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Originally published In Press as doi:10.1074/jbc.M704651200 on August 8, 2007

J. Biol. Chem., Vol. 282, Issue 40, 29424-29430, October 5, 2007
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Design of a Specific Activator for Skeletal Muscle Sodium Channels Uncovers Channel Architecture*Formula

Lior Cohen{ddagger}, Nitza Ilan{ddagger}, Maya Gur{ddagger}, Walter Stühmer§, 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 §Department of Molecular Biology and Neuronal Signaling, Max Planck Institute of Experimental Medicine, D-37075 Göttingen, Germany

Received for publication, June 6, 2007 , and in revised form, August 6, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Gating modifiers of voltage-gated sodium channels (Navs) are important tools in neuroscience research and may have therapeutic potential in medicinal disorders. Analysis of the bioactive surface of the scorpion beta-toxin Css4 (from Centruroides suffusus suffusus) toward rat brain (rNav1.2a) and skeletal muscle (rNav1.4) channels using binding studies revealed commonality but also substantial differences, which were used to design a specific activator, Css4F14A/E15A/E28R, of rNav1.4 expressed in Xenopus oocytes. The therapeutic potential of Css4F14A/E15A/E28R was tested using an rNav1.4 mutant carrying the same mutation present in the genetic disorder hypokalemic periodic paralysis. The activator restored the impaired gating properties of the mutant channel expressed in oocytes, thus offering a tentative new means for treatment of neuromuscular disorders with reduced muscle excitability. Mutant double cycle analysis employing toxin residues involved in the construction of Css4F14A/E15A/E28R and residues whose equivalents in the rat brain channel rNav1.2a were shown to affect Css4 binding revealed significant coupling energy (>1.3 kcal/mol) between F14A and E592A at Domain-2/voltage sensor segments 1–2 (D2/S1-S2), R27Q and E1251N at D3/SS2-S6, and E28R with both E650A at D2/S3-S4 and E1251N at D3/SS2-S6. These results show that despite the differences in interactions with the rat brain and skeletal muscle Navs, Css4 recognizes a similar region on both channel subtypes. Moreover, our data indicate that the S3-S4 loop of the voltage sensor module in Domain-2 is in very close proximity to the SS2-S6 segment of the pore module of Domain-3 in rNav1.4. This is the first experimental evidence that the inter-domain spatial organization of mammalian Navs resembles that of voltage-gated potassium channels.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Among the superfamily of voltage-gated ion channels, sodium channels (Navs)3 play a central role in the generation of action potentials in excitable cells and are the target of a large variety of neurotoxins, drugs, and insecticides (1, 2). Although the structure of homo-tetrameric voltage-gated potassium channels (Kvs) was recently resolved (3, 4), the structure of Navs has not yet been determined.

Eukaryotic Navs are constituted from one large protein that forms four homologous, non-identical domains. As for Kvs, each domain of the Nav comprises two functional modules. The pore module consists of transmembrane segments S5 and S6 connected by a membrane-associated re-entrant loop (SS1-SS2). The voltage-sensing module, which consists of segments S1-S4, undergoes a conformational alteration in response to changes in the membrane potential, enabling channel activation (1, 5). Estimation of the distances between transmembrane segments of the homo-tetrameric bacterial Nav, NaChBac, suggested an intra-subunit organization similar to that of Kvs in a lipid bilayer (6), but the inter-domain organization of the pore and voltage-sensing modules in eukaryotic Navs have not been determined.

A variety of mammalian Navs, encoded by at least nine genes, have been described (1, 7). Expression of these channels varies greatly across tissues and developmental stages (1). Nav1.1–1.3 and Nav1.6 are expressed in the central nervous system, Nav1.6 and Nav1.7 are expressed in the peripheral nervous system, Nav1.8 and Nav1.9 are expressed in sensory neurons, and Nav1.4 and Nav1.5 are expressed in skeletal and cardiac muscle, respectively. Many genetic disorders leading to abnormal function of these channels have been described in humans (e.g. Refs. 8–12). For example, mutations in the SCN4A gene, encoding for the human skeletal muscle channel Nav1.4, lead to various types of periodic paralysis, paramyotonia congenital, and Nav myotonias (reviewed in Ref. 8). Several mutations rendering hypokalemic periodic paralysis (hypoPP) (8, 13–15) were identified in the voltage sensor segment S4 of Domain 2 (D2/S4) of the human skeletal muscle sodium channel, hNav1.4, causing a positive shift in the voltage dependence of channel activation and gating pore currents at resting membrane potential (13–16). Action potentials evoked from human hypoPP muscle samples are sluggish and smaller than those obtained in healthy muscle tissue (15). Gating modifiers that facilitate channel activation could potentially restore the properties of defective Navs, but to be therapeutically applicable they have to be highly specific for hNav1.4.

