Correspondence to: Eduardo Marbán, Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, 720 Rutland Avenue/Ross 844, Baltimore, MD 21205. Fax:410-955-7953 E-mail:marban{at}jhmi.edu.
Released online: 28 December 1999
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Abstract |
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The deep regions of the Na+ channel pore around the selectivity filter have been studied extensively; however, little is known about the adjacent linkers between the P loops and S6. The presence of conserved charged residues, including five in a row in domain III (D-III), hints that these linkers may play a role in permeation. To characterize the structural topology and function of these linkers, we neutralized the charged residues (from position 411 in D-I and its homologues in D-II, -III, and -IV to the putative start sites of S6) individually by cysteine substitution. Several cysteine mutants displayed enhanced sensitivities to Cd2+ block relative to wild-type and/or were modifiable by external sulfhydryl-specific methanethiosulfonate reagents when expressed in TSA-201 cells, indicating that these amino acids reside in the permeation pathway. While neutralization of positive charges did not alter single-channel conductance, negative charge neutralizations generally reduced conductance, suggesting that such charges facilitate ion permeation. The electrical distances for Cd2+ binding to these residues reveal a secondary "dip" into the membrane field of the linkers in domains II and IV. Our findings demonstrate significant functional roles and surprising structural features of these previously unexplored external charged residues.
Key Words: sodium channel, outer pore, cysteine mutagenesis, sulfhydryl modification, single-channel recording
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INTRODUCTION |
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Voltage-gated Na+ channels are responsible for initiating action potentials in excitable tissues including heart, muscle, and nerve by selectively transporting Na+ ions across the surface membrane (
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While the functional and structural aspects of this deep region of the Na+ channel pore have been extensively studied, little is known about the flanking regions of the P loops. Inspection of the primary sequence of Na+ channels reveals that the linkers between the P segments and S6 on the carboxyl-terminal side of the selectivity filter contain a number of charged residues; in particular, the domain III linker contains five charges in a row (Figure 1). Most of these charged residues are highly conserved from jellyfish to human. This striking pattern leads to several immediate questions. Do these charged residues play similar structural and functional roles as those located deeper in the pore, or are they simply peripheral residues of little functional importance? Do these residues participate in the process of ion permeation; e.g., by increasing the local effective Na+ concentration at the external mouth of the pore and/or by affecting ionic selectivity? To address these questions, we neutralized each of the charged residues in these P loopS6 (P-S6)1 linkers (from position 411 in D-I and its analogues in D-II, III, and IV to the putative start sites of S6, see also Figure 1) individually by cysteine substitution. Side-chain accessibility of cysteine-substituted mutants was probed by sensitivity to Cd2+ blockade and by reactivity to sulfhydryl-specific methanethiosulfonate (MTS) reagents using whole-cell patch-clamp recordings. Single-channel recordings were performed to probe changes in channel conductance with charge neutralization and to assess the electrical distance for Cd2+ binding to the cysteine-substituted charged residues. Our results demonstrate unsuspected functional and structural features of this previously unexplored region of the Na+ channel.
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MATERIALS AND METHODS |
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Site-directed Mutagenesis and Heterologous Expression
Mutagenesis was performed on the rat skeletal muscle sodium channel subunit (µ12;
Wild-type (WT) and mutated channels were expressed in TSA-201 cells (a transformed HEK 293 cell line stably expressing the SV40 T-antigen) by addition to the cells of 1 µg/60-mm dish of DNA encoding the subunit using the Lipofectamine Plus transfection kit (GIBCO-BRL). Transfected cells were incubated at 37°C in a humidified atmosphere of 95% O25% CO2 for 4872 h for channel protein expression before electrical recordings. Given that the
subunit suffices for permeation, ß1 subunits were not routinely coexpressed. Nevertheless, we verified that E1551C, a representative domain IV mutant, was unaltered in its selectivity, Cd2+ blocking affinity, or MTS susceptibility when coexpressed with ß1 subunit.
