Distinct Sites Regulating Grayanotoxin Binding and Unbinding to D4S6 of Nav1.4 Sodium Channel as Revealed by Improved Estimation of Toxin Sensitivity*

Hiroshi MaejimaDagger , Eiji Kinoshita§, Issei Seyama, and Kaoru Yamaoka§||

From the Dagger  Institute of Health Sciences and the § Department of Physiology, School of Medicine, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551 and the  Faculty of Human Life Science, Hiroshima Jogakuin University, Ushita Higashi 4-13-1, Higashi-ku, Hiroshima 732-0063, Japan

Received for publication, November 29, 2002, and in revised form, January 10, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Grayanotoxin (GTX) exerts selective effects on voltage-dependent sodium channels by eliminating fast sodium inactivation and causing a hyperpolarizing shift in voltage dependence of channel activation. In this study, we adopted a newly developed protocol that provides independent estimates of the binding and unbinding rate constants of GTX (kon and koff) to GTX sites on the sodium channel protein, important in the molecular analysis of channel modification. Novel GTX sites were determined in D2S6 (Asn-784) and D3S6 (Ser-1276) by means of site-directed mutagenesis; the results suggested that the GTX receptor consists of the S6 transmembrane segments of four homologous domains facing the ion-conducting pore. We systematically introduced at two sites in D4S6 (Nav1.4-Phe-1579 and Nav1.4-Tyr-1586) amino acid substituents with residues containing hydrophobic, aromatic, charged, or polar groups. Generally, substitutions at Phe-1579 increased both kon and koff, resulting in no prominent change in dissociation constant (Kd). It seems that the smaller the molecular size of the residue at Nav1.4-Phe-1579, the larger the rates of kon and koff, indicating that this site acts as a gate regulating access of toxin molecules to a receptor site. Substitutions at Tyr-1586 selectively increased koff but had virtually no effect on kon, thus causing a drastic increase in Kd. At position Tyr-1586, a hydrophobic or aromatic amino acid side chain was required to maintain normal sensitivity to GTX. These results suggest that the residue at position Tyr-1586 has a more critical role in mediating GTX binding than the one at position Phe-1579. Here, we propose that the affinity of GTX to Nav1.4 sodium channels might be regulated by two residues (Phe and Tyr) at positions Phe-1579 and Tyr-1586, which, respectively, control access and binding of GTX to its receptor.

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INTRODUCTION
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Biological toxins that act selectively on the voltage-dependent sodium channels have been used to elucidate molecular mechanisms of sodium channel gating processes. Batrachotoxin (BTX),1 grayanotoxin (GTX), veratridine, and aconitine, classified as Site 2 toxins (1), are endowed with some characteristics in common (2-5); 1) they bind to the sodium channel in its open state, 2) the modified sodium channel lacks the inactivation process, and 3) the activation potential of the modified sodium channel is shifted in the direction of hyperpolarization. Among the well characterized toxins, GTX has distinct advantages in that the relevant interaction sites on the GTX molecule with the sodium channel are known as a result of extensive analyses of its structure-activity relationship (6-8). The methyl group of the beta -surface of the molecule at C-10 and the hydroxyl groups of its beta -surface at C-3, C-5, and C-6 are essential to the pharmacological action of GTX and the hydrophobic microenvironment of the alpha -surface, of which C-14S is most critical, greatly contribute to increased GTX potency (see Fig. 2A). Moreover, the GTX action has a unique dependence on the state of the sodium channel; although BTX binds to and dissociates from the channel in its open state (9), GTX binds only to the open channel and dissociates from the closed channel (10).

