Clockwise Domain Arrangement of the Sodium Channel Revealed by µ-Conotoxin (GIIIA) Docking Orientation*

Ronald A. LiDagger, Irene L. Ennis§, Robert J. French||, Samuel C. Dudley Jr.**DaggerDagger, Gordon F. Tomaselli, and Eduardo Marbán§§

From the Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, the  Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada, and the ** Departments of Medicine and Physiology, Emory University, Atlanta, Georgia 30322

Received for publication, December 1, 2000, and in revised form, January 10, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

µ-Conotoxins (µ-CTXs) specifically inhibit Na+ flux by occluding the pore of voltage-gated Na+ channels. Although the three-dimensional structures of µ-CTXs are well defined, the molecular configuration of the channel receptor is much less certain; even the fundamental question of whether the four homologous Na+ channel domains are arranged in a clockwise or counter-clockwise configuration remains unanswered. Residues Asp762 and Glu765 from domain II and Asp1241 from domain III of rat skeletal muscle Na+ channels are known to be critical for µ-CTX binding. We probed toxin-channel interactions by determining the potency of block of wild-type, D762K, E765K, and D1241C channels by wild-type and point-mutated µ-CTXs (R1A, Q14D, K11A, K16A, and R19A). Individual interaction energies for different toxin-channel pairs were quantified from the half-blocking concentrations using mutant cycle analysis. We find that Asp762 and Glu765 interact strongly with Gln14 and Arg19 but not Arg1 and that Asp1241 is tightly coupled to Lys16 but not Arg1 or Lys11. These newly identified toxin-channel interactions within adjacent domains, interpreted in light of the known asymmetric toxin structure, fix the orientation of the toxin with respect to the channel and reveal that the four internal domains of Na+ channels are arranged in a clockwise configuration as viewed from the extracellular surface.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

µ-Conotoxins (µ-CTX)1 are receptor site I sodium channel blockers (1) isolated from the sea snail Conus geographus (2-5) that inhibit Na+ flux in voltage-gated Na+ channels by physically occluding the pore (2, 6-9). They are peptides consisting of 22 amino acid residues with six cysteines that form three intramolecular disulfide bonds, imparting backbone rigidity (2-7, 10-11). Their three-dimensional structures are well defined (12-14), making µ-CTXs useful probes to study the Na+ channel pore structure. Unlike tetrodotroxin and saxitoxin, whose smaller sizes render their receptor sites more compact (15-20), high-affinity binding of µ-CTXs results from the summed effects of numerous weaker toxin-channel interactions (9, 21-23). In contrast to charybdotoxin (ChTx) or agitoxin (AgTx), whose binding to the homotetrameric K+ channels can assume any one of four orientations with respect to the channel (24-26), µ-CTX binds to Na+ channels presumably in only one (as yet undetermined) favorable orientation because of intrinsic pore asymmetry (9, 18, 27-30). This notion is supported by the findings that critical channel residues for µ-CTX block identified to date are mostly clustered in domain II (DII), while "homologous" residues in other domains often have less or no effect (9, 30-32). Previously, Chang et al. (32) demonstrated that the toxin residue Arg13, which is required to inhibit current flow, interacts intimately with the DII pore residue Glu758. Based on this observation, the estimated dimensions of the channel pore, and the known structure of the toxin, these authors proposed that µ-CTX docks like an inverted pyramid with Arg13 reaching the deep pore region. The experiments that we describe here were designed to determine the actual µ-CTX docking orientation around the central axis.

Despite the detailed knowledge regarding the three-dimensional structure of µ-CTX, the molecular configuration of its channel receptor remains uncertain. Unlike K+ channels, which consist of four identical monomeric subunits, the pore-forming subunits of Na+ and Ca2+ channels are single polypeptides composed of four nonidentical homologous domains connected by cytoplasmic linkers (Fig. 1A). Although various lines of evidence have demonstrated that the four Na+ channel domains are structurally asymmetrical (9, 18, 27-30), the fundamental question of whether these domains are arranged in a clockwise or counter-clockwise configuration (when viewed from the extracellular side) remains unanswered (Fig. 1B). Indeed, review articles from different laboratories continue to depict the channels in either clockwise (33) or counterclockwise (1) topology, reflecting the paucity of data favoring one model over the other.