Electrophysiology
Electrophysiological recordings were performed using the whole-cell or cell-attached single-channel variants of the patch clamp technique ( when filled with the internal recording solution (see below). All recordings were performed at room temperature.
Single-channel currents were measured in the presence of 20 µM fenvalerate (Dupont) to promote long channel openings ( and coated with Sylgard.
Solutions
Whole-cell currents were recorded in a bath solution containing (mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH adjusted to 7.4 with NaOH. The pipette solution contained (mM): 35 NaCl, 105 CsF, 1 MgCl2, 10 HEPES, 1 EGTA, pH adjusted to 7.2 with CsOH. Appropriate amounts of blockers or covalent modifiers [methanethiosulfonate ethylsulfonate (MTSES) and methanethiosulfonate ethylammonium (MTSEA); Toronto Research Chemicals] were added to the bath when required. For single-channel recordings, the bath contained (mM): 140 KCl, 1 BaCl2, 10 HEPES, pH adjusted to 7.4 with KOH. The pipette solution contained (mM): 140 NaCl, 1 BaCl2, 10 HEPES, pH adjusted to 7.4 with NaOH. All chemicals were purchased from Sigma Chemical Co. unless otherwise specified.
Data Analysis and Statistics
Half-blocking concentrations (IC50) for Cd2+ were determined by least-square fits of the doseresponse data to the following binding isotherm using the Levenberg-Marquardt algorithm: I/IO = 1/{1 + ([Cd2+]/I)n}, where I and IO are the peak currents measured from a step depolarization to -10 mV from a holding potential of -100 mV before and after application of Cd2+, respectively, and n is the Hill coefficient (assumed to equal 1 for a single binding site for Cd2+).
Currentvoltage relationships were obtained by holding cells at -100 mV and stepping from -60 to +50 mV in 10-mV increments. Reversal potentials were calculated by fitting the currentvoltage relationship to a Boltzmann distribution function: I = [(Vt - Vrev) * Gmax]/{1 + exp[(Vt-V1/2)/k]}, where I is the peak INa at a given test potential Vt, Vrev is the reversal potential, Gmax is the maximal slope conductance, V1/2 is the half point of the relationship, and k is the slope factor.
For single-channel analysis, amplitude histograms were fitted to the sum of Gaussians using a nonlinear least squares method. Slope (single-channel) conductance was obtained by linear fit of the currentvoltage relationship. The fraction of the transmembrane electric field that Cd2+ traversed (i.e., electrical distance, ) to reach its binding site was estimated by making a logarithmic plot of the ratio of unblocked and blocked unitary current amplitudes as a function of membrane potential followed by linear fits (
Steady state activation (m) curves were derived from the relation m
= g/gmax, where the conductance g was obtained from the currentvoltage relationship by scaling the peak current (I) by the net driving force using the equation g = I/(Vt - Erev), where Vt is the test potential. For steady state inactivation (h
), we recorded the current in response to a test depolarization to -20 mV (Itest), which immediately followed a 500-ms prepulse to a range of voltages. h
was estimated as a function of the prepulse voltage by the ratio Itest/I, where I is the current measured in the absence of a prepulse. Steady state gating parameters were estimated by fitting data to the Boltzmann functions using the Marquardt-Levenberg algorithm in a nonlinear-squares procedure: m
or h
= 1/{1 + exp[(Vt - V1/2)/k]}, where Vt is the test potential, V1/2 is the half point of the relationship, and k (= RT/zF) is the slope factor.
Data reported are mean ± SEM. Statistical significance was determined using paired Student's t test at the 5% level.