Scorpion beta-toxins (e.g. the anti-mammalian Css2 and Css4 from Centruroides suffusus suffusus) are short polypeptides reticulated by four disulfide bonds that target Navs and modulate their gating properties (17). They are typified by the shift they induce in the voltage dependence of channel activation to more negative membrane potentials upon binding to receptor site 4 (17), shown to be associated with Domain 2 of the Nav (18–21). Four conserved residues in D2/S1-S2 and S3-S4 have been implicated in the interaction of the beta-toxin Css4 with mammalian Navs (19, 20), and two residues in D3/SS2-S6 have recently been linked to the preference of the beta-toxin Tz1, from the scorpion Tityus zulianus, for the muscle channel rNav1.4 over the brain channel rNav1.2a (22). These data, and the fact that Css4 is more potent at hNav1.2a than hNav1.4 (23), have suggested that the set of residues comprising receptor site 4 on both channels are not identical.

As the binding of beta-toxins is independent of membrane potential and because a depolarizing prepulse is required to observe their effect, it was suggested that prebound beta-toxins trap the D2/S4 segment in its outward activated position, leading to enhanced channel activation upon subsequent depolarizations (19). Despite the accumulated knowledge about their mode of interaction with the Nav, complete elucidation of neurotoxin receptor site 4 and the extent of its conservation in various channel subtypes remained to be described.

Toxins with high preference for Nav subtypes are useful for identifying distinct channels and studying their mechanism of action and may also be instrumental in development of new drugs for treating neurological and muscular disorders. Moreover, as the receptor binding sites for toxins incorporate residues of different, not necessarily adjacent, extracellular loops (1, 20, 22), by identifying interacting residues of toxins and channels and by relying on the known toxin structures we may elucidate the spatial organization of the channel extracellular face.

Mutagenic dissection of scorpion beta-toxins that affect both mammalian (Css4) and insect (the excitatory toxin Bj-xtrIT from Buthotus judaicus and the depressant toxin LqhIT2 from Leiurus quinquestriatus hebraeus) Navs revealed a spatially similar cluster of bioactive residues (24–26). Moreover, we have suggested that a spatially conserved Glu residue in beta-toxins (position 28 in Css4) forms a "hot spot" for interaction with receptor site 4 (25).

Here we show profound variations in the bioactive surface of Css4 toward the rat muscle and brain Navs, which we used to construct a Css4 triple mutant that exclusively binds and affects rNav1.4. This mutant toxin can restore most of the gating properties of a rNav1.4 mutant designed according to a mutation found in hypoPP patients. We also identified Css4-rNav1.4 residue pairs that exhibit coupling energies indicating close proximity of specific extracellular channel loops. These data enabled the first estimation of the distance between extracellular loops of the gating and pore modules of two adjacent Nav domains and provide evidence that the inter-domain architecture of Navs resembles that of Kvs.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Mutagenesis of Css4—PCR-driven mutagenesis, expression in Escherichia coli, in vitro folding, and purification of Css4 derivatives have been described in detail elsewhere (25).

Expression of Sodium Channels in Oocytes—The pNa200 vectors bearing the genes encoding rNav1.2a, rNav1.3, and rNav1.6 were a gift from Dr. A. Goldin (University of California, Irvine). The pAlter vectors bearing the genes encoding rNav1.4 and hNav1.5 were a gift from Dr. R. G. Kallen (University of Pennsylvania, Philadelphia, PA). These genes and the auxiliary hbeta1 were transcribed in vitro using T7 RNA polymerase and the mMESSAGE mMACHINETM system (Ambion, Austin, TX) (27, 28) and injected into Xenopus laevis oocytes as previously described (21).

