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Originally published In Press as doi:10.1074/jbc.M603212200 on May 23, 2006

J. Biol. Chem., Vol. 281, Issue 30, 20673-20679, July 28, 2006
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Direct Evidence That Receptor Site-4 of Sodium Channel Gating Modifiers Is Not Dipped in the Phospholipid Bilayer of Neuronal Membranes*

Lior Cohen{ddagger}, Nicolas Gilles§, Izhar Karbat{ddagger}, Nitza Ilan{ddagger}, Dalia Gordon{ddagger}1, and Michael Gurevitz{ddagger}2

From the {ddagger}Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel and the §Commissariat à l'Energie Atomique, Department d'Ingenierie et d'Etudes des Proteines, C.E. Saclay, F-91191 Gif Sur Yvette Cedex, France

Received for publication, April 4, 2006 , and in revised form, May 9, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
In a recent note to Nature, R. MacKinnon has raised the possibility that potassium channel gating modifiers are able to partition in the phospholipid bilayer of neuronal membranes and that by increasing their partial concentration adjacent to their receptor, they affect channel function with apparent high affinity (Lee and MacKinnon (2004) Nature 430, 232–235). This suggestion was adopted by Smith et al. (Smith, J. J., Alphy, S., Seibert, A. L., and Blumenthal, K. M. (2005) J. Biol. Chem. 280, 11127–11133), who analyzed the partitioning of sodium channel modifiers in liposomes. They found that certain modifiers were able to partition in these artificial membranes, and on this basis, they have extrapolated that scorpion beta-toxins interact with their channel receptor in a similar mechanism as that proposed by MacKinnon. Since this hypothesis has actually raised a new conception, we examined it in binding assays using a number of pharmacologically distinct scorpion beta-toxins and insect and mammalian neuronal membrane preparations, as well as by analyzing the rate by which the toxin effect on gating of Drosophila DmNav1 and rat brain rNav1.2a develops. We show that in general, scorpion beta-toxins do not partition in neuronal membranes and that in the case in which a depressant beta-toxin partitions in insect neuronal membranes, this partitioning is unrelated to its interaction with the receptor site and the effect on the gating properties of the sodium channel. These results negate the hypothesis that the high affinity of beta-toxins for sodium channels is gained by their ability to partition in the phospholipid bilayer and clearly indicate that the receptor site for scorpion beta-toxins is accessible to the extracellular solvent.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Neurotoxins have been used extensively as molecular probes for defining the functional architecture of voltage-gated sodium and potassium channels (Navs and Kvs) (1–4). Voltage-gated ion channels are composed of two functionally linked yet structurally independent domains (or modules) (2, 5). The pore domain, encompassing trans-membrane segments S5–S6, is responsible for selective ion permeation, whereas a voltage-sensing domain (trans-membrane segments S1–S4) alters the conformation of the channel in response to changes in the membrane potential enabling channel activation. Structural and mechanistic models derived from the crystal structures of the archaebacterial channel KvAP (6, 7) and the mammalian channel Kv1.2 (8, 9) led to the suggestion that the positively charged S4 segment and parts of S3 form a stable hairpin (voltage-sensor paddle) located at the periphery of the channel within the membrane (6). Comparison of KvAP and Kv1.2 crystal structures (8, 9) has suggested that the two channels are similar in structure with a very modest difference in the orientation of their voltage sensor (10). Although Kvs are tetramers of four subunits, each containing six trans-membrane segments, sodium and calcium channels consist of a single large protein of four homologous domains (D1–D4, pseudo-subunits), each containing S1–S6 segments (2).