The negatively charged residues Asp762 and Glu765 located in the DII P-S6 linker (30) and Asp1241 of the DIII P-loop (9) of the rat skeletal muscle (µ1) Na+ channel are important for µ-CTX binding. These DII and DIII channel residues are exposed to the aqueous phase of the pore and influence single channel conductance (27, 29). Despite the significant roles of these residues in µ-CTX binding, no site of interaction on the toxin has been reported to date. Identification of the interacting partners, on the toxin, of these DII and DIII channel residues will not only reveal the docking orientation of µ-CTX when bound but will also help to define the internal domain arrangement (Fig. 1B). To identify the toxin-channel interaction sites, we synthesized five GIIIA-based toxin derivatives: R1A, K11A, Q14D, K16A, and R19A. These toxin mutants were selected because their side chains protrude from different faces of the toxin molecule (Fig. 2), enabling a rigorous dissection of toxin interactions with the channel residues in domains II and III. We first examined the IC50 for block of wild-type (WT), D762K, E765K, and D1241C mutant channels by both WT and point-mutated toxins and then applied mutant cycle analysis to quantitate the individual interaction (or coupling) energies between these channel and toxin sites. Our results indicate that Asp762 and Glu765 interact with Gln14 and Arg19, and Asp1241 interacts with Lys16. These results constrain the toxin binding orientation and reveal that the four Na+ channel domains are arranged in a clockwise configuration (when viewed from the extracellular face).



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Fig. 1.   A, schematic representation of the putative transmembrane topology of the Na+ channel alpha -subunit. The regions between the fifth and sixth transmembrane segments (S5 and S6) form the P-loops. The approximate location of the DII P-S6 linker (thick red lines) where Asp762 and Glu765 are located is shown. Asp1241 (red star) is located in the ascending limb of the DIII P-loop. B, schematic representation of a clockwise (left panel) and a counter-clockwise (right panel) domain arrangement of the four homologous domains of the Na+ channel as viewed from the extracellular side. Identification of toxin-channel interacting points in different domains will allow determination of the correct configuration of domain arrangement. Suppose residue A (red triangle) of the blocker is known to interact with DII; evidence showing interaction of residue B (cyan triangle) with DIII would suggest a clockwise domain arrangement. In contrast, interactions of DIII with residue C (purple triangle) would indicate a counter-clockwise arrangement.



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Fig. 2.   Three-dimensional structure of µ-CTX-GIIIA. Top panel, toxin residues studied in this report (i.e. Arg1, Lys11, Gln14, Lys16, and Arg19) and Arg13 are indicated with one-letter amino acid codes. Arg13 has been demonstrated to interact strongly with the DII pore residue Glu758. Since the side chains of these toxin residues protrude from different faces of the toxin, identification of novel channel/toxin contact points will reveal information regarding the orientation of µ-CTX when it is bound to the channel, thereby providing insights to the pore structure. Bottom panel, µ-CTX displayed in ribbon format. The helical face consisting of Arg13, Gln14, Lys16, Hyp17, and Arg19 is highlighted (magenta).



    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Site-directed Mutagenesis and Heterologous Expression-- µ1 sodium channel (34) mutants were created using polymerase chain reaction as previously described (29, 35). Na+ channels were heterologously expressed in tsA-201 cells using the LipofectAMINE Plus transfection kit (Life Technologies, Inc.). Transfected cells were incubated at 37 °C in a humidified atmosphere of 95% O2, 5% CO2 for 48-72 h for channel protein expression before electrical recordings.