The main structural component of sodium channels is the pore-forming alpha -subunit, whose molecular mass is more than 250 kDa. The alpha -subunit consists of four homologous domains (D1-D4), each containing six alpha -helical transmembrane segments (S1-S6) (11, 12). The regions between S5 and S6 in each of the four domains form pore loops that dip into the membrane to create a narrow selectivity filter at the external end of the ion-conducting pore. The remainder of the pore is formed by each S6 segment of the four domains in close apposition to one another (13, 14). Recent site-directed mutagenesis studies show that specific amino acid residues within each of the four S6 segments are important determinants of action of Site 2 toxins (15-23). Previously, we reported that D1S6 and D4S6 segments (but not D2S6 and D3S6) are required for GTX binding to the sodium channel (19-21). Unexplored GTX sites in D2 and D3 have been examined in this study. Within these segments, a number of putative binding sites on the rat skeletal muscle sodium channel isoform, Nav1.4 (Ile-433, Asn-434, Leu-437, Ile-1575, Phe-1579, and Tyr-1586), may interact with the alpha -surface of the GTX molecule, whose pharmacological action is effected within a hydrophobic microenvironment (8). Residue Tyr-1586 in D4S6 has been found to provide a unique binding site for GTX, which excludes BTX and veratridine. Moreover, we have attributed the difference in GTX sensitivity of the sodium channel isoforms, Nav1.4, and the rat cardiac channel, Nav1.5, to a critical residue (Ser-251) in the intracellular linker of D1S4-S5 (19). Kimura et al. (19) deduced particular GTX binding sites by making several chimeras targeting the heterologous amino acid residues of Nav1.4 and Nav1.5; these chimeras showed reduced sensitivity to GTX. Most recently, Maejima et al. (24) found novel binding sites both in D1S4-S5 and D4S4-S5 linkers of Nav1.4 and Nav1.5 through an alanine-scanning method.

Recently, Yuki et al. (10) developed a novel method to evaluate the sensitivity of sodium channels to GTX; this method independently determines the binding and unbinding rate constant (kon and koff) of GTX to sodium channels. The sensitivity of sodium channels to GTX is indicated by the dissociation constant (Kd), i.e. the ratio of koff to kon. Here, using this new technique, we reevaluated already-known GTX sites, hoping to uncover novel roles of these sites in GTX binding. At the sites in D4S6, we made systematic amino acid substitutions to further clarify their roles in more detail.

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Construction of Point Mutants of the Rat Skeletal Muscle Sodium Channel-- Point mutants of the sodium channel were constructed using the cDNA clone encoding the rat skeletal muscle sodium channel alpha -subunit, Nav1.4 (25). To construct the mutants by substitutions with various amino acids, we followed a PCR-based site-directed mutagenesis method as described previously (19-21, 24, 26). All of the resulting mutants were confirmed with restriction mapping and sequencing using an ABI PRISMTM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).

Transient Transfection and Cell Culture-- The constructed point-mutated cDNA clones were inserted into a mammalian expression vector pCI-neo (Promega Corp., Madison, WI) or pcDNA3.1 (Invitrogen) and were then transiently co-transfected with CD8 cDNA into HEK 293 (human embryonic kidney) cells using the SuperFect transfection reagent (Qiagen, Hilden, Germany). The cells were grown to 50% confluence in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (BioWhittaker, Walkersville, MD), 30 units/ml penicillin G (Meiji Corp., Tokyo, Japan), and 30 µg/ml streptomycin (Meiji Corp.) in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. The transfected cells were used for electrophysiological experiments as late as 3-4 days after being replated in 35-mm tissue culture dishes. Transfection-positive cells were identified by Immunobeads (CD8-Dynabeads, Dynal, Oslo, Norway) before Na+ current (INa) recording.

Electrophysiological Recording-- Macroscopic INa from the transfected cells was measured using the whole-cell patch clamp method. The bath solution contained 70 mM NaCl, 67 mM N-methyl-D-glucamine, 1 mM CaCl2, 1.5 mM MgCl2, 10 mM glucose, and 5 mM HEPES, pH 7.4. The pipette solution contained 70 mM CsF, 60 mM CsCl, 12 mM NaF, 5 mM EGTA, and 5 mM HEPES, pH 7.4. To assess the effects of GTX on whole-cell INa, 300 µM GTX I (a GTX analogue) was added to the pipette solution because GTX is known to act intracellularly (27), and this concentration is sufficient to evaluate sensitivity of the mutated channels to GTX (19).

    RESULTS
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Comparison of the Sensitivity to GTX among Wild Type Nav1.4 and Its Mutant Channels-- Because GTX binds to sodium channels exclusively in their open state (10, 19-21, 24, 28), repetitive depolarizing pulses were applied to induce GTX modification. After these repetitive conditioning pulses, test pulses to various membrane potentials induced a characteristic sustained current (Fig. 1, A2, B2, and C2). Without repetitive prepulses, the majority of channels were unmodified by GTX so that the recorded currents in the presence of 300 µM GTX (in the internal medium) exhibited the typical behavior of unmodified channels. However, closer examination revealed a residual non-inactivating component, indicating that a very small fraction of the channels was modified during the test pulse itself without repetitive preconditioning pulses (Fig. 1, A1, B1, and C1).