Regions of these channels involved in the gating mechanism have been shown to be associated with receptor sites for gating modifier toxins derived from venoms of scorpions (11), sea anemones (11, 12), and spiders (6, 13, 14). Interestingly, these gating modifier toxins appear to interact with an equivalent region (S3–S4 loop), albeit at different domains of voltage-gated ion channels (1, 11, 14, 15). Analysis of the activity of VSTX1, a novel potassium channel gating modifier from the spider Grammostola spatulata, on the archaebacterial channel KvAP has led Lee and MacKinnon (16) to suggest that its apparent high affinity is gained by partitioning in the phospholipid bilayer, leading to an increase of toxin concentration near its receptor site (17, 18). Based on the fact that such a modifier interacts with the S3–S4 loop (13) and relying on the x-ray structure of KvAP (6), they claimed that this loop is dipped within the phospholipid bilayer. This hypothesis has attracted the minds of other pharmacologists (19, 20) and motivated experiments in which the ability of toxins that affect Nav gating to partition in liposomes was examined (20). The results of these experiments led to generalization of the MacKinnon hypothesis (16) with the claim that if such toxins were able to partition in liposomes, they might also be able to partition in neuronal membranes and thus reach their target receptor site. However, this suggestion has never been verified experimentally on intact neuronal membranes.

Sodium channel gating modifiers derived from scorpion venom appear to interact with the S3–S4 loops (1) and are suitable for examining the validity of MacKinnon's hypothesis (16) directly on neuronal membranes. These toxins are divided into {alpha} and beta classes according to their mode of action and binding properties (21, 22). {alpha}-Toxins (e.g. Lqh{alpha}IT and Lqh2 from Leriurus quinquestriatus hebraeus and Lqq5 from L. quinquestriatus quinquestriatus) prolong action potentials by inhibition of the fast inactivation of Navs upon binding to receptor site-3, which was partially mapped to loop D4/S3–S4 (11, 23–25). beta-Toxins shift the voltage dependence of channel activation to more negative membrane potentials upon binding to receptor site-4, assigned mainly to loop D2/S3–S4 in mammalian and insect Navs (1, 26–28). This class is divided into: 1) anti-mammalian toxins (e.g. Css2 and Css4 from Centruroides suffusus suffusus (21, 22); 2) toxins that affect insects and mammals (e.g. Ts1 from Tityus serrulatus and Lqhbeta1 (29, 30); 3) anti-insect selective depressant toxins (e.g. LqhIT2 and Lqh-dprIT3 (31, 32); and 4) anti-insect selective excitatory toxins (e.g. AahIT from Androctonus australis hector (33) and Bj-xtrIT from Buthotus judaicus (34)), which differ from the depressant toxins in mode of action and structure (35, 36).

Since in their experiments the scorpion {alpha}-toxin Lqq5 did not partition in phospholipid vesicles, whereas the beta-toxin Css2 did, Blumenthal and colleagues (20) have suggested that beta-toxins access their D2/S3–S4 binding site via a similar process proposed for Kv gating modifiers, namely partition in the phospholipid bilayer and then lateral diffusion toward the binding site on the sodium channel. The unusual number of hydrophobic residues that appear on the surface of site-4 gating modifiers seemingly supports this assumption (37). This suggestion constitutes the underpinning of the idea that receptor site-4, which is associated with D2/S3–S4, is dipped within the membrane, whereas receptor site-3, which is associated with D4/S3–S4, faces the extracellular solvent (20).

We have challenged this revolutionary idea by analyzing the mode of interaction of a number of radiolabeled beta-toxins with receptor site-4 on insect and mammalian neuronal membrane preparations and bring direct evidence that negates this hypothesis. We show that scorpion beta-toxins bind with a fast association rate to a single channel receptor site and that most of them do not partition in neuronal membranes. We show that although some beta-toxins, such as the depressant toxin LqhIT2, partition in insect neuronal membranes, this partition is not related to the toxin binding to receptor site-4 and effect.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Toxins—Bj-xtrIT, LqhIT2, Css4, and Lqhbeta1 were produced in a recombinant form as was described previously (34, 38, 39).