Site-directed Mutagenesis of rNav1.4—pAlter containing the entire rat skeletal muscle sodium channel {alpha}-subunit was used for oligonucleotide-based mutagenesis. The PCR-amplified fragment containing the mutations E592A, H599Q/D601S/N602S, E650A, L653A, Q657E, G658N, and R666G was cleaved by BsiwI and BssHII and inserted into the corresponding sites of the vector. E1251N and H1257K were cleaved by EcoNI and inserted into the corresponding sites of the vector, which was further used for transcription after DNA sequence verification.

Two Electrode Voltage Clamp Recording and Data Analysis— Currents were measured 1–2 days after injection using a two electrode voltage clamp and a Gene Clamp 500 amplifier (Axon Instruments, Union City, CA). Data were sampled at 10 kHz and filtered at 5 kHz. Toxins were diluted with bath solution and applied directly to the bath to achieve the desired final concentration. Data acquisition was controlled by a Macintosh PPC 7100/80 computer equipped with an ITC-16 analog/digital converter (Instrutech Corp., Port Washington, NY), using Synapse (Synergistic Systems). The bath solution contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, and 5 mM 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 psi.

Capacitance transients and leak currents were removed by subtracting a scaled control trace using a P/6 protocol (29). For the GV analysis, mean conductance (G) was calculated from the peak current/voltage relationship using the equation G = I/(V - Vrev), where I is the peak current, V is the membrane potential, and Vrev is the reversal potential. The normalized conductance/voltage relationship was fit with either a one- or two-component Boltzmann distribution according to Equation 1

Formula 1

where V11/2 and V21/2 are the respective membrane potentials for two populations of channels for which the mean conductance is half maximal, k1 and k2 are their respective slopes, and A defines the proportion of the second population (amplitude) with respect to the total. For fits in which only one population of channels was apparent, A was set to zero. The voltage dependence of steady-state fast inactivation was described using a single Boltzmann distribution as shown in Equation 2

Formula 2

where I is the peak current measured during the test depolarization step, Imax is the current without a preceding conditioning step, V is the membrane potential of the conditioning step, V1/2 is the membrane potential at which half-maximal inactivation is achieved, k is the slope factor, {alpha}0 is the remaining normalized peak current at highly depolarizing conditioning potentials, and {alpha}1 is the normalized amplitude (30). For mutant double cycle analysis, the dose response for Css4 effects on rNav1.4 was determined based on the current induced by the toxin following a test pulse to a voltage of -35 mV from the V0.5 of activation determined for each channel mutant (Table 1). The free energy change in toxin binding to the wild-type/mutant channel pair ({Delta}G) was calculated as the difference of the average -RTln (EC50) for the wild type and mutant, where R is the gas constant and T stands for temperature in 0K. {Delta}{Delta}G was taken as the difference of the {Delta}Gs for Css4 and the toxin mutant: {Delta}{Delta}G = (GWT, native - Gmutant, native) - (GWT, mutant - Gmutant, mutant), where the first and second subscript positions refer to the channel and the toxin, respectively. RT = 0.59 kcal/mol.


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  		TABLE 1
Effects of Css4 mutants on the activation of various Navs and rNav1.4 mutants The G/V relations in the presence of Css4 mutants exhibit two components: a minor negative shift in the V0.5 of activation of the entire channel population (upper number), and a stronger shift in the V0.5 (lower number) of a fraction (in parentheses) of the channel population. Data represent the mean ± S.E. of at least six independent experiments. The EC50 of Css4E15A was 0.46 and 0.88 µM for rNav1.2a and rNav 1.4, respectively. Current-voltage relations were determined as described in Fig. 2. ND, not determined.