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Fig. 1.   GTX modification of sodium channels. Differences in sensitivity to GTX I between wild type Nav1.4 and mutants (F1579A, Y1586A, and N784K). A-D, INa families and I-V relations for unmodified peak INa and for GTX-modified steady-state INa in wild type Nav1.4 (A), mutant F1579A (B), Y1586A (C), and N784K (D). Control currents (unmodified currents) were taken in the presence of intracellular GTX. The currents were scarcely modified without preconditioning pulses even in the presence of 300 µM GTX, as shown in panels A1, B1, and C1. Modified currents (A2, B2, and C2) were induced by a train of 100 conditioning prepulses (pulse potential = -20 mV; pulse duration = 6 ms, interpulse interval = 100 ms; holding potential = -120 mV). I-V relationships (A3, B3, C3, and D3) of unmodified and modified currents were obtained using 160-ms test pulses to a variable potential (-140 mV to +50 mV in 10-mV steps); unmodified currents (filled squares) were measured as peak INa, and modified currents (open circles) were measured as the steady-state current at the end of the pulse.

To permit comparison of data from different cells, we standardized the currents by computing their maximum chord conductance from current-voltage curves (19-21, 24, 28). The I-V relationships for unmodified INa through wild type Nav1.4 channels and its mutant forms2 (Nav1.4-F1579A and Nav1.4-Y1586A) are given in Fig. 1, A3, B3, and C3. The continuous line was fitted to peak INa at membrane potentials from 0 to +60 mV, and the chord conductance was estimated from the slope. For GTX-modified currents, application of 100 conditioning pulses evoked an entirely different family of sustained currents through modified sodium channels (Fig. 1, A2, B2, and C2). Modified INa at the end of a 160-ms test pulse was plotted against the membrane potential, and the chord conductance of the modified channel population was estimated from the slope of the I-V relationship between -50 and +50 mV (dashed line in Fig. 1, A3, B3, and C3). We routinely used the ratio of maximal chord conductances of GTX-modified to unmodified channels as a measure of channel sensitivity to GTX modification, which is referred to as the relative chord conductance (RCC). Our previously reported RCC values for wild type Nav1.4 and various mutant channels have been provided for comparison with present results in Table I. As for the F1579A mutant, our previously reported RCC value of 0.75 (20) was not confirmed in the present study, which yielded a value of only 0.22. 

                              
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Table I
RCC, kon, koff, and Kd in wild type Nav1.4 and its point mutants
NA means that the GTX modification was so small that it was not available to measure relative chord conductance, kon or koff. NE means functional channels were not expressed in these mutants. Dash denotes data are not available because experiments were not done in the mutant and exhibited the same value of RCC as wild type or no functional mutants.

Novel Binding Sites of GTX in Transmembrane Segment D2S6 and D3S6 of Nav1.4 Sodium Channel-- In our previous studies, it was confirmed that Ile-433, Asn-434, and Leu-437 in D1S6 and Ile-1575, Phe-1579, and Tyr-1586 in D4S6 of sodium channels were critical sites for GTX modification (19-21). Recently, it was reported that Asn-784 and Leu-788 in D2S6 and Ser-1276 and Leu-1280 in D3S6 of Nav1.4 were important in BTX action (15, 16). For this reason we substituted Asn-784 and Leu-788 in D2S6 and Ser-1276 and Leu-1280 in D3S6 of Nav1.4 with Ala or Lys. The resultant RCC values are shown in Table I. RCC values of Nav1.4-N784K in D2S6 decreased from 0.35 to 0 compared with the wild type (Fig. 1D and Table I). Similarly, in D3S6, RCC values of Nav1.4-S1276A and Nav1.4-S1276K also decreased to 0.19 and 0, respectively (Table I). Mutant channels of Nav1.4-N784A, -L788A, -L788K, -L1280A, and -L1280K did not exhibit measurable INa. Thus, we could confirm that Asn-784 in D2S6 and Ser-1276 in D3S6 are also critical in GTX action, much like their role in BTX action, thus indicating that the sites in all S6 transmembrane segments may cooperatively contribute to GTX binding. GTX sites in Nav1.4 identified in our studies are schematically summarized in Fig. 2B.