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RESULTS |
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Changes in Cd 2+ Sensitivity of Na+ Channels by Cysteine Substitutions of P-S6 Linker Charged Residues
We assessed the side-chain accessibility of P-S6 residues to the aqueous phase by examining the Cd2+ sensitivity of single cysteine mutants. 12 of 16 single cysteine mutants expressed functional channels. D1248C, R1250C, K1252C, and E1259C did not express in 510 rounds of transfection, with and without exposure to 10 mM dithiothreitol to exclude a spontaneous internal disulfide bridge that might render the channels nonconducting () in four instances (Table 1). Given the small magnitude of these changes, we next turned our attention to permeation. Figure 2 summarizes the half-blocking concentration for Cd2+ of each of the functional cysteine mutants. All mutated channels but three (K415C, E1251C, E1254C) showed enhanced Cd2+ sensitivity (P < 0.05) when compared with WT rSkM1 channels. Because Cd2+ is presumably binding to the introduced sulfhydryls, thereby blocking Na+ flux through the pore physically and/or electrostatically, the observation of enhanced Cd2+ block indicates that the side chains of these residues line the aqueous lumen of the pore (
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Modification by MTS Reagents
One explanation for the unaltered Cd2+ sensitivity of K415C, E1251C, and E1254C is that the side chains of these residues are buried within the channel protein and are not exposed to the aqueous phase. However, it is also possible that Cd2+ indeed binds to the substituted cysteines of these "Cd2+-insensitive" mutants, but that such binding does not reduce Na+ flux due to the relatively small size of Cd2+ as a blocker (ionic radius = 1.1 Å). To distinguish between these possibilities, we employed the hydrophilic sulfhydryl-reactive methanethiosulfonate derivatives MTSEA (positively charged) and MTSES (negatively charged) as molecular probes. These agents introduce bulky adducts via a mixed disulfide bond (MTSEA, 66 Å3; MTSES, 90 Å3) such that successful modification of an accessible substituted cysteinyl near the pore is more likely to influence permeation (
Figure 3 summarizes the effects of MTS reagents on peak sodium currents (INa) of WT and cysteine mutant channels. In these experiments, saturating concentrations of MTS reagents (2.5 mM MTSEA or 10 mM MTSES) were applied to the channels by external perfusion for 1015 min followed by washout. Consistent with previous reports, WT channels were modified by neither MTSEA nor MTSES, indicating that an accessible cysteine is required for these agents to be effective (
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Figure 4 depicts the time course of sulfhydryl modification of cysteine-substituted channels upon addition of MTSEA or MTSES. Representative current traces before () and after () modifications are also shown (Figure 4, left). Application of MTSEA (Figure 4 A) or MTSES (B) decreased or enhanced INa of E765C channels, respectively. MTS modifications were irreversible even after extensive washout of the reagents (510 min with ~50 ml control bath solution). To further verify that sulfhydryl modification was complete, we also examined the sensitivity of INa to Cd2+ blockade after treatment with MTSES, since Cd2+ is known to bind with much higher affinity to free sulfhydryls than to oxidized sulfhydryls (
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Interestingly, application of MTSEA to the Cd2+-insensitive K415C channels led to complete elimination of sodium current (Figure 4 C), suggesting that this residue is indeed accessible from the external medium. In contrast, the addition of MTSES to this construct did not affect INa (Figure 4 D). Unlike K415C, the Cd2+-insensitive E1251C and E1254C channels were not modified by either MTSEA or MTSES. It is possible that MTS agents may have reacted with the substituted cysteines of these channels without producing any functional consequences. However, we are unable to distinguish these changes from side-chain effects per se. Cd2+ sensitivity of these mutants after MTS modification was not assessed as they were by themselves insensitive to Cd2+ block (Figure 2).
Single-Channel Conductance
One putative role of the superficial negative charges studied in this report is that these residues may constitute another outer cluster of vestibular charge that functions to increase the local effective Na+ concentration at the external pore mouth, thereby supplementing the rings of charge closer to the selectivity filter (
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Fractional Electrical Distances for Cd2+ Binding of Cd2+-sensitive Cysteine Mutants
The voltage dependence of unitary current blockade by Cd2+ has been used to estimate the relative depths of substituted cysteines in the pore () for Cd2+ block of Cd2+-sensitive mutants. The addition of Cd2+ to R411C, E765C, E1253C, and E1551C channels led to rapid unresolved blocking events appearing as reductions in unitary current (Figure 5 A). Figure 5 B shows the corresponding currentvoltage relationships recorded in the presence of Cd2+ (
). Logarithmic plots of the ratio of unblocked and blocked unitary current amplitudes of these channels as a function of the membrane potential allows estimation of their electrical distances (Figure 5 C) (
) are summarized in Figure 6.