 
Binding Experiments—Skeletal muscle membranes and brain synaptosomes were prepared from adult albino Wistar rats (~300 g, laboratory-bred) as previously described (31, 32). The rats were sacrificed according to the rules of the Animal Care Unit at Tel Aviv University (permit number L-03-54) following National Institutes of Health guidelines. Radio-iodination of Css4, purification of monoiodotoxin, and binding assays were performed as previously described (25). Nonspecific toxin binding was determined in the presence of 1 µM unlabeled toxin and consisted typically of 15–30% of total binding. Each experiment was performed in duplicate and repeated at least three times as indicated (n). Data are presented as the mean ± S.D. for independent experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Comparison of Css4 Interactions with Rat Skeletal Muscle and Brain Navs and Construction of a Specific Modifier for Nav1.4—To clarify the difference in Css4 potency at Nav1.2a versus Nav1.4 we sought for the toxin bioactive surface toward the skeletal muscle Nav and compared it with the reported surface toward brain Navs (25). We have previously shown that substitution of Tyr-4, Thr-10, Phe-14, Leu-19, Tyr-24, Arg-27, Glu-28, Gln-32, Tyr-40, Tyr-42, Phe-44, Trp-47, Trp-58, and Lys-63 for Ala considerably affected Css4 binding affinity for rat brain synaptosomes (supplemental Table S1 and Ref. 25). Most of these substitutions also decreased the binding affinity for rat muscle membranes, except for those at Thr-10, Phe-14, Glu-28, Gln-32, and Lys-63 (supplemental Table S1 and Fig. 1). These results raised a subset of residues that are involved in the surface of interaction of the toxin with both rat brain and skeletal muscle Navs and highlighted a few residues whose roles differ. This difference is notable in light of the CD alteration rendered by T10A, F14A, and K63A and the fact that Glu-28 and Gln-32 have been assigned to the "hot spot" in the surface of interaction of Css4 with the rat brain channel (25). Thus, receptor site-4, which pertains to the interface between Css4 and the Nav, differs in mammalian brain versus muscle Navs, especially in respect to Phe-14 and Glu-28 (supplemental Table S1), which laid the groundwork for construction of a mutant toxin specific for Nav1.4. Because the affinity of mutant E15A for both channel types exceeded 10-fold that of the unmodified toxin (supplemental Table 1 and Fig. 1), we constructed Css4 mutants bearing various combinations of substitutions F14A and E28R in the background of E15A. The most significant among these constructs was the triple mutant Css4F14A/E15A/E28R, whose affinity for the brain channel dropped 254-fold whereas its affinity for the skeletal muscle channel was similar to that of Css4E15A (supplemental Table S1), thus providing a highly selective ligand for muscle channels.


Figure 1
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  		FIGURE 1.
Competition of wild-type and mutant Css4 toxins on binding to rat brain and skeletal muscle membrane preparations. Membranes were incubated with 0.1 nM 125I-His-Css4 and increasing concentrations of the indicated mutants at 22 °C for 60 min. Nonspecific binding, determined in the presence of 1 µM His-Css4, was subtracted. The Ki values (in nM, n ≥ 3) for rat brain synaptosomes and rat skeletal muscle membranes, respectively, were Css4, 0.98 ± 0.1, 3.9 ± 1.17; Css4E15A, 0.07 ± 0.01, 0.3 ± 0.1; Css4F14A, 141 ± 18, 3.7 ± 0.4; Css4R27A, 31 ± 6.3, 56.5 ± 10.5; Css4E28A, 635 ± 98, 6.2 ± 2.3. The curves are from representative experiments.

 
We then analyzed the effects of these mutants on a variety of mammalian Navs co-expressed in Xenopus oocytes with the beta1 subunit, using Css4E15A as the first point of reference. Css4E15A was active at rNav1.2a and rNav1.4 but had no effect at rNav1.3, hNav1.5, and rNav1.6 even at a concentration of 5 µM (Table 1 and supplemental Fig. S1). Hence, the preference of Css4E15A for the mammalian Nav subtypes was similar to that of Css4 (19, 23, 33), yet the potency of Css4E15A increased ~10-fold (EC50 = 0.46 for rNav1.2a and 0.88 µM for rNav1.4) compared with the unmodified toxin (25). Css4E15A induced a -25-mV shift in the threshold of activation for both rNav1.2a and rNav1.4 (at 0.5 and 1 µM, respectively) (Fig. 2 and supplemental Fig. S1). The conductance-voltage relation (G/V) curves for rNav1.2a and rNav1.4 in the presence of Css4E15A exhibit two components with a minor negative shift in the voltage for half activation (V0.5) of the entire channel population and a stronger shift in the V0.5 of a fraction of the channel population (Fig. 2 and Table 1, values in parentheses). The effects of Css4E15A on channel activation resembled those of Css4 in the requirement for a depolarizing prepulse of similar duration and the rates of association and dissociation at rNav1.2a and rNav1.4 (supplemental Fig. S1) (19, 20, 23).