Synthesis of Point-mutated µ-CTX-- The GIIIA form of µ-CTX was used in this study. Toxin derivatives were synthesized as previously described (21, 32). Briefly, solid phase synthesis was performed on a polystyrene-based Rink amide resin using 9-fluorenylmethoxycarbonyl chemistry. Synthesized peptides were air-oxidized and purified by high pressure liquid chromatography. Peptide composition was verified by quantitative amino acid analysis and/or mass spectroscopy. One-dimensional proton NMR spectra of the synthesized toxin were compared with those of native CTX to ensure proper folding of the point-mutated toxin.

Electrophysiology and Data Analysis-- Electrophysiological recordings were performed using the whole-cell patch clamp technique (36) with an integrating amplifier (Axopatch 200A; Axon Instruments). Transfected cells were identified by epifluorescence microscopy. Pipette electrodes had a final tip resistances of 1-3 megaohms. Recordings were performed at room temperature in a bath solution containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, pH adjusted to 7.4 with NaOH. Only cells expressing peak Na+ currents between 1 and 5 nA were used to ensure good current resolution while maintaining adequate voltage control. Designated toxin amounts were added to the bath when required. The internal recording solution contained 35 mM NaCl, 105 mM CsF, 1 mM MgCl2, 10 mM HEPES, 1 mM EGTA, pH adjusted to 7.2 with CsOH. All chemicals were purchased from Sigma unless otherwise specified.

Toxin was superfused continuously during the experiment. Flow rate was maintained at ~5 ml/min (bath volume was 150 µl). Wash-out began only after the peak currents had reached a steady-state level. Raw current records were analyzed using custom-written software. Equilibrium half-blocking concentrations (IC50) for µ-CTX were determined from the following binding isotherm,
I/I<SUB>o</SUB>=(1)/(1+([<UP>toxin</UP>]<UP>/IC<SUB>50</SUB></UP>)) (Eq. 1)
where IC50 represents the half-blocking concentration, and IO and I are the peak currents measured from a step depolarization to -10 mV from a holding potential of -100 mV before and after application of the toxin, respectively.

For mutant cycle analysis, coupling/interaction energies (Delta Delta G) for various mutant toxin-channel pairs were calculated from the equilibrium IC50 values using the following equations,
&Dgr;&Dgr;G=&Dgr;G<SUB>1</SUB>−&Dgr;G<SUB>2</SUB> (Eq. 2)

&Dgr;G<SUB>1</SUB>=G<SUP><UP>Mutated toxin-WT channel</UP></SUP>−<UP>G</UP><SUP><UP>WT toxin-WT channel</UP></SUP>=RT (<UP>ln IC<SUB>50</SUB></UP><SUP><UP>Mutated toxin-WT channel</UP></SUP>−<UP>ln IC<SUB>50</SUB></UP><SUP><UP>WT toxin-WT channel</UP></SUP>) (Eq. 3)

&Dgr;G<SUB>2</SUB>=G<SUP><UP>Mutated toxin-Mutated channel</UP></SUP>−G<SUP><UP>WT toxin-Mutated channel</UP></SUP>=RT (<UP>ln IC<SUB>50</SUB></UP><SUP><UP>Mutated toxin-Mutated channel</UP></SUP>−<UP>ln IC<SUB>50</SUB></UP><SUP><UP>WT toxin-Mutated channel</UP></SUP>) (Eq. 4)
where R is the gas constant and T is the Kelvin temperature. The S.E. values for Delta Delta G were estimated by dividing the square root of the sum of the variances of the RT ln IC50 means by the square root of the number of degrees of freedom.