Binding Experiments—Locust synaptosomes, prepared from dissected brains and ventral nerve cords of adult Locusta migratoria, were prepared by established methods (40). Neuronal membranes from cockroach were prepared from whole heads of adult Periplaneta americana according to a previously described method (34). Mammalian brain synaptosomes were prepared from adult albino Sprague-Dawley rats (~300 g, laboratory-bred), as was described previously (41). Membrane protein concentration was determined using a Bio-Rad protein assay by using bovine serum albumin as standard. All toxins were radioiodinated by lactoperoxidase (Sigma; catalog number L8257; 7 units/60 µl of reaction mix) using 10 µg of toxin and 0.5 mCi of carrier-free Na125I (Amersham Biosciences) following a published protocol (42). The monoiodotoxin was purified using a Resource Analytical reverse phase high pressure liquid chromatography column (6.4 x 100 mm, 15-µm particle size; Amersham Biosciences). The concentration of the radiolabeled toxin was determined according to the specific activity of the 125I corresponding to 2500–3000 dpm/fmol monoiodotoxin, depending on the age of the radiotoxin and by estimation of its biological activity (usually 70–80% (43)). The composition of the media used in the binding assays and termination of the reactions were described previously (41, 43). Nonspecific toxin binding was determined in the presence of 1–30 µM unlabeled toxin and is indicated in the figure legends. Equilibrium competition binding assays were performed using increasing concentrations of unlabeled toxin in the presence of a constant low concentration of 125I-toxin and analyzed by the computer program KaleidaGraph (Synergy Software, Reading, PA) using a non-linear Hill equation (for IC50 determination). The Ki values were calculated by the equation Ki = IC50/(1+(L*/Kd)), where L* is the concentration of radioiodinated toxin and Kd is its dissociation constant. Each experiment was performed in duplicate and repeated at least three times as indicated (n). Data are presented as mean ± S.D. of number of independent experiments (41, 43).

The kinetic data for ligand association and dissociation rates were subjected to analysis by LIGAND (Elsevier Biosoft, Cambridge, UK) by using Kinetic Analysis. Each curve was subjected to multislope analysis to determine whether there are one or two slopes. For LqhIT2 binding, kinetic analysis of single and double exponent curves was performed by IGOR Pro (wave-Metrics Inc., Lake Oswego, OR). Toxin dissociation was induced by the addition of excess cold toxin, and the dissociation rate constant (koff) was determined directly from a first order plot of toxin dissociation versus time. The time constant ({tau} = ln2/koff) was estimated directly from the binding curves. The rate of toxin association (kon) was determined from the equation kon = kobs([RL]e/([L][RL]max), where [L] is the ligand concentration, [RL]e is the concentration of the complex at equilibrium, [RL]max is the maximum number of receptors present (determined in a parallel saturation experiment), and kobs is the slope of the pseudo-first order plot ln([RL]e/([RL]e – [RL]t)) versus time (44). Each experiment was performed at least three times. Data are presented as mean ± S.E. of the number (n) of independent experiments.

Expression of Sodium Channels in Oocytes and Two-electrode Voltage Clamp Experiments—cRNAs encoding the Drosophila melanogaster para (DmNav1) sodium channel {alpha}-subunit and the auxiliary TipE subunit (kindly provided by Drs. J. Warmke (Merck) and M. S. Williamson (Institute for Arable Crops Research-Rothamsted, Harpenden, Hertfordshire, UK), respectively), rNav1.2a (plasmid pNa200, a gift from Dr. A. Goldin, University of California, Irvine, CA), and the auxiliary human beta1 were transcribed in vitro using T7 RNA-polymerase and the mMESSAGE mMACHINETM system (Ambion, Austin, TX (45, 46)) and were injected into Xenopus laevis oocytes as was described previously (28).


Figure 1
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  		FIGURE 1.
Effects of LqhIT2 and Lqhbeta1 on Nav activation. The time course of the effect of 500 nM Lqhbeta1on rNav1.2 currents is shown in the upper panel, and the time course of Lqhbeta1 effect on DmNav1 currents is shown in the inset. The time course of the effect of 1 µM LqhIT2 on DmNav1 currents is presented in the lower panel. The kinetics of shift in activation was measured at –40 mV after a 50-ms priming depolarizing pulse to +60 mV as indicated by the voltage protocol, which was applied once every 1 s. The recorded developing currents upon toxin application are shown on the right-hand side of both panels. The solid lines indicate the period of toxin presence.