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Fig. 2.   Structure of GTX and GTX sites in Nav1.4 sodium channel. A, structure of GTX I. Numerals indicate number of carbon atoms in the GTX I molecule. B, summary of GTX sites in the Nav1.4 sodium channel. The indicated point mutations in Nav1.4 caused moderate (squares) or marked suppression (squares with bold borders) of GTX action. For reference, amino acid sequences (numbered from the N terminus) for the S4-S5 linkers in domains D1 and D4 and GTX sites in D1S6 and D4S6 (19-21, 24) are also displayed.

Protocol That Derives kon and koff of GTX Binding-- In a previous communication, we presented a method that enables us to independently measure rate constants kon and koff for toxin binding and unbinding to sodium channels (10). GTX modifies sodium channels in proportion to the time integral of the unmodified sodium channel conductance (ToNa; Fig. 3B). The IGTX-ToNa relationship can be readily obtained since ToNa varies as a function of the potential of the three conditioning pulses employed to open unmodified sodium channels (Fig. 3A). Increment of IGTX brought about by the three conditioning pulses is plotted against integrated currents evoked by the three conditioning pulses divided by the driving force (Fig. 3B). The slope of the observed linear relationship (Fig. 3B) gives kon according to Equation 1 (see Yuki et al. (10) for further details),
I<SUB><UP>GTX</UP></SUB>≈C×k<SUB><UP>on</UP></SUB>×<LIM><OP>∫</OP></LIM><FR><NU>I<SUB><UP>Na</UP></SUB></NU><DE>×(E<SUB><UP>test</UP></SUB>−E<SUB><UP>Na</UP></SUB>)</DE></FR> <UP>dt</UP>=C×k<SUB><UP>on</UP></SUB>×<UP>T</UP>o<SUB>Na</SUB> (Eq. 1)
where IGTX represents current through GTX-modified sodium channels, INa is current through unmodified sodium channels, Etest is the voltage applied during test pulses to evoke IGTX, ENa is sodium reversal potential, and C is a constant of 1.61 × 10-3 that is invariant over sodium channel isoforms (19) and derived from sodium channel properties such as single channel conductance and open probability of unmodified or GTX-modified sodium channels. Briefly, numbers of GTX-modified sodium channels change according to Equation 2 because GTX only binds to open state of the sodium channel,
<FR><NU><UP>d</UP>G<UP>*</UP></NU><DE><UP>d</UP>t</DE></FR><UP> = </UP>[<UP>GTX</UP>]<UP> × </UP>k<SUB><UP>on</UP></SUB><UP> × </UP>O<UP> − </UP>k<SUB><UP>off</UP></SUB><UP> × </UP>G<UP>*</UP> (Eq. 2)

G*=<FR><NU>I<SUB><UP>GTX</UP></SUB></NU><DE>(E<SUB><UP>test</UP></SUB>−E<SUB><UP>Na</UP></SUB>)×g<SUB><UP>GTX</UP></SUB>×Po<SUB><UP>GTX</UP></SUB></DE></FR> (Eq. 3)

O=<FR><NU>I<SUB><UP>Na</UP></SUB></NU><DE>(E<SUB><UP>cond</UP></SUB>−E<SUB><UP>Na</UP></SUB>)×g<SUB><UP>Na</UP></SUB></DE></FR> (Eq. 4)
where O and G* represent the number of open unmodified and GTX-modified sodium channels, respectively, [GTX] is the concentration of GTX, gNa and gGTX are the single channel conductances of unmodified and GTX-modified sodium channels, respectively, Po GTX is the open probability of GTX-modified sodium channels, and Econd is the voltage applied during conditioning pulses to induce GTX-modification. Because G* and O can be obtained from experimental data using Equations 3 and 4, Equations 2, 3, and 4 can be combined and solved for IGTX to yield Equation 5,
I<SUB><UP>GTX</UP></SUB>=C×k<SUB><UP>on</UP></SUB>×To<SUB><UP>Na</UP></SUB>−k<SUB><UP>off</UP></SUB>×<LIM><OP>∫</OP></LIM>I<SUB><UP>GTX</UP></SUB><UP>d</UP>t (Eq. 5)
However, because best-fitting values of koff were negligible compared with the term C × kon (i.e. koff = 0.046 and C × kon = 6.24 in the presence of 300 µM GTX (10)), we simplified Equation 5 to Equation 1 by setting koff = 0, which thus provides an acceptable estimate of IGTX. In fact, a linear relationship between ToNa and IGTX, passing through the origin, is invariably obtained (Fig. 3B).