All data are reported as mean ± S.E. Statistical significance was determined using a paired Student's t test at the 5% level.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Molecular Interactions between µ-CTX and the DII P-S6 Residues Asp762 and Glu765-- We initially looked for molecular interactions between the DII P-S6 channel residues (i.e. Asp762 and Glu765) and µ-CTX variants using mutant cycle analysis. Prediction of interactions (attractive or repulsive) between channel and toxin residues using this analysis requires determinations of blocking affinity of all of the following combinations: WT channel-WT toxin, mutant channel-WT toxin, WT channel-mutant toxin, and mutant channel-mutant toxin (see "Experimental Procedures" and Ref. 37 for review). Therefore, we first determined the half-blocking concentrations (IC50 values) for block of WT µ1 Na+ channels by both WT µ-CTX and the mutant toxin derivatives R1A, Q14D, and R19A. Fig. 3A shows representative whole-cell sodium currents recorded from cells expressing WT channels in the absence and presence of the toxin variants. The corresponding dose-response curves for binding of these toxins are shown in Fig. 3B. Consistent with previous results (11, 13, 21), the toxin derivatives R1A, Q14D, and R19A displayed significantly reduced affinity for block of WT Na+ channels compared with WT µ-CTX. Using the same strategy, we then determined the IC50 for block of the DII P-S6 mutants D762K and E765K by the same toxins. The IC50 values for these toxin-channel pairs are summarized in Fig. 4. Whereas both D762K and E765K channels were blocked by WT or R1A toxins with reduced potency, their block by Q14D and R19A was augmented compared with WT channels (Fig. 4). From the measured IC50 values, we estimated coupling or interaction energies (Delta Delta G) between these toxin and channel sites (Fig. 5). In general, the patterns of interactions for D762K and E765K were similar. Both DII-PS6 residues had the strongest interactions with Q14D (D762K/Q14D = 1.5 ± 0.1 kcal/mol; E765K/Q14D = 1.3 ± 0.1 kcal/mol), followed by R19A (D762K/R19A = 0.9 ± 0.1 kcal/mol; E765K/R19A = 1.0 ± 0.1 kcal/mol); in contrast, using the conventional cut-off of 0.5 kcal/mol for significance (38), there was no clear interaction with R1A (D762K/R1A = 0.5 ± 0.1 kcal/mol; E765K/R1A = 0.5 ± 0.1 kcal/mol). These results might be explained if Asp762 and Glu765 are close to Gln14, farther from Arg19, and distant from Arg1 in the toxin-bound state.



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Fig. 3.   A, representative raw current traces of WT channels elicited by depolarization to -10 mV from a holding potential of -100 mV in the absence and presence of WT µ-CTX, R1A, Q14D, or R19A as indicated. Control peak amplitudes were normalized for comparison. Their magnitudes were 2.6, 4.9, 2.6, and 5.0 nA for WT GIIIA, R1A, Q14D, and R19A, respectively. B, the dose-response relationship for block of WT channels by WT µ-CTX and its derivatives. Normalized peak Na+ currents at -10 mV were plotted as a function of extracellular µ-CTX concentrations. Data points were fitted with a binding isotherm to estimate the IC50 for µ-CTX block of each channel (see "Experimental Procedures"). Data are plotted as mean ± S.E.



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Fig. 4.   A, representative sodium currents from D762K and E765K channels recorded in the absence and presence of WT, R1A, Q14D, or R19A µ-CTX as indicated. Control peak amplitudes for D762K channels were 2.6, 2.6, 4.1, and 3.6 nA for block by WT CTX, R1A, Q14D, and R19A, respectively. For E765K channels, they were 2.9, 3.1, 5.0, and 1.9 nA, respectively. B, bar graphs summarizing the half-blocking concentrations (IC50) for block of WT, D762K, and E765K channels by WT and mutant µ-CTX derivatives R1A, Q14D, and R19A. Data presented were averaged data from 3-10 individual determinations.



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Fig. 5.   Estimated interaction energies (Delta Delta G) of D762K and E765K with R1A, Q14D, and R19A. Q14D and R19A but not R1A interact significantly (see text) with the DII P-S6 residues D762K and E765K. Interactions of D762K and E765K were stronger with Q14D than with R19A, suggesting that these channel residues are more closely associated with Gln14 than with Arg19 when the toxin molecule is bound to the channel.