Figure 2
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  		FIGURE 2.
Conductance-voltage relations for rNav1.2a and rNav1.4 in the presence of various Css4 mutants. A, rNav1.4. B, rNav1.2a. Concentrations of the mutant toxins and activation parameters (V0.5) are shown in Table 1. The current-voltage relations were determined as described in supplemental Fig. S1 using the voltage protocol with a depolarizing prepulse.

 
Css4F14A/E15A, Css4E15A/E28R, and Css4F14A/E15A/E28R had no effect on the voltage dependence of activation of the neuronal channels rNav1.2a, rNav1.3, and rNav1.6 and the cardiac channel hNav1.5. However, they shifted the voltage dependence of rNav1.4 activation equally as well as Css4E15A, demonstrating complete specificity for the skeletal muscle channel (Table 1 and Fig. 2).

Effect of Css4F14A/E15A/E28R on the Gating Properties of rNav1.4R666G, an Equivalent of the Genetic Disorder hNav1.4R672G—An R672G mutation in D2/S4 of the human Nav1.4 was identified in the SCN4A gene of patients with hypoPP (8) and shown to generate an 8-mV rightward shift in the voltage dependence of activation and a 5-mV leftward shift in the steady-state fast inactivation (13). Because Css4F14A/E15A/E28R induced a hyperpolarizing shift in the voltage dependence of rNav1.4 activation (Fig. 2), we examined its effects on an identical mutation constructed in the equivalent position of the rat Nav1.4 (rNav1.4R666G). We found that the voltage dependence of channel activation indeed was right-shifted by 8 mV (V0.5 = -17 ± 1.3 mV) relative to the unmodified channel (V0.5 = -24.9 ± 0.3 mV, Fig. 3A), as well as its steady-state fast inactivation that was left-shifted by 5 mV (V0.5 = -55.5 ± 0.7 mV) relative to the unmodified channel (V0.5 =-49 ± 0.6 mV, Fig. 3B). In a concentration of 1 µM, Css4F14A/E15A/E28R shifted the voltage dependence of activation of rNav1.4R666G to V0.5 = -25 ± 0.7 mV, and less than 15% of the mutant channel population was activated at more negative membrane potentials (Fig. 3A). In addition, the steady-state fast inactivation of rNav1.4R666G was right-shifted, providing a V0.5 = -50.4 ± 0.7 mV (Fig. 3B). These effects by Css4F14A/E15A/E28R demonstrated its ability to restore most of the altered gating properties of rNav1.4R666G, which under the influence of this specific modulator performed much like the unmodified rNav1.4 under control conditions.


Figure 3
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  		FIGURE 3.
Characterization of rNav1.4R666G and Css4F14A/E15A/E28R effects on channel gating properties. A, conductance-voltage relations of the unmodified rNav1.4 (V0.5 = -24.9 ± 0.3 mV) and of rNav1.4R666G in the absence (V0.5 = -17 ± 1.3 mV) and presence of 1 µM Css4F14A/E15A/E28R (V0.5 = -25 ± 0.7 and -34.2 ± 1.3 mV for 13% of the channel population). The current-voltage relations were determined as described in Fig. 2. B, steady-state inactivation of rNav1.4 fits a Boltzmann function (see "Experimental Procedures", and Equation 2) with V0.5 = -49 ± 0.6 and V0.5 = -55.5 ± 0.7 mV for rNav1.4R666G and V0.5 = -50.4 ± 0.7 mV for rNav1.4R666G in the presence of 1 µM Css4F14A/E15A/E28R. Steady-state fast inactivation was determined using a 50-ms prepulse to +60 mV followed by a hyperpolarizing pulse to -100 mV and a series of 50-ms prepulses from -90 to -20 mV in 5-mV increments before the test pulse of -20 mV (see inset).