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Fig. 3.   Measurement of kon and koff. A, protocol for the measurement of kon. An initial pulse of 40 ms (from a holding potential of -120 mV to -80 mV) evoked modified current (IFirst) after various prepulses induced channel modification. Additional modification was produced by three subsequent conditioning pulses that opened unmodified sodium channels. The increment in modified current (IGTX) caused by the three additional pulses (ISecond) was assessed at -80 mV as IGTX = ISecond - IFirst. Increments in modified currents caused by trains of three conditioning prepulses to various potentials (-70 to 0 mV in 10-mV step; see the inset in A) are shown in B as a plot of ToNa (time integral of unmodified currents during the three conditioning prepulses divided by the driving force) against IGTX. kon was measured as the slope of the fitted line (see Equation 1 for further details). C, to measure koff, the time constant (tau ) for decay of modified sodium currents was measured at -80 mV; for this experiment, channel modification was induced by 100 prepulses (pulse potential, 0 mV; holding potential, -120mV). A single exponential curve was fitted to the decay of modified sodium current. The reciprocal of this time constant is equal to koff; i.e. koff = 1/tau (see "Results"). The dissociation constant (Kd) is calculated from the ratio koff:kon, i.e. Kd = koff/kon.

To obtain koff we measured the decay time constant of IGTX at a potential of -80 mV (Fig. 3C). At this potential, unmodified sodium channels will not open, and hence, no further channel modification can occur. At the same time, because GTX-modified channels do not inactivate and in fact maintain a constant open probability at this potential, decay of IGTX can only represent dissociation of GTX from its binding site. Thus, the reciprocal of this time constant can be regarded as koff.

Measurements of kon, koff, and Kd of GTX Binding to Wild Type Nav1.4 and Its Mutant Sodium Channels-- Technically, it is impossible to measure kon and koff in mutant channels that have no sensitivity to GTX. Thus, we measured kon and koff of the mutant channels whose sensitivity was decreased partially, and data are summarized in Table I and Fig. 4. As an overview, these data have four prominent features. 1) Mutations at Phe-1579 give the largest effect on kon. 2) Mutations at Tyr-1586 give the largest effect on koff. 3) The change in koff produced by mutations at Phe-1579 are balanced by the change in kon, resulting in relatively constant Kd. 4) Mutations at Tyr-1586 have relatively minor effects on kon, resulting in a significant increase in Kd. These data indicate that association and dissociation of GTX with its binding site must be governed by different molecular entities.


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Fig. 4.   Normalized values of kon (A), koff (B), and Kd (C). Values of kon, koff, and Kd of mutant channels are normalized to those of wild type Nav1.4 and are displayed separately in A, B, and C, respectively.

Effects of Various Mutations at Phe-1579 and Tyr-1586 on kon, koff, and Kd-- As described above, we found that kon was increased most markedly by mutations at Phe-1579 and koff was increased most markedly by mutations at Tyr-1586. Thus, it is reasonable to assume that Phe-1579 and Tyr-1586 have particularly important roles in GTX binding. We introduced systematic mutations at these sites using substituent amino acids with hydrophobic, aromatic, charged, and polar side groups. Specifically, we substituted Phe-1579 with Lys, Ser, Gly, Ala, His, Cys, Met, Val, Tyr, and Trp and substituted Tyr-1586 with Glu, Lys, Ser, Gln, Ala, Cys, Met, Ile, Phe, and Trp. We could not detect measurable INa in mutations F1579E, F1579Q, F1579I, F1579L, F1579P, Y1586D, Y1586R, Y1586N, Y1586T, Y1586H, and Y1586P. Resultant RCC, kon, koff, and Kd values are summarized in Table II. kon, koff, and Kd values normalized to those of the wild type Nav1.4 are also graphically displayed in Fig. 4. The order of amino acids presented is based on the degree of hydrophobicity (29-31). Only one mutant of Y1586K in the systematic substitution experiment lost the sensitivity to GTX, so that kon and koff could not be estimated.