Electrostatic Interactions between µ-CTX and Asp1241-- We and others have shown that neutralization of the charge on the DIII P-loop residue Asp1241 significantly destabilizes µ-CTX binding (9, 32, 38). However, the role of this residue in µ-CTX's interactions with the Na+ channel is undefined. Since µ-CTX carries a net positive charge and Asp1241 is negatively charged and exposed to the aqueous pore (9, 27), the toxin and this channel residue may interact electrostatically. To define the toxin interaction with this residue, we introduced the point mutations D1241C for charge neutralization, D1241K for charge reversal, and D1241E for charge conservation; the effects on µ-CTX block are summarized in Fig. 6. Consistent with previous results (9), D1241C (IC50 = 3.3 ± 1.5 µM, n = 3) exhibited reduced block by µ-CTX compared with WT channels. As expected for an electrostatic effect, the charge-reversed mutation D1241K (IC50 = 9.4 ± 3.4 µM, n = 3) further destabilized toxin affinity compared with the charge-neutralized cysteine mutant. In contrast, the charge-conserving mutation D1241E partially preserved sensitivity to µ-CTX (IC50 = 110.0 ± 20.1 nM, n = 4); D1241E channels were only ~3-fold less sensitive than WT channels. Taken together, these results support the notion that electrostatic interactions exist between Asp1241 and µ-CTX.



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Fig. 6.   Effects of charge neutralization, reversal, and restoration at position DIII-D1241 on block by µ-CTX. A, representative Na+ current tracings of WT, D1241C, D1241K, and D1241E channels elicited by depolarization to -10 mV from a holding potential of -100 mV in the absence and presence of µ-CTX as indicated by the arrows. Toxin-free peak currents were normalized for ease of comparison. Their magnitudes were 2.6, 4.2, 3.2, and 3.5 nA, respectively. B, bar graphs summarizing the half-blocking concentrations (IC50) for µ-CTX of WT and the same channels shown in A. D1241C displayed a ~100-fold reduction in µ-CTX affinity compared with WT channels. The charge-reversed mutation D1241K further destabilized toxin binding. The charge-conserved mutation D1241E only partially restored wild-type µ-CTX sensitivity. Its IC50 was 3-fold higher than WT. Data are all presented as mean ± S.E.

Interaction of Asp1241 with Lys16-- To further constrain the binding orientation, we studied the interactions of DIII-D1241 with µ-CTX, looking for couplings between this channel residue and Arg1, Lys11, and Lys16 of the toxin. These toxin sites were chosen because their side chains protrude from different faces of µ-CTX compared with Gln14 and Arg19 (see Fig. 2 for locations of these residues). Fig. 7A shows the IC50 values for block of D1241C channels by the µ-CTX derivatives R1A, K11A, and K16A. Interestingly, the potency of block by K16A was significantly greater in D1241C channels than in WT channels, consistent with a stronger interaction between the D1241C mutant channel and K16A than between WT channels and K16A. This change in interaction energy could reflect a gain of attractive and/or a relief of repulsive forces, leading to increased potency of the K16A derivative. In contrast, the potencies of block by either R1A or K11A were reduced when applied to D1241C channels, in a similar manner to that seen with the WT channels, indicating an additive effect of these channel and toxin mutations. Fig. 7B shows the coupling energies for D1241C with R1A, K11A, and K16A. D1241C displays an exceptionally strong positive interaction with Lys16 (3.2 ± 0.2 kcal/mol), implying a close association between these toxin and channel sites in the toxin-channel complex. Less significant interactions were identified with Arg1 (0.5 ± 0.2 kcal/mol) and with Lys11 (0.7 ± 0.2 kcal/mol).



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Fig. 7.   Interactions of D1241C with Arg1 and Lys16. A, bar graphs summarizing the IC50 values of WT (open bars) and D1241C channels (closed bars) for block by R1A, K11A, and K16A. Data presented are mean ± S.E. B, interaction energies (Delta Delta G) of D1241C with R1A, K11A, and K16A calculated from the experimental IC50 values shown in A. D1241C displays a substantial interaction with K16A and a marginal interaction with K11A.



