao, JZTX-I, JZTX-III, and JZTX-XI. Similar with JZTX-II, JZTX-I inhibits the fast inactivation but its selectivity is relatively poor [37,38]. In contrast to JZTX-II, JZTX-III preferentially interacts with Nav1.5 and inhibits Nav1.5 activation by docking to receptor site 4 and binding to the extracellular DIIS3–S4 linker, which produces a positive shift of the sodium conductance and decreases the peak current without affecting the rate of inactivation [26,39]. However, the effect of JZTX-XI does not conform to either typical ␣-scorpion or ␤-scorpion toxins but is similar to that of ␦-ACTXs [27], which have a conspicuous effect on Nav1.5 inactivation and decrease the peak sodium currents [15,30]. These data indicate that the toxins from the single venom can affect Nav1.5 by different action mechanisms, revealing the huge biodiversity of spider venom peptides. In conclusion, JZTX-II is a selective gating modifier of hNav1.5, which inhibits the fast inactivation of the channel through targeting on site 3. This toxin had some different properties when inhibiting Nav1.5 compared with other Nav1.5 toxins identified in our previous studies. Acknowledgements This work was supported by the National Basic Research Program of China (973 Program), grant No. 2010CB529801; the

Please cite this article in press as: Huang Y, et al. Molecular basis of the inhibition of the fast inactivation of voltage-gated sodium channel Nav1.5 by tarantula toxin Jingzhaotoxin-II. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.03.012

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Please cite this article in press as: Huang Y, et al. Molecular basis of the inhibition of the fast inactivation of voltage-gated sodium channel Nav1.5 by tarantula toxin Jingzhaotoxin-II. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.03.012

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