 


Figure 4
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  		FIGURE 4.
Effects of Css4E15A on rNav1.4 mutants. A, alignment of D2/S1-S2, D2/S3-S4, and D3/SS2-S6 Nav regions of a number of Navs (Swissprot accession numbers are P15390 for rNav1.4, P04775 for rNav1.2a, P04774 for rNav1.1, P08104 for rNav1.3, Q14524 for hNav1.5, CAA70364 for rNav1.6, O08562 for rNav1.7). Numbers in superscript provide the position of the indicated residues in the channel sequence. B, differences in conductance-voltage relations of rNav1.4 mutants in the presence of Css4E15A. Open circles designate control and closed circles the results obtained at various concentrations of Css4E15A (see Table 1). The current-voltage relations were determined as described in Fig. 2.

 
Aside from the therapeutic potential arising from the specificity of Css4F14A/E15A/E28R for rNav1.4, our results have raised the question of whether Css4 recognizes a similar region in rNav1.2a and rNav1.4. Therefore, we examined the effects of mutations in both the toxin and the channel on their interaction.

Css4E15A Effects on rNav1.4 Mutants—Based on recent reports about substitutions introduced to rNav1.2a (E779Q in D2/S1-S2; E837Q, L840C, and G845N in D2/S3-S4) and rNav1.4 (G658N in D2/S3-S4; E1251N and H1257K in D3/SS2-S6) that reduced the effects of the beta-toxins Css4 (19, 20) and Tz1 (22), we constructed rNav1.4 mutants E592A in D2/S1-S2; E650A, L653A, and G658N in D2/S3-S4; and E1251N and H1257K in D3/SS2-S6 (see Fig. 4A for sequence alignment). In addition, four residues that differ between rNav1.2a and rNav1.4 at D2/S1-S2 and S3-S4 were substituted at rNav1.4 with their rNav1.2a equivalents (H599Q/D601S/N602S and Q657E). Analysis of the eight channel mutants in the presence of Css4E15A revealed a similar negative shift in the G/V relations for rNav1.4E592A, rNav1.4H599Q/D601S/N602S, rNav1.4E650A, and rNav1.4Q657E and the unmodified channel. In contrast, channel mutations L653A and G658N abolished the Css4E15A effect as indicated by the unaffected G/V relations measured with up to 10 µM toxin (Table 1 and Fig. 4). The G/V relations of rNav1.4E1251N and rNav1.4H1257K were affected by Css4E15A, but with lower potency (EC50 = 1.91 and 5.3 µM, respectively) (Tables 1 and 2 and Fig. 4B). These results suggest that Leu-653 and Gly-658, and to a lesser extent Glu-1251 and His-1257, are involved in the interaction of Css4E15A with rNav1.4.


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  		TABLE 2
EC50 for activation of mutant rNav 1.4 channels by Css4 mutants EC50 values (µM) were determined as described in supplemental Fig. S2. The data represent the mean ± S.E. for the independent experiments (number in parentheses). ND, not determined.

 
Mutant Double Cycle Analysis of Css4 against rNav1.4—The high activity of Css4F14A/E15A, Css4E15A/E28R, and especially Css4F14A/E15A/E28R at the skeletal muscle channel and the lack of effect at the brain channel indicate that Phe-14 and Glu-28 are two major points of difference in the interface between Css4 and the two types of Navs. Based on the general conservation of mammalian Navs and the elucidation of a subset of common residues involved in the interaction of Css4 with the two channel types, we compared the effects of Css4E15A, Css4F14A/E15A, Css4E15A/E28R, and Css4F14A/E15A/E28R on the activation properties of the eight rNav1.4 mutants. In addition, we included in this comparative analysis Css4E15A/R27Q because of the proximity of Arg-27 to Glu-28 and the similar effect of its substitution on both rNav1.2a and rNav1.4 (supplemental Table S1 and Table 1) (25). Where a significant change in activity was obtained (Table 1), the EC50 values were determined, enabling mutant double cycle analysis (Table 2 and supplemental Fig. S2). The EC50 of Css4F14A/E15A at rNav1.4E592A was 20-fold higher than its EC50 at the unmodified channel (19.2 ± 2.1 versus 0.93 ± 0.15 µM; Table 2), indicating a coupling energy of -1.65 kcal/mol between F14A of the toxin and E592A of the channel. Css4E15A/R27Q hardly affected rNav1.4 (EC50 = 26.2 ± 2.9 µM), indicating the importance of Arg-27 for Css4 interaction with its rNav1.4 receptor site (Table 2). Of the eight channel mutations, E1251N significantly increased the ability of Css4E15A/R27Q to produce a negative shift in the conductance-voltage relations of the channel (Tables 1 and 2). Indeed, the potency of Css4E15A/R27Q at rNav1.4E1251N was very similar to that of Css4E15A (Table 2). This indicates that R27Q has a significant positive coupling energy with E1251N ({Delta}{Delta}G = 1.89 kcal/mol). Most intriguing were the effects of Css4E15A/E28R on the channel mutants. Whereas the EC50 of Css4E15A/E28R for the unmodified channel was very similar to that of Css4E15A (0.98 ± 0.03 µM), it increased prominently for rNav1.4E650A and rNav1.4E1251N (29.8 ± 3.1 and 23.9 ± 2.7 µM, respectively) (Tables 1 and 2 and Fig. 4B). Hence, a single Css4 residue (Glu-28) exhibits negative coupling energy with two channel residues ({Delta}{Delta}G =-1.8 and -1.41 kcal/mol for E650A and E1251N, respectively) positioned on two distinct extracellular loops in Domains 2 and 3 (D2/S3-S4 and D3/SS2-S6).