    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

µ-Conotoxin Macrosite-- Consistent with previous studies, our experiments with alanine-substituted µ-CTX GIIIA derivatives on WT µ1 channels show significant reductions in toxin-binding affinity, suggesting that the positively charged toxin residues Arg1, Lys11, Lys16, and Arg19 are critical for its biological activity. These results were qualitatively similar to previous studies using either glutamine or alanine substitution for charge neutralization at these toxin positions, when assessed using a rat diaphragm bioassay or bilayer-incorporated single Na+ channels, respectively (11, 21). The reduction in blocking affinity reported in these earlier studies, and in our own data, was in the order Arg19 > Lys16 > Arg1 ~ Lys11. Since the side chains of these toxin residues are spread over a wide surface and protrude in different directions around the toxin molecule, these results are most consistent with the notion that the µ-CTX binding site, unlike the ones for tetrodotroxin and saxitoxin, covers a more extensive area of the channel surface. Additionally, the highly site-specific pattern of interactions among the various toxin and channel sites further implies that intimate toxin-channel interactions are localized and argue against global disruptions of either the channel or the toxin by the mutations introduced.

Charge manipulations at position 1241 in DIII by point mutations support an electrostatic role for this residue in µ-CTX binding, since the charge reversal mutation (D1241K) further destabilized toxin block compared with the charge-neutralized mutant (D1241C), whereas conservation of charge (D1241E) partially restored wild-type toxin affinity. This incomplete restoration by the glutamate substitution suggests that an aspartate is required at position 1241 to confer wild-type µ-CTX blocking behavior. This observation could be attributed to steric hindrance, since the glutamate side chain is one methyl group longer than that of aspartate. Overall, the data suggest that the charges, as well as their precise location within the pore, are critical for toxin-channel interaction.

The Arg13-Gln14-Lys16-Hyp17-Arg19 Toxin Face Recognizes the Channel Receptor-- Inspection of the three-dimensional structures of GIIIA µ-CTX reveals that Arg13, Gln14 (or Arg14 for µ-CTX GIIIB, whose three-dimensional structure is essentially the same as GIIIA) (14), Lys16, Hyp17, and Arg19 are clustered on one helical face of the toxin molecule (Fig. 2, bottom panel) (11-14). We and others have demonstrated that Arg13, Lys16, Hyp17, and Arg19 are essential residues for the biological activity of the toxin (see Refs. 11, 13, 21, 32, 39, and 40, and this report). Here we further show that Gln14 and Arg19 of the toxin interact strongly with the DII P-S6 channel residues Asp762 and Glu765. Previously, Chang et al. (32) showed that Arg13 associates closely with the DII P-loop residue Glu758. However, even with the assumption that Arg13 is directed inward, approximately parallel to the conducting axis of the pore, these findings still do not completely define the docking orientation of µ-CTX with respect to all channel domains, because the data are limited to toxin-domain II interactions. We therefore studied also the interactions of DIII-D1241 with the toxin. Our results indicate that Asp1241 interacts strongly with Lys16 but not Arg1 and Lys11 (Fig. 7). Taken together, these findings highly suggest that the toxin Arg13-Gln14-Lys16-Hyp17-Arg19 helical face interacts with the channel receptor site (13-14). Fig. 8 shows a schematic representation of a possible docking orientation of µ-CTX based on the pattern of specific toxin-channel interactions observed. According to this model, the Arg13-Gln14-Lys16-Hyp17-Arg19 helical face associates closely with the P-segment (including the P-S6 linker) of domain II. Arg13 projects into the pore and is closest to Glu758. Asp762 and Glu765 both closely interact with Gln14 as well as Arg13 but are capable of interacting also with Arg19. The proposed docking orientation of µ-CTX predicts that Arg1 faces the opposite direction and is remote from DII, consistent with our experimental observations (cf. Fig. 5). In addition, our model also predicts that Lys11 protrudes in a direction away from the DII P-loop and therefore, like R1, also should not interact strongly with any of the DII residues.