values of selected pore residues that are known to be located deeper in the pore close to the selectivity filter region (domain I: E403; II: I757, E758; III: D1241; IV: D1532) are also shown for reference (
;
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Ionic Selectivity
Certain P-loop residues have been identified as critical determinants of ionic selectivity (
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DISCUSSION |
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We have previously combined electrophysiological and mutagenesis techniques to explore functional and topological features of the Na+ channel pore on both the amino- and carboxyl-terminal sides of the immediate putative DEKA "selectivity" ring. In brief, we demonstrated that the Na+ channel pore structure is highly asymmetrical (
Accessibility of Cysteine Mutants to Cd2+ and MTS Reagents
All of the functional P-S6 linker cysteine mutants studied but three exhibited heightened sensitivity to current blockade by the group IIB metal Cd2+ relative to WT. However, these Cd2+-sensitive mutants (two- to fivefold enhancements) were generally not as sensitive as those located putatively deeper in the pore that, when mutated to cysteine, often display 10100-fold increased sensitivity (
Reductions in INa by MTSEA modification of many mutants and the increase in INa by MTSES observed in D762C, E765C, and D1547C channels could result from simple electrostatic effects on the permeation pathway as a result of charge restoration or reversal, respectively; in contrast, the complete elimination of current by MTSEA modification (a charge restoration) of R411C, K415C, and R1558C and the lack of effects of MTSES (a charge reversal) on INa of these constructs cannot be explained by simple electrostatic theory. In addition, the differential responses (or lack thereof) of negatively charged neutralized mutants other than D762C, E765C, and D1547C to MTSES, despite the susceptibility of the same mutants to the smaller MTSEA, also require more complex interpretations, as discussed below.
Covalent modification of channel proteins by MTS compounds with alteration of the current magnitude is dependent on a number of factors, including the size and charge of the agent (
Cysteine scanning mutagenesis has the advantage of allowing assessment of side-chain accessibility as well as post-translational protein modifications at specific sites. However, this technique also makes certain basic assumptions that critically influence data interpretation. Firstly, it is assumed that the side chain of the substituted cysteine lies in an orientation similar to that of the native wild-type residue. Addition of aqueous-limited sulfhydryl-specific modifying agents should therefore react more readily with ionized cysteine sulfhydryls exposed to the aqueous phase (i.e., the lumen of the channel) than with nonionized sulfhydryls buried within the lipid membrane or protein. Any changes in current or channel function upon such reaction are then used as an indication of whether the residue in question is accessible. Nevertheless, it is possible that application of sulfhydryl modifiers could result in trapping of "abnormal" or atypical channel states. Also, successful modification may not necessarily lead to changes in function, as mentioned earlier. Cysteine-scanning mutagenesis also assumes that amino acid replacements do not result in global or nonspecific alterations of the structure and function of the protein of interest and that any elevation in Cd2+ sensitivity of the substituted channels arises entirely from the inserted cysteine. However, mutations may expose endogenous cysteine(s) that is (are) inaccessible in the native channel, which in turn may underlie changes in sensitivity to Cd2+ blockade and sulfhydryl modification observed in some mutant channels (
Functional Roles in Ion Permeation
The Na+ channel pore is known to contain two rings of charge: an inner NH2-terminal or DEKA ring (I:D400, II:E755, III:K1237, and IV:A1529 in rSkM1) and an outer COOH-terminal ring (I:E403, II:E758, III:D1241, and IV:D1532 in rSkM1). These charge rings are separated by three to four neutral residues in the ascending portion of the P loops or the so-called SS2 region (
Structural Inferences from Single-Channel Recordings