                              
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Table II
RCC, kon, koff, and Kd in point mutants of Nav1.4 at Phe-1579 or Tyr-1586
NA means that the GTX modification was so small that it was not available to measure relative chord conductance, kon, or koff.

In mutating Phe-1579, we could not correlate the observed changes in kon and koff with side chain properties of amino acids such as their hydrophobic, aromatic, or polar nature or their ionic charge. However, we did find a prominent increase in kon especially with amino acid substituents having small side chains (Gly, Ala, Ser, and Cys). Large molecular dimension or ionic charge of side chains might be a hindrance to GTX binding at Phe-1579. On the other hand, we found that substituents with extremely hydrophilic residues (Glu, Lys, and Ser) at Tyr-1586 significantly reduced the sensitivity to GTX. Hydrophobicity (Y1586A, Y1586C, Y1586M, and Y1586I) may help to maintain sensitivity to GTX, but aromatic residues (Tyr, Phe, and Trp) are most effective in minimizing the Kd value. One of neutral residues, Gln, at Tyr-1586 could not be a hindrance to GTX binding.

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In this study, we utilized a new protocol that independently determines kon and koff values for GTX in sodium channel mutants. We, thus, uncovered new kinetic aspects of GTX sites in Nav1.4 channels. This protocol was remarkably effective in revealing the kinetic properties of GTX, because it detected changes in the response of sodium channel mutants to GTX when Kd of these mutants was not altered compared with the wild-type control. Accordingly, we found two functional sites for GTX action, Phe-1579 and Tyr-1586, which specifically affect GTX binding rate constants kon and koff, respectively. Systematic amino acid substitutions within these two sites revealed that hydrophobic or aromatic amino acids in Nav1.4-Tyr-1586 are required to maintain GTX binding, whereas mutations at Nav1.4-Phe-1579 do not alter the apparent GTX binding affinity (Kd) but in fact induce simultaneous changes in both kon and koff. In addition to previously identified GTX sites, we have discovered novel GTX sites in D2S6 (Asn-784) and D3S6 (Ser-1276), indicating that the GTX receptor consists of the S6 transmembrane segments of four homologous domains facing the ion-conducting pore.

Advantages of Measuring kon and koff in the Analysis of GTX Responses of Point Mutants-- Advantages of our newly developed method to independently measure kon and koff of GTX include the following. We could detect latent mutants that affect GTX binding. For example, many of the Phe-1579 mutants having Kd values similar to the wild type (see Fig. 4 and Table II) could be classified as GTX sites with functions either to control access of toxin to the receptor site or to serve as the toxin binding site per se. The most prominent mutation-induced increases of kon (F1579S or F1579G) and koff (Y1586E) occurred at distinct sites. More precisely, systematic mutations at these two sites (Fig. 4, A and B) revealed that mutations at Nav1.4-Phe-1579 essentially increase kon, and those in Nav1.4-Tyr-1586 essentially increase koff. This for the first time gives an indication that binding and dissociation of GTX might be regulated at two different sites within the sodium channel protein.

Assessment of Newly Derived Parameters kon and koff-- We previously adopted the convention of RCC to assess the sensitivity of sodium channels to GTX (19-21, 24, 28). In the present study, we estimated Kd directly from kon and koff as an index of channel sensitivity to GTX. As shown in Fig. 5, a linear regression was found between Kd and RCC, indicating that both indices reliably report the GTX sensitivity. However, data points derived from mutants of the 1579 residue scattered well away from the regression line (Fig. 5). This is probably due to underestimation of RCC, because unmodified INa without preconditioning pulses in 1579 mutants was overestimated due to a residual non-inactivating current component; such a residual component was relatively large in 1579 mutants and disappeared when GTX was depleted from internal solution (data not shown). Without preconditioning pulses, infrequent depolarizing pulses did not normally induce detectable GTX modification having the described non-inactivating component, but very large increases in kon in these mutants enabled rapid channel modification to occur during the first pulse (see the data from F1579A in Fig. 1, B1). Indeed, the mutants having small molecule amino acids (Gly, Ser) at Phe-1579 exhibited measurable non-inactivating component at the first depolarization (data not shown). On the other hand, F1579Y was exceptional; its behavior deviated in a direction markedly different from all other 1579 mutants. The F1579Y mutant exhibited a peculiar phenomenon, i.e. both peak current and GTX-modified sustained currents were spontaneously increased during the course of experiments by a factor of 1.5 relative to the unmodified peak current obtained at the beginning of the experiments. By the RCC protocol, relative chord conductance of the GTX-modified current (with prepulses) was obviously overestimated, because unmodified INa was only sampled at the beginning of experiments when the amplitude of unmodified INa was 1.5 times smaller than that of the unmodified INa that was actually present during subsequent measurement of GTX-modified INa. We do not know why F1579Y mutant exhibited this spontaneous increase in INa (Fig. 5). The newly developed method, which allows direct evaluation of kon and koff, has the virtue of being less sensitive than the RCC convention to perturbations such as spontaneous variations in INa or unexpectedly rapid development of GTX modification.