Figure 5
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  		FIGURE 5.
Schematic presentation of the inter-domain arrangement of the voltage-sensing and pore modules in rNav1.4. A, schematic Css4 structural model (in ribbon) (23) based on the known structure of the beta-toxin Cn2 (>90% similarity in sequence) (35) (PDB accession 1cn2) highlighting the three residues shown here to have coupling energies with channel residues derived from three distinct extracellular loops of Domains 2 (yellow) and 3 (blue). B, external view of the proposed inter-domain arrangement of the transmembrane segments (S1-S6) in the mammalian Nav based on panel A and following the proposed similarity in intra-domain arrangement between NaChBac and Kv1.2 (3, 6). The dashed line illustrates a Css4 projection in its putative bound form to demonstrate the size relations of the toxin and the channel.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The design of Css4F14A/E15A/E28R, a specific activator of rNav1.4, was based on systematic analysis of the binding of a large collection of Css4 mutants to Navs in rat brain and rat skeletal muscle membrane preparations and the finding that substitution of Phe-14 and Glu-28 for Ala markedly decreased activity at the brain channel without affecting activity at the muscle channel. Despite these differences, the substitution of a number of other Css4 residues had a similar impact on the two channel subtypes, suggesting that the toxin recognizes a similar region on both channels. The "similar/non-similar" face of toxin interaction with these channels raised two experimental avenues that could exploit Css4F14A/E15A/E28R. The first was to assess the therapeutic potential of such a specific Nav activator, and the second was to examine its interaction with the skeletal muscle channel.

The Putative Therapeutic Potential of Css4F14A/E15A/E28R in Neuromuscular Disorders—Over thirty mutations in SCN4A, the gene encoding the skeletal muscle Nav, have been linked to neuromuscular disorders such as hypo- and hyperkalemic periodic paralyses, paramyotonia congenital, Nav myotonias, and congenital myasthenic syndrome (8). Three of these mutations were localized to D2/S4 of hNav1.4 (R669H, R672G/H/S, and R675G/Q/W) (reviewed in Ref. 8), and one was localized to D3/S4 of hNav1.4 (R1132Q) (14). Skeletal muscle fibers from a patient heterozygous for R672G displayed depolarization and weakness in low potassium extracellular solution (15). Both the increased inactivation and the impaired voltage dependence of activation caused by the R672G mutation may contribute to the reduction of Nav performance and reduced membrane excitability (14). Sternberg et al. (34) reported a very severe hypoPP phenotype in a family carrying the R672G mutation where the frequency and severity of attacks increased in response to treatment with acetazolamide. The ability of Css4F14A/E15A/E28R to restore the gating properties of rNav1.4R666G (Fig. 3), which mimicked the hypoPP mutation hNav1.4R672G, demonstrates a putative therapeutic potential when seeking a remedy to the defective SCN4A gene product. The specificity of Css4F14A/E15A/E28R for the skeletal muscle Nav suggests that it merits investigation as a possible treatment for hypoPP and perhaps other neuromuscular disorders with symptoms of reduced muscle excitability.