The electrical distances of domain II pore residues reveal a striking pattern: they ascend (I757 and E758), and then descend (D762 and E765) back into the pore. Assuming that no significant structural or conformational changes of the pore are induced by the mutations and upon Cd2+ binding to the substituted cysteine, one possibility for this observation is that this region of the pore (i.e., the DII linker) may reverse direction and dip back into the membrane. Figure 7 demonstrates a schematic representation of such possible orientations of the domain II P-S6 linker. This could occur by forming a partial ring that extends horizontally at a tilted angle. One should, however, recognize that electrical distances do not directly translate into physical distances, particularly in regions where the transmembrane electric field gradient is not linear. Nevertheless, our data raise new possibilities about the local topology of the domain II P segment since many of the previous pore mutations studied in this domain either did not express or were inaccessible, making its topology relatively uncertain (
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Similar to DII, residues in DIV also show a similar "reverse" pattern. Residues in this segment ascend (D1532, D1545, and D1547), and then descend (E1551 and R1558). Further mutagenesis experiments are required to determine whether the same pattern is also observed in domains I and III. It is also noteworthy that only a total of nine residues (from I757 to E765) in DII span an electrical distance of ~0.15, whereas 27 residues (from D1532 to R1558) in DIV span a relatively short electrical distance of ~0.08, suggesting the former are more extended. Such domain-specific topological arrangements provide further evidence for the asymmetrical structure of the Na+ channel pore (
Contribution to Gating
The Na+ channel outer pore may undergo conformational changes in some forms of slow inactivation (
Toxin Pharmacology
Guanidinium toxins such as tetrodotoxin (TTX), saxitoxin, and µ-conotoxin (µ-CTX), whose 3-D structures are known, are useful molecular tools to investigate the Na+ channel pore structure. These toxins are site I Na+ channel blockers that block Na+ ion flux by physically occluding the pore (
Summary
In summary, the negatively charged residues located in the P-S6 linkers are critical for determining the wild-type channel conductance, possibly by enriching the local effective Na+ concentration at the external pore mouth. These residues also play significant roles in toxin binding and modulation of channel gating. We conclude that this unexplored outermost region, previously thought to be remote from the pore, contributes significantly to both structural and functional properties of the Na+ channel.
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Footnotes |
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1 Abbreviations used in this paper: D-I, -II, -III, and -IV, domains I, II, III, and IV; MTS, methanethiosulfonate; MTSEA, MTS ethylammonium; MTSES, MTS ethylsulfonate; P-S6 linker, P loopS6 linker; WT, wild type.
Dr. Vélez's current address is Department of Physiology, Faculty of Sciences, University of Valparaíso, Valparaíso, Chile. Dr. Chiamvimonvat's current address is Division of Cardiology, University of Cincinnati, Cincinnati, OH 45267-0542.
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Acknowledgements |
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We thank Ailsa Mendez-Fitzwilliam and Dr. Irene Ennis for construction of sodium mutant channels.
This work was supported by the National Institutes of Health (P50 HL-52307 to E. Marbán and R01 HL-50411to G.F. Tomaselli). R.A. Li is the recipient of a fellowship award from the Heart and Stroke Foundation of Canada. E. Marbán holds the Michel Mirowski, M.D., Professorship of Cardiology of the Johns Hopkins University.
Submitted: 8 October 1999
Revised: 3 December 1999
Accepted: 6 December 1999
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References |
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---|
Akabas, M.H., Kauffmann, C., Archdeacon, P., Karlin, A. 1994a. Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the alpha-subunit. Neuron. 13:919-927[Medline].
Akabas, M.H., Kauffmann, C., Cook, T.A., Archdecon, P. 1994b. Amino acid residues lining the chloride channel of the cystic fibrosis transmembrane regulator. J. Biol. Chem. 269:14865-14868
Backx, P., Yue, D., Lawrence, J., Marban, E., Tomaselli, G. 1992. Molecular localization