 
Two-electrode Voltage Clamp Recording—Currents were measured 1–2 days after injection using a two-electrode voltage clamp and a GeneClamp 500 amplifier (Molecular Devices, Downingtown, PA). Data were sampled at 10 kHz and filtered at 5 kHz. Data acquisition was controlled by a Macintosh PPC 7100/80 computer equipped with an ITC-16 analog/digital converter (Instrutech Corp., Port Washington, NY) utilizing Synapse (Synergistic Systems). Capacitance transients and leak currents were removed by subtracting a scaled control trace utilizing a P/6 protocol (46). Bath solution contained (in mM): 96 NaCl, 2 KCl, 1 MgCl2, 2 CaCl2, 5 HEPES, pH 7.85. Oocytes were washed with bath solution flowing from a BPS-8 perfusion system (ALA Scientific Instruments, Westbury, NY) with a positive pressure of 4 p.s.i. Toxins were diluted with bath solution and applied directly to the bath to the final desired concentration.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
We have examined the hypothesis that the binding site of scorpion beta-toxins on the Navs resides in the phospholipid milieu of neuronal membranes by using different experimental approaches. We analyzed the effects of a number of toxins on the gating properties to assess whether they develop in rates that could be correlated with the rates of partitioning and lateral diffusion presented by Lee and MacKinnon (16) and compared them with the kinetic parameters of their binding interaction with their channel receptor site. We have also analyzed directly whether these toxins are capable of partitioning in neuronal membranes.

Effects of Scorpion beta-Toxins on the Activation Properties of NaVs—We compared the effects of the anti-insect selective depressant toxin LqhIT2 on the insect para DmNav1 channel with those induced by the anti-mammalian beta-toxin Css4 on the rat brain rNav1.2a channel (27), as well as analyzed the effects of the multipotent toxin Lqhbeta1 on both channels expressed in Xenopus laevis oocytes. The three beta-toxins induced the typical effect of shift in voltage dependence of channel activation to more negative membrane potentials (Fig. 1) (27, 30)). The apparent association rates of LqhIT2 and Lqhbeta1 to DmNav1 were determined by the currents induced by the depolarization step to –40 mV (Fig. 1), as was determined for Css4 (27). The currents developed in a similar fast rate ({tau} = 1.53 ± 0.29 and 14.3 ± 3.3 s, for LqhIT2 and Lqhbeta1, respectively; Fig. 1), as was shown for Css4 on rNav1.2 (27). Thus, the beta-toxin association rate to receptor site-4, which leads to the modification of the Nav gating properties, is 2 orders of magnitude faster than the relatively slow rates of partitioning and lateral diffusion of VSTX1 to its channel receptor in artificial phospholipid membranes (16).

Kinetic Parameters of the Binding of beta-Toxins to Neuronal Membranes—The kinetic parameters of Bj-xtrIT, Css4, and LqhIT2 binding to neuronal membranes were determined using radiolabeled ligands. This analysis revealed that the association rate constant (kon) of the 125I-Bj-xtrIT to cockroach neuronal membranes was 20.4 ± 5.3 x 106 M–1 s–1 (n = 3), and the kon of 125I-Css4 to rat brain synaptosomes was 12.6 ± 3.2 x 106 M–1 s–1 (Fig. 2), which were comparable with the kon of the classical scorpion {alpha}-toxin Lqh2 to rat brain synaptosomes (12.0 ± 4.0 106 M–1 s–1 (41)). The dissociation rate constants (koff) of Bj-xtrIT and Css4 from their respective receptor sites were also similar, 0.91 ± 0.25 x 10–3 s–1 (n = 4) and 1.66 ± 0.4 x 10–3 s–1 (n = 3), respectively. The equilibrium dissociation constants (Kd) calculated from the kinetic parameters were in agreement with those obtained in equilibrium binding studies (Fig. 2, insets; Kd = 0.09 ± 0.03 nM (n = 4) and Kd = 0.73 ± 0.11 nM (n = 4) for Bj-xtrIT and Css4, respectively). These results indicate that these toxins bind in a reversible manner to a single receptor site in neuronal membranes.


