Correspondence to: Samuel C. Dudley, Jr., Assistant Professor of Medicine and Physiology, Division of Cardiology, Emory University/VAMC, 1670 Clairmont Road, Room 111B, Decatur, GA 30033. Fax:(404) 329-2211 E-mail:sdudley{at}emory.edu.
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Abstract |
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Voltage-gated Na+ channels underlie the electrical activity of most excitable cells, and these channels are the targets of many antiarrhythmic, anticonvulsant, and local anesthetic drugs. The channel pore is formed by a single polypeptide chain, containing four different, but homologous domains that are thought to arrange themselves circumferentially to form the ion permeation pathway. Although several structural models have been proposed, there has been no agreement concerning whether the four domains are arranged in a clockwise or a counterclockwise pattern around the pore, which is a fundamental question about the tertiary structure of the channel. We have probed the local architecture of the rat adult skeletal muscle Na+ channel (µ1) outer vestibule and selectivity filter using µ-conotoxin GIIIA (µ-CTX), a neurotoxin of known structure that binds in this region. Interactions between the pore-forming loops from three different domains and four toxin residues were distinguished by mutant cycle analysis. Three of these residues, Gln-14, Hydroxyproline-17 (Hyp-17), and Lys-16 are arranged approximately at right angles to each other in a plane above the critical Arg-13 that binds directly in the ion permeation pathway. Interaction points were identified between Hyp-17 and channel residue Met-1240 of domain III and between Lys-16 and Glu-403 of domain I and Asp-1532 of domain IV. These interactions were estimated to contribute -1.0 ± 0.1, -0.9 ± 0.3, and -1.4 ± 0.1 kcal/mol of coupling energy to the native toxinchannel complex, respectively. µ-CTX residues Gln-14 and Arg-1, both on the same side of the toxin molecule, interacted with Thr-759 of domain II. Three analytical approaches to the pattern of interactions predict that the channel domains most probably are arranged in a clockwise configuration around the pore as viewed from the extracellular surface.
Key Words: electrophysiology, site-directed mutagenesis, molecular models, kinetics, binding sites
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INTRODUCTION |
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Knowledge of the structure of the voltage-gated Na+ channel is necessary to understand its various functions and to optimize the pharmacokinetics of antiarrhythmic, local anesthetic, and anticonvulsant drugs. Although the K+ channel pore-forming tetramer complex has been successfully analyzed by diffraction techniques (
µ-Conotoxin GIIIA (µ-CTX)1 is a 22amino acid peptide toxin, originally isolated from piscivorous cone snails, that binds Na+ channels (
Previously, we have shown interactions of the critical guanidinium group on Arg-13 of µ-CTX with predominantly two acidic residues of the adult rat skeletal muscle Na+ channel (µI) outer vestibule, Glu-403 and Glu-758 (
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Specific channeltoxin interactions can be inferred by mutant cycle analysis (
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MATERIALS AND METHODS |
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The methods are similar to those previously used and have been described in detail (
µ-CTX Mutations
µ-CTX mutations were made by solid phase synthesis using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry (95% pure, as determined by analytical HPLC. The total peptide content of the samples was determined by quantitative amino acid analysis. Variance in weighing was estimated to lead to no more than a 10% error in calculating the toxin concentration.
Reagents were obtained from the following sources: Fmoc amino acids from Calbiochem-Novabiochem">Calbiochem-Novabiochem, Bachem, Genzyme, and Richelieu Biotechnologies; Rink amide resin was from Calbiochem-Novabiochem">Calbiochem-Novabiochem; and the coupling reagents were from Richelieu and Sigma-Aldrich.
Na+ Channel Mutagenesis
Oligonucleotide-directed point mutations were introduced into the rat adult skeletal muscle Na+ channel cDNA. DNA sequencing of the entire polymerized regions insured that only the intended mutations were present. Stage V and VI Xenopus oocytes from female frogs (NASCO or Xenopus 1) were injected with 50100 ng of cRNA. Oocytes were incubated at 16 for 1272 h before examination.