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Fig. 8.   Clockwise arrangement of channel domains. A graphical representation is shown for the interactions between µ-CTX, the DII P-loop, and DIII D1241 when bound to the Na+ channel pore. The model depicts the helical face of µ-CTX, which comprises Arg13, Gln14, Lys16, Hyp17, and Arg19, interacting closely with the critical DII channel residues Glu758, Asp762, and Glu765. The back bone loop and placement of residues Asp762 and Glu765 are generally consistent with the electrical distance data of Li et al. (29), but there are no specific data that constrain the turn in the DII backbone to be either right- or left-handed. In the actual channel-toxin complex, side chain configurations may differ from those in the solution structure, adding some uncertainty to the predictions of positions of pore-lining residues. Arg1 and Lys11 face different sides of the channel (cf. Fig. 2) and do not interact with DII. DIII-D1241 associates closely with Lys16. The channel domains are arranged in a clockwise configuration when viewed from the extracellular surface.

Clockwise Domain Arrangement-- The strong coupling between the toxin residue Lys16 and the channel residue Asp1241 in DIII (Fig. 7) place these toxin and channel residues in close proximity with each other in the toxin-bound state. According to the toxin orientation proposed above, Lys16 lies above Arg13, implying that Asp1241 is located shallower within the pore than Glu758. This is consistent with fractional electrical distances estimated from single channel recordings (27). This arrangement of the toxin molecule, channel domains, and residues also explains the somewhat marginal interaction of Asp1241 with Lys11, since this toxin residue may not directly face Asp1241 (or DIII). Based on the known structure of µ-CTX and the proposed toxin docking orientation, our results suggest a clockwise arrangement of the channel domains when viewed from the external surface (Fig. 8). This conclusion is consistent with that proposed recently by Dudley et al. (39), although in general their conclusion was based on entirely different lines of evidence and interpretation. The models of toxin-channel docking in the two studies differ in several important details. In contrast to our model, Dudley et al. place Lys16 between DI and DIV. They detected little binding energy between Lys16 and Met1240 in DIII using the mutant pair K16A and M1240A and propose that Arg1 is adjacent to DII using R1A and T759I. Their arguments were based on analysis of possible interactions with all four domains, which, in general, were energetically weaker than the key interactions reported here.

Conclusion-- In summary, we have demonstrated that the toxin residues Gln14 and Arg19 interact with the DII P-S6 channel residues Asp762 and Glu765 and that Lys16 interacts strongly with DIII-D1241. These data suggest a unique docking orientation of µ-CTX. Based on these results and the known three-dimensional structure of µ-CTX, we further conclude that the four Na+ channel domains are arranged in a clockwise configuration when viewed from the extracellular side.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants R01 HL-52768 (to E. M.) and R01 HL-50411 (to G. F. T.) and by operating funds from the Medical Research/Canadian Institutes of Health Research (to R.J.F.).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.

Dagger Recipient of a fellowship award from the Heart and Stroke Foundation of Canada.

§ Supported by a fellowship award from Universidad Nacional de La Plata, Argentina.

|| Recipient of salary support as a Medical Research Council/Canadian Institutes of Health Research Distinguished Scientist and an Alberta Heritage Foundation for Medical Research Medical Scientist.

Dagger Dagger Recipient of salary support from a Scientist Development Award from the American Heart Association and National Institutes of Health Grant HL-64828.

§§ Holder of the Michel Mirowski, M.D. Professorship of Cardiology of The Johns Hopkins University. To whom correspondence should be addressed: Inst. of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, 720 Rutland Ave./Ross 844, Baltimore, MD 21205. Tel.: 410-955-2776; Fax: 410-955-7953; E-mail: marban@jhmi.edu.

Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M010862200


    ABBREVIATIONS

The abbreviations used are: µ-CTX, µ-conotoxin; WT, wild-type; DII, DIII, and DIV, domain II, III, and IV, respectively; Hyp, hydroxyproline.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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