Figure 2
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  		FIGURE 2.
Bj-xtrIT and Css4 binding properties. Upper panel, binding of 125I-Bj-xtrIT to cockroach neuronal membranes (10 µg/ml). Lower panel, binding of 125I-Css4 to rat brain synaptosomes (41 µg of protein/ml). In both experiments, the membranes were incubated at 22 °C in the presence of 0.1 nM radiolabeled toxin up to 45 min to allow maximal binding. Then excess cold toxin (1 µM) was added, and the displacement of the radiolabeled toxin was monitored. Analysis of the association and dissociation rates provided the following values: for Bj-xtrIT, kon = 20.4 ± 5.3 x 106 M–1 s–1 (n = 3), and koff = 0.91 ± 0.25 x 10–3 s–1 (n = 4), and for Css4, kon = 12.6 ± 3.2 x 106 M–1 s–1 (n = 3), and koff = 1.66 ± 0.4 x 10–3 s–1 (n = 3). Scatchard plots of the equilibrium binding curve obtained with increasing concentrations of unlabeled toxins incubated 1 h with 0.1 nM radiolabeled toxins appear in the insets (B/F indicates bound over free). Nonspecific binding, determined in the presence of 1 µM toxin (20 and 15% of total Bj-xtrIT and Css4 binding, respectively) was subtracted. The binding properties obtained are as follow: for Bj-xtrIT, Kd = 0.09 ± 0.03 nM (n = 4) and Bmax = 0.23 ± 0.03 pmol/mg (n = 4), and for Css4, Kd = 0.73 ± 0.11 nM (n = 4) and Bmax = 0.82 ± 0.08 pmol/mg (n = 4). A representative experiment is shown for both toxins.

 
The interaction of the depressant toxin LqhIT2 with cockroach neuronal membranes was, however, more complex, and therefore the kon value could not be determined directly. LqhIT2 binds to two distinct, non-interacting binding sites (47) (Fig. 3), of which the high affinity site is of low capacity (48) (Ki = 0.9 ± 0.66 nM; Bmax = 2.4 ± 0.02 pmol/mg, (n = 3); Fig. 3, inset) and is similar to that of excitatory toxins, e.g. Bj-xtrIT (Fig. 2, inset) (48). The high affinity site was shown to be blocked by anti-peptide antibodies directed against external sodium channel regions (47), and its capacity was reported to be similar to that of saxitoxin receptors on the same neuronal membranes (47–49). These results indicate that the high affinity binding site for LqhIT2 resides on the sodium channel. The low affinity site of LqhIT2 (Ki = 307 ± 88 nM, n = 3) is of a capacity that is too high to be related to Navs(Bmax = 68 ± 9.3 pmol/mg), and its nature is still unknown (47). Since the low affinity/high capacity binding site observed for LqhIT2 is evident when the high affinity site is saturated by an excitatory toxin, such as AahIT or Bj-xtrIT (34, 47) (Fig. 3), and the radiolabeled toxin can be displaced by LqhIT2 and Lqhbeta1 (Fig. 3), it does not represent partition in the phospholipid membrane. Partition in the membrane should have infinite capacity (i.e. non-displaceable and not dependent on concentration) and relatively slow kinetics, as reported for the binding of the spider gating modifier toxin VSTX1 to phospholipid membranes (16).


Figure 3
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  		FIGURE 3.
Binding properties of depressant beta-toxins. Cockroach neuronal membranes (40 µg/ml) were incubated 1 h at 22°C in the presence of 0.1 nM 125I-LqhIT2 and the indicated concentrations of unlabeled toxins. The two Ki values obtained for LqhIT2 were 0.9 ± 0.66 and 307 ± 88 nM (n = 3). Lqhbeta1 displaced 125I-LqhIT2 from both sites with apparent Ki values of 0.4 ± 0.26 and 276 ± 65 nM (n = 3), whereas Bj-xtrIT inhibited 125I-LqhIT2 binding only to its high affinity site with a Ki value of 0.5 ± 0.13 nM (n = 3). The level of the nonspecific binding, determined in the presence of 30 µM LqhIT2 (corresponding to ~50% of total binding), is indicated by the dashed line. The binding properties derived from the Scatchard plot of the equilibrium binding shown in the inset are: Kd = 1.1 ± 0.3 nM (n = 2) and Bmax = 2.4 ± 0.02 pmol/mg (n = 2); Kd = 475 ± 64 nM (n = 2) and Bmax = 68 ± 9.3 pmol/mg (n = 2). B/F indicates bound over free.