Derivation of Channel Architecture from Css4-rNav1.4 Mutant Double Cycle Analysis—The interaction of Css4 and rNav1.4 was examined by mutant double cycle analysis, focusing on residues employed in the design of the selective activator and on rNav1.4 channel residues whose equivalents were either proposed to be involved in the interaction of Css4 with rNav1.2a (20) or whose substitution was shown to affect the interaction of the toxin Tz1 with rNav1.4 (22). Four conserved residues in the external loops of rNav1.2a have been implicated in the interaction with Css4: Glu-779 in D2/S1-S2 and Glu837, Leu-840, and Gly-845 in D2/S3-S4 (Fig. 4A) (20). The decrease in the ability of Css4E15A to modulate channel activation following substitutions of L653A and G658N in rNav1.4 (equivalent positions of Leu-840 and Gly-845 in rNav1.2a) (Table 1 and Fig. 4A) suggests that these residues belong to a conserved region of receptor site 4. However, the lack of effect of substitutions E592A and E650A in rNav1.4 (equivalent positions in rNav1.2a are Glu-779 and Glu-837) on Css4E15A action indicates that despite their conservation in all mammalian Navs these residues do not belong to the common receptor for scorpion beta-toxins on Navs (Table 1 and Fig. 4A).

Overall, the substantial variations in the receptor site for Css4 in rNav1.2a and rNav1.4 are consistent with the results of binding assays of Css4 mutants on rat brain and muscle Navs and are further demonstrated by the difference in the face of interaction between the two channels and the triple mutant Css4F14A/E15A/E28R (supplemental Table S1). In light of the general conservation of mammalian Navs, the high specificity of the triple Css4 mutant for Nav1.4 suggests that the toxin residues Phe-14 and Glu-28 encounter a different face upon binding to rNav1.2a versus rNav1.4. This prompted us to examine by mutant cycle analysis Css4-rNav1.4 interacting pairs. We focused on the toxin residues Phe-14 and Arg-28, whose substitution abolished the activity toward rNav1.2a, but not rNav1.4, and Arg-27, whose substitution affected both channel types (Tables 1 and 2 and Fig. 2). In the channel we selected those residues whose substitution was shown to influence the effect of scorpion beta-toxins (20, 22). The significant coupling energies obtained between F14A (toxin) and E592A at D2/S1-S2 (channel), as well as E28R (toxin) and the two channel residues E650A at D2/S3-S4 and E1251N at D3/SS2-S6 (Table 2), along with the three-dimensional model of Css4 (Fig. 5) (25, 35) enabled to estimate the distances between the toxin channel interacting pairs. As the distance between Cbeta of Phe-14 and Cbeta of Glu-28 is 6–8 Å, Glu-592 of D2/S1-S2 is likely to reside <10 Å from Glu-650 of D2/S3-S4. Because E28R of the toxin demonstrated a negative energy of interaction with both E650A of D2/S3-S4 and E1251N of D3/SS2-S6, we conclude that the two channel residues are very close to one another (Fig. 5). This conclusion is further corroborated by the high positive coupling energy between R27Q of the toxin and E1251N of the channel (Tables 1 and 2). Based on these data and in the absence of a three-dimensional structure of the Nav, we suggest that loop S3-S4 of the voltage sensor module in Domain 2 is in very close proximity with loop SS2-S6 of the pore module in Domain 3. Although substantiation of this suggestion requires resolution of the channel three-dimensional structure, the proposed architecture resembles that reported for Kvs, where the voltage-sensing module of each domain is in close proximity to the pore module of the adjacent domain, in a clockwise orientation (Fig. 5) (3).


   FOOTNOTES
 
* This work was supported by German-Israeli Foundation for Scientific Research and Development Grant G-770-242.1/2002 (to D. G. and W. S.), 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 National Institutes of Health Grant 1 U01 NS058039-01 (to M. 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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Table S1. 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. E-mail: mamgur{at}post.tau.ac.il.

3 The abbreviations used are: Nav, voltage-gated sodium channel; Css4, Centruroides suffusus suffusus beta-toxin 4; hypoPP, hypokalemic periodic paralysis. Back


   ACKNOWLEDGMENTS
 
We thank Prof. F. Frolow, Tel Aviv University, for help with the illustration of the toxin-channel interaction.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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