Electrophysiology
Recordings were made by two-electrode voltage clamp at room temperature (2022°C). The oocytes were placed in a bath chamber with a solution exchange time sufficiently short to resolve toxin blocking kinetics. The standard bath solution consisted of the following (in mM): 90 NaCl, 2.5 KCl, 1 CaCl2, 1 MgCl2, and 5 HEPES, titrated to pH 7.2 with 1 N NaOH. Recordings of the peak currents were made every 20 s upon step pulses from -100 to 0 mV. Only oocytes with between 1 and 10 µA of peak current were studied. The change in peak INa with time was fitted by single exponential functions and was used to estimate kinetic rate constants. The 50% inhibitory concentration (IC50) for toxin binding was calculated from the ratio of peak currents in the absence and presence of toxin (
The free energy change in toxin binding to a wild-type/mutant channel pair (G) was calculated as the difference of the average RTln(IC50) for the wild-type and mutant, where R is the gas constant and T is temperature. The standard errors (SEMs) for
G were estimated as the square of the variance of the RTln(IC50) averages divided by the square root of the sum of the number of observations.
G was taken as the difference of the
Gs for µ-CTX and the toxin mutant (
G = (GWT,native - Gmutant,native) - (GWT,mutant - Gmutant,mutant), where the first and second subscript positions refer to the channel and the toxin, respectively, and the standard error of this number was reported as the square root of the sum of the variances of the RTln(IC50) averages (
G may be positive or negative, both representing a coupling interaction. The negative values represent less coupling energy between the mutant pair as compared with the native residue pair. A positive
G indicates that the introduced pair has more coupling energy after mutation relative to the native pair. This might occur as a result of relief of a preexisting repulsion or by the creation of a novel attraction between the new pair. Data are presented as means ± SEM. Statistical comparisons of
Gs and
Gs were performed using two-tailed t tests assuming unequal variances (Excel 97; Microsoft Corp.).
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RESULTS |
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Our experimental goal was twofold: (1) to expand our understanding of the binding interactions of µ-CTX, and (2) to draw structural inferences about the channel by determining points of interaction between the DQHypK collar amino acids directly above the critical Arg-13 residue (Fig 1).
The Effect of Mutations on Toxin Blocking Efficacy
Fig 2 (top) shows toxin blocking efficacy of the µ-CTX derivatives with the wild-type channel. The native µ-CTX affinity for the wild-type µI Na+ channel was in the range of values reported by others for the rat skeletal muscle channel (
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The effects of channel mutations from each of the four domain pore-forming loops on native µ-CTX affinity are shown in Fig 2 (bottom). The choice of channel residues mutated was based upon previously predicted collarchannel interactions (
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Determination of ToxinChannel Couplings
Mutant cycle analysis was used to determine coupling between µ-CTX and the outer vestibule. Results for all combinations of channeltoxin mutants tested are shown in Table 2. The µ-CTX mutant Q14D showed a significant coupling to channel residue T759I (Fig 3 A). Mutant cycles incorporating this toxin mutation showed a domain-specific pattern consistent with coupling between Gln-14 and the domain II residue Thr-759 (Fig 3 B). The G for the Q14DT759I interaction relative to the native complex was statistically different (P < 0.01) from the
G values for the Q14D/N404R, Q14D/E403Q, and Q14D/D1532N.
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Hyp-17 of µ-CTX showed a domain-specific interaction exclusively with Met-1240 of domain III (Fig 3 B). This interaction is consistent with the S of Met acting as a hydrogen bond receptor for the
-OH of Hyp. The energy of this type of interaction has been shown to be
1.1 kcal/mol (
G) of
1.5 kcal/mol. Therefore, most of this loss could be explained by elimination of the Hyp-17/Met-1240 interaction. The Hyp-17 interaction appeared specific for Met-1240 of domain III. No interaction was identified between the adjacent Asp-1241 and Hyp-17 (
G = 0.2 ± 0.1 kcal/mol), even though D1241A had a large effect on native µ-CTX affinity, and no other significant interactions were identified between Hyp-17 and channel residues of the other domains.
Lys-16 is opposite Gln-14 in the µ-CTX collar tetrad, and the K16A mutation resulted in the next largest reduction in blocking efficacy among the collar tetrad derivatives tested. As anticipated from the structure of µ-CTX and the interaction of Gln-14 with domain II, the toxin 16 site showed the strongest interaction with domain IV Asp-1532 (Fig 3 B). However, the Lys-16 interaction was not confined to Asp-1532. An interaction of lesser energy was demonstrated with the domain I channel residue, Glu-403 (P < 0.01). Of less clear significance, an energetically opposite K16AT759I interaction was observed that was statistically different from zero (P < 0.01).
No strong Asp-12 interactions were resolved by these experiments (Fig 3 B). Because of the previously demonstrated interactions, the expectation was that Asp-12 would be adjacent to channel residues of domain I. The coupling with Asn-404 was small but statistically significant. Asp-12 showed no coupling with residues in domain II (Thr-759) or domain III (Met-1240).