 
LqhIT2 Partitioning in the Phospholipid Bilayer of Insect Neuronal Membranes—Blumenthal and colleagues (20) have shown that the beta-toxin Css2 was able to partition in an artificial phospholipid bilayer (liposomes), whereas the scorpion {alpha}-toxin Lqq5 did not. Therefore we examined in a direct approach whether beta-toxins are capable of partitioning in the phospholipid bilayer of neuronal membranes. We followed after the direct binding of LqhIT2 in low (0.1 nM) and high concentrations (30 µM) to locust and cockroach neuronal membranes using tracer amounts of 125I-LqhIT2. Since a high LqhIT2 concentration occupies all displaceable binding sites on these neuronal membrane preparations (Fig. 3), any measurable 125I-LqhIT2 associated with the membranes would imply interaction with a site of infinite capacity, that is, the phospholipid bilayer. In a similar manner, we measured the binding interaction of 125I-Bj-xtrIT and 125I-Css4 with the neuronal membranes of cockroach and rat brain, and as a control, we used the {alpha}-toxin 125I-Lqh{alpha}IT, which binds to receptor site-3 on insect Navs.

The nonspecific binding of 125I-Bj-xtrIT, 125I-Css4, and 125I-Lqh{alpha}IT, each determined in the presence of 1 µM corresponding unlabeled toxin, consisted of only a small fraction of the total binding and was time-independent (Fig. 4). In contrast, the binding of 125I-LqhIT2 to the cockroach membranes in the presence of 30 µM LqhIT2 was time-dependent and reached equilibrium after ~50 min (Fig. 4). Similar results were obtained in binding assays of 125I-LqhIT2 on locust neuronal membranes (Fig. 4). These results suggested that the LqhIT2 fraction that bound to the insect membranes in a non-displaceable yet time-dependent manner was associated with the phospholipid bilayer. The LqhIT2 slow non-displaceable association, which constitutes ~50% of total binding, is considered as nonspecific binding in the equilibrium competition experiments (Fig. 3). It is noteworthy that the ability of LqhIT2 to partition in liposomes composed of phosphatidylcholine/phosphatidylethanolamine was demonstrated previously (50). Together with this observation, we conclude that LqhIT2 non-specific time-dependent binding to locust and cockroach neuronal membranes (Fig. 4) is most likely a result of the toxin slow partitioning in the phospholipid bilayer.


Figure 4
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  		FIGURE 4.
Time dependence of the nonspecific binding of beta-toxins. Neuronal membrane preparations from cockroach (40 µg/ml), locust (160 µg/ml), and rat brain (41 µg/ml) were incubated for upto 2 h at 22°C with 0.1 nM 125I-toxin in the presence of a high concentration of the unlabeled toxin. The cold toxin concentration was 1 µM for Bj-xtrIT, Css4, and Lqh{alpha}IT and 30 µM for LqhIT2. Each data point for LqhIT2 represents the mean ± S.D. of three independent experiments.

 


Figure 5
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  		FIGURE 5.
Time dependence of LqhIT2 binding to cockroach neuronal membranes. The membranes (40 µg/ml) were incubated up to 2 h at 22 °C with 0.1 nM 125I-LqhIT2 to determine total binding and then under identical conditions but in the presence of 5 µM LqhIT2 to determine the nonspecific binding. Analysis of the association rates performed by IGOR Pro provided one exponential fit for the nonspecific binding ({tau}nonspecific = 1527 ± 155s(n = 3)) and a double exponential fit for total binding ({tau}fast = 74 ± 33 s; {tau}slow = 1363 ± 220s(n = 3)). Subtraction of the nonspecific binding from total binding provided a curve with a single exponential fit of the specific binding ({tau} = 108 ± 36s(n = 3)). A representative experiment is shown. The inset shows the same experiment with a linear time scale.