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Fig. 5.   Correlation between RCC and binding constant (K). The parameter K is the reciprocal of Kd. Open circles indicate values (RCC and parameter K) obtained from mutants of position Phe-1579, and filled squares indicate those of wild type Nav1.4 and the indicated mutants. The regression line is through filled squares, including wild type Nav1.4. Note that the data points for Phe-1579 mutants scatter away from this line.

Specific Amino Acid Requirements in the Sites of Nav1.4-Phe-1579 and Nav1.4-Tyr-1586 in Achieving Interaction with GTX Molecules-- We introduced systematic amino acid substitutions for Phe with Lys, Ser, Gly, Ala, His, Cys, Met, Val, Tyr, and Trp in Nav1.4-Phe-1579 and for Tyr with Glu, Lys, Ser, Gln, Ala, Cys, Met, Ile, Phe, and Trp in Nav1.4-Tyr-1586 consisting of hydrophilic, hydrophobic, aromatic, and polar amino acid side chains.

At the Nav1.4-Phe-1579 site there was no clear indication that hydrophobic, polar, or aromatic properties of residue side chains play a critical role in increasing either kon or koff. It seems that larger molecular size of the amino acid side chains predicted smaller rate constants. Thus, the emerging picture is that the smaller the molecular size of the side chain, the greater the access of GTX molecules to the receptor site. As for the site at Tyr-1586, it is distinct from the site at Phe-1579 in that the GTX sensitivity (Kd = koff/kon) was extensively altered by the mutations. Furthermore, we could categorize the effect of mutations into three groups according to the resultant Kd values. Aromatic residues (Tyr, Phe, Trp) produce the highest sensitivity of the three groups, although an exception was found in the case of Gln. Hydrophilic residues (Glu, Lys, Ser) could not maintain a suitable microenvironment to permit GTX binding. Hydrophobic residues (Ala, Cys, Met, Ile) improved the efficiency of GTX binding compared with hydrophilic residues.

It has been reported that specific amino acids are required in BTX or local anesthetic binding to a putative receptor site in D4S6 (Nav1.3-F1710) corresponding to Nav1.4-Phe-1579 (9, 32). Stable BTX binding to the channel protein requires a polar or aromatic residue at this receptor site to maintain the ion-dipole or cation-pi interaction (9). Tetracaine requires an aromatic residue at its receptor site to maintain electrostatic interactions between the positive charge on the protonated tertiary amine of this local anesthetic and the electron-rich pi  face of the aromatic residue (32). As for GTX, Yakehiro et al. (8) show that the hydrophobic moiety must be preserved on the alpha -surface of GTX molecules for effective GTX action because the introduction of a hydroxyl group into C-14S facing the alpha -surface of the GTX analog markedly reduced GTX potency (8). Thus, it is supposed that the hydrophobic environment around the receptor site is important in GTX action. In this study, we argued that an aromatic or hydrophobic side chain at residue Tyr-1586 in Nav1.4 is required to preserve sensitivity to GTX. This is consistent with the requirement for hydrophobicity on the alpha -surface of GTX in maintaining GTX potency. Direct interaction between the alpha -surface of GTX and Tyr-1586 may be necessary for normal GTX action.

Although Tyr-1586 has a determinant role in GTX action as discussed above, Phe-1579 may have a rather regulatory function. Mutations at Nav1.4-Phe-1579 produced only a marginal change in Kd values. Furthermore, F1579K was the only mutant whose sensitivity was not totally eliminated by Lys substitution (see Table I). It is reasonable to assume that the charged side chain in F1579K interferes with access of the hydrophobic alpha -surface of GTX molecules because we observed an exceptionally small kon when Lys was substituted (see Fig. 4A).