The structure of the toxin (
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DISCUSSION |
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In this study, interactions between four residues of µ-CTX and several residues in the rat skeletal muscle Na+ channel outer vestibule were demonstrated by mutant cycle analysis. These interactions help define important elements for toxinchannel high affinity recognition and binding. Furthermore, the combination of the structure of µ-CTX and these demonstrated interactions establish constraints on the domain organization of the Na+ channel.
Consistency with Previous Results
For wild-type channels, the IC50s of µ-CTX mutations D12A, Q14D, K16A, and Hyp17P were 0.6-, 31-, 24-, and 14-fold change, respectively, from the native toxin IC50. These changes are consistent with previous reports. The slight increase in blocking efficacy seen with D12A is consistent with all other reports of the effect of neutralization of the negative charge at this site (21-fold change in the dissociation constant seen for K16Q in bilayers incorporating rat skeletal muscle Na+ channels (
The effects of channel mutants seen in this study were generally consistent with previous reports. The mutation E403Q increased the native µ-CTX IC50 by 8.9-fold, similar to a 4-fold increase reported previously for the identical mutation (
Interpretation of Interaction Energies
Toxins of known structure have proven extremely useful in probing the outer vestibule of K+ channels (
The major potential source of error with mutant cycle analysis arises from the possibility of structural changes in either of the interacting molecules as the direct result of mutations, or resulting secondarily from changes in the nature of the ligandprotein interaction. Allosteric effects can often be identified by generalized disruption of normal function of the channel. The mutated channels in this study were evaluated functionally, and they showed no significant alterations in the macroscopic gating behavior or reversal potential. Several toxin mutants have been screened for major structural changes by nuclear magnetic resonance with negative results. All toxinchannel pairs show blocking interactions, suggesting that the interacting surfaces were not grossly altered. Furthermore, the pattern of domain- and residue-specific Gs supported the specificity of interactions.
Of the interactions noted above, four showed negative Gs, and there was at least one positive
G. In performing mutant cycle analysis, mutations are usually chosen to eliminate interactions without inducing new ones. We defined the interaction energy such that negative energies of interaction would represent a loss of binding energy in the mutant pair complex compared with the native, bound complex. This idea is consistent with the negative
Gs in the R1A/T759I, K16A/D1532N, K16A/E403Q, and Hyp17P/Met1240A pairs. Alternatively, a negative
G could arise from new repulsions resulting from the substituted residues as compared with the native ones. In either event, an interaction exists, implying that the residues are near enough to interact with each other in the toxinchannel complex. This dependence of
G on the choice of substituted residue was predicted by
The Q14DT759I interaction showed a positive G. Since the overall effect of the Q14D mutation was to decrease toxin blocking efficacy, the Gln-14/Thr-759 interaction is not sufficient to explain the entire Q14D effect. This could be the result of the double mutant complex eliminating a repulsion in the native complex or, less likely, adding a new attractive force between the toxin Asp and the channel Ile relative to the Gln/Thr pair. The overall effect of T759I on native toxin binding is small, which is consistent with the opposing effects. It is plausible that there could be electrostatic repulsion between the substituted Asp-14 of the toxin and the negative residues of the vestibule, but Glu-403 and Asp-1532 must be too far away to contribute significantly. Consistent with this possibility, Li and his co-workers (Li, R.A., I.L. Ennis, S.C. Dudley Jr., R.J. French, G.F. Tomaselli, and E. Marban, manuscript submitted for publication) have noted significant interactions between Gln-14 and channel residues Asp-762 and Glu-765. This observation supports the conclusion that Gln-14 is oriented toward domain II.
The failure to show a Gln-14/Glu-403 interaction is consistent with the implication derived from the data of G = -0.3 kcal/mol).
Our failure to demonstrate a strong interaction of Asp-12 with the channel does not preclude the possibility that Asp-12 is near domain I. In the BarstarBarnase complex wherethe crystal structure could be determined,
The Lys-16 interaction pattern was more difficult to interpret. At a level considered to identify confidently interactions (Gs with Asp-1532 of domain IV and Glu-403 of domain I, suggesting that Lys-16 might be located between these two domains. Multidomain interactions suggest that caution should be used when interpreting the effects of charge-changing mutations. In this case, the largest energy associated with the Lys-16/Asp-1532 interaction hints that Lys-16 might be closest to domain IV.