 
In light of these results, we were able to estimate the association rate of LqhIT2 to its high affinity site on Navs in cockroach neuronal membranes, which could not be determined directly due to the toxin partitioning in the membrane. Association kinetics of 0.1 nM 125I-LqhIT2 provided two slopes with small and high time constants ({tau}fast = 74 ± 33 s; {tau}slow = 1363 ± 220 s; Fig. 5), indicating fast and slow kinetic rates. In contrast, the association kinetics of 125I-LqhIT2 in the presence of high concentration of the unlabeled toxin (5 or 30 µM) was monotonous with a single slope of high time constant ({tau}nonspecific = 1527 ± 155 s, Fig. 5; see also Fig. 4), which represented LqhIT2 partitioning in the phospholipid bilayer. Subtraction of the two curves provides an apparent specific binding association curve with a single slope of a small time constant ({tau} = 108 ± 36 s; Fig. 5), indicating that LqhIT2 binding to its high affinity site is of fast kinetics. Such kinetics has been shown for Bj-xtrIT and Css4 ({tau} = 134 and 118 s, respectively; Fig. 2). These toxins bind to a single receptor site on insect or rat brain Navs (Fig. 2) and do not interact with the phospholipid bilayer (Fig. 4). Thus, the kinetics of LqhIT2 partitioning in the phospholipid bilayer is more than 10-fold slower when compared with the kinetics of its interaction with receptor site-4 (Fig. 5). On the basis of these results as well as the ability of LqhIT2 to affect DmNav1 activation within a few seconds (Fig. 1), we conclude that the slow partitioning of LqhIT2 in the membrane does not contribute to its interaction with receptor site-4 or to its physiological effect.

Receptor Site-4 on the NaV Is Accessible from the Extracellular Side of Neuronal Membranes—Using different experimental approaches, we show that scorpion beta-toxins affect Navs gating independent of their ability to partition in the phospholipid bilayer. We first demonstrate that the beta-toxins Css4 (very similar to Css2 (51)) and Bj-xtrIT do not partition in rat brain or insect neuronal membranes (Fig. 4) and that their rates of association to receptor site-4 (Fig. 2) (27, 52) are 2 orders of magnitude faster than that measured for VSTX1 partition and lateral diffusion (16). The difference between our data and those of Blumenthal and colleagues (20) may be attributed to the phospholipid composition and the presence of cholesterol in the neuronal membranes used in our study. Not only has the lipid composition of membranes been shown to influence the partition coefficient of polypeptides (53–55), but it was also shown that specific phospholipids were required for reconstitution of functional rat brain Navs in liposomes and for binding of the scorpion {alpha}-toxin Lqq5 to receptor site-3 (56). Our results are corroborated by previous reports on 125I-Css2 binding to Navs in electroplaque membranes (57) and 125I-Css4 binding to reconstituted rat brain sodium channels in liposomes (58), in which the nonspecific binding was time-independent, suggesting that the two toxins did not interact with the phospholipid bilayer. We further show that the slow partitioning of the beta-toxin LqhIT2 in neuronal membranes (Figs. 4 and 5) is unrelated to its binding to receptor site-4, whereas its fast, high affinity binding correlates well with the fast development of toxin effect on channel activation (Fig. 1) (27). Since {alpha}-toxins do not seem to interact with phospholipids upon binding to receptor site-3 (20) (Fig. 4), we conclude that both receptor site-4 and receptor site-3 of Nav gating modifiers are not dipped in the neuronal membrane. These receptor sites involve loops of S3–S4 in domains 2 and 4 of the channel, which suggests that at least in part, these loops protrude out of the phospholipid bilayer to the extracellular aqueous solution.


   FOOTNOTES
 
* This research was supported by United States-Israel Binational Agricultural Research and Development Grant IS-3480-03 (to M. G. and D. G.); by Israeli Science Foundation, Grants 733/01 (to M. G.) and 1008/05 (to D. G.); and by the German-Israeli Foundation for Scientific Research and Development Grant G-770-242.1/2002 (to D. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence may be addressed. Tel.: 972-3-6409844; Fax: 972-3-6406100; E-mail: dgordon{at}post.tau.ac.il. 2 To whom correspondence may be addressed. Tel.: 972-3-6409844; Fax: 972-3-6406100; E-mail: mamgur{at}post.tau.ac.il.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Cestele, S., and Catterall, W. A. (2000) Biochimie (Paris) 82, 883–892
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