Properties That Differentiate GTX from Other Site 2 Toxins or Local Anesthetics-- GTX shares common binding sites with the Site 2 toxins BTX and veratridine, namely, Ile-433, Asn-434, and Leu-437 in D1S6 and Ile-1575 and Phe-1579 in D4S6 of Nav1.4 (17-23). Other GTX-specific sites were further documented in D4S6 (Tyr-1586) of Nav1.4 (20). Several GTX sites were also found in D1 and D4 (see Fig. 2B) (19-21, 24). This study has now added two novel sites in D2S6 (Asn-784) and D3S6 (Ser-1276) to the list of binding sites common to Site 2 toxins. However, Tyr-1586 is GTX-specific, because mutations at this position did not affect BTX binding (17, 22). This might explain the difference in binding kinetics between GTX and BTX. BTX dissociates from the open state of the sodium channel (9), whereas GTX exclusively dissociates from the closed state (10). It is supposed that BTX dissociates from its receptor site in the ion-conducting pore and shifts to a cytoplasmic membrane site during the open state of the channel, as if it were reversing its itinerary (9). On the other hand, because Tyr-1586 is GTX-specific among Site 2 toxins and mutation of this site dramatically increases unbinding (koff) of GTX, it is possible that this site contributes to the GTX-specific binding-unbinding reaction by trapping GTX in the open channel and releasing GTX from the closed channel.

Local anesthetics like lidocaine, tetracaine, QX314, and QX222 modify channel activation and block sodium current. These local anesthetics also share common binding sites at Asn-434 and Leu-437 in D1S6, Ser-1276 in D3S6, Ile-1575, Phe-1579, and Tyr-1586 in D4S6 of Nav1.4 with GTX (16, 19-21, 33, 34). As in GTX binding, local anesthetic binding is affected by introduction of amino acid substitutions at both Phe-1579 and Tyr-1586. However, there are also differences in mode of reactions between GTX and local anesthetics. In contrast to GTX reactions, Phe-1579 is thought to be the major local anesthetic binding site rather than Tyr-1586, which merely regulates access of local anesthetics to the Phe-1579 site (32).

Other Critical Sites for GTX Action in S6 Transmembrane Segments-- Much as we determined that sites Phe-1579 and Tyr-1586 in D4S6 are critical to GTX action on the basis of drastic changes in kon or koff, we are likely to find similarly important sites in other domains. However, in this study, mutants in other domains did not produce a prominent change in either kon or koff. There are two reasons for failure to detect these sites; 1) we cannot determine kon or koff in mutants that are completely insensitive to GTX, thus exhibiting RCC values of 0, as observed especially with Lys mutations at Ile-433, Asn-434, Leu-437, Asn-784, and Ser-1276 and 2) mutations such as I433V do not necessarily change the chemical properties of relevant amino acids (i.e. both Ile and Val are hydrophobic residues). Thus, application of systematic mutation to these unexplored sites with measurement of kon and koff might be a productive avenue for future research. Ile-433 of D1S6 could be one of the most promising sites, because mutations of this site affect binding not only of GTX and other Site 2 toxins but also of pyrethroids (35). It is interesting to note the common functional effects of GTX and pyrethroids on sodium channel kinetics; both GTX and pyrethroids markedly prolong channel open time and remove or slow sodium inactivation (36-38).

    ACKNOWLEDGEMENTS

We express our sincere gratitude to Dr. Stephen M. Vogel (Department of Pharmacology, University of Illinois at Chicago, College of Medicine) for critical reading of the manuscript. We thank the Research Center for Molecular Medicine, Faculty of Medicine, Hiroshima University for the use of their facilities.

    FOOTNOTES

* This work was supported by Ministry of Education and Culture of Japan Grants 11470011 and 14370013 (to K. Y.) and 11770023 and 14770014 (to E. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed. Tel.: 81-82-257-5123; Fax: 81-82-257-5124; E-mail: kyamaok@hiroshima-u.ac.jp.

Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M212133200

2 Each mutant channel will be referenced by the original amino acid followed by its number and introduced amino acid.

    ABBREVIATIONS

The abbreviations used are: BTX, batrachotoxin; D, domain; GTX, grayanotoxin; INa, Na+ current; I-V, current-voltage; S, segment; RCC, relative chord conductance.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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