Because the M1240C mutation did not change the IC50 of native µ-CTX binding significantly, Backx and his coworkers (G was calculated for the Hyp17Pro/M1240A interacting pair. This suggests that the net effect of Met-1240 mutations is the sum of at least two interactions with opposing energetic effects. Our study supports previous demonstrations that Asp-1241 is important for µ-CTX binding (
Structural Implications of Coupling Data
Our experimentally derived coupling data are most consistent with a circumferentially sequential, clockwise arrangement of the domains around the ion permeation pathway. Gln-14, Hyp-17, and Lys-16 are arranged at approximately right angles to each other in a plane perpendicular to the axis of the pore. These toxin residues interact most strongly with residues of domains II, III, and IV, respectively. These interactions are best explained if the domains are arranged in the clockwise pattern, as shown in Fig 4. In the structure of µ-CTX, Arg-1 is on the same side of the toxin as Gln-14, and its coupling with a domain II residue also supports the clockwise domain arrangement.
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Three different approaches to the interpretation of our observations all lead to this conclusion. First, an intuitive approach in which the strongest coupling of each collar residue is considered to dominate and define the orientation in the pore indicates a clockwise arrangement as shown in Fig 4 A. Second, to systematically take into account all of our coupling data, rather than make conclusions using selectively identified strong interactions, we devised a novel analysis based on defining a resultant interaction vector for each of the collar tetrad residues. Within the data set, this analysis provides an unbiased summary of statistically significant multidomain interactions, such as those of the charged residue Lys-16, which, when considered in isolation, would suggest couplings in several directions. Shown in Fig 4 B (see figure legend for details), this analysis argues for a clockwise domain orientation. Determination of additional couplings may affect quantitatively the interaction vectors, but is unlikely to alter the basic clockwise conclusion. Finally, the common conclusion, a clockwise domain arrangement, is further supported by a statistical analysis of the collected interaction energy data. The sums of Gs for each of the eight possible sequential clockwise and counterclockwise configurations of the domaincollar interactions were made and the variances were calculated. The most favorable clockwise configuration was as shown in Fig 4 A. This was tested against the two most favorable counterclockwise conformations. In both comparisons, the clockwise configuration was favored, with P < 0.001.
There are a limited number of toxinchannel interactions that can be elucidated with a single toxin, so it is important to test multiple toxins of differing shapes to constrain models adequately. A multiple toxin approach minimizes the possibility of being misled by allosteric changes produced by mutagenesis. The conclusions about channel architecture inferred here from µ-CTXchannel interactions are similar to those derived from STXchannel interactions (Penzotti, J.L., G.M. Lipkind, H.A. Fozzard, and S.C. Dudley Jr., manuscript submitted for publication). Interactions derived using neoSTX and µ-CTX, two toxins with significantly different structures and chemical interactions, support the validity of the general features of the model in Fig 4 A, and set the stage for further tests of outer vestibule structural predictions.
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Footnotes |
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1 Abbreviations used in this paper: G, the change in free energy of binding;
G, interaction energy; Hyp, hydroxyproline; IC50, 50% inhibitory concentration; koff, off rate; kon, on rate; µ-CTX, µ-conotoxin GIIIA; µI, rat adult skeletal muscle Na+ channel; neoSTX, neosaxitoxin; STX, saxitoxin;
off, exponential time constant for relief of toxin block;
on, exponential time constant for toxin block; TTX, tetrodotoxin.
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Acknowledgements |
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Ian Glaaser, Colleen Parrett, Junxiu Ling, and Bin Xu provided technical assistance. The authors thank Dr. Jack Kyle and Dr. Steven Chang for their assistance, Dr. Denis McMaster for synthesizing peptides, Dr. D. McIntyre and P. Hwang for NMR data, Dr. Kwokyin Hui for testing some toxin derivatives by single channel recording, and Drs. R.A. Li, I.L. Ennis, G.F. Tomaselli, and E. Marban for sharing submitted manuscripts. We thank Dr. Richard Horn for helpful discussions.
This research was supported by an American Heart Association, Southeast Affiliate Beginning Grant-in-Aid (to S.C. Dudley), by a Program Project Award from the National Institutes of Health (to H.A. Fozzard, No. P01-HL20592), and by an operating grant from the Medical Research Council of Canada (to R.J. French). S.C. Dudley is supported by a Scientist Development Award from the American Heart Association, a Procter and Gamble University Research Exploratory Award, and National Institutes of Health Grant (HL64828). N.S. Chang was supported by the training grant TO1-GM07019. R.J. French received salary support as an Alberta Heritage Foundation for Medical Research Medical Scientist and a Medical Research Council Distinguished Scientist.
Submitted: 4 April 2000
Revised: 13 September 2000
Accepted: 15 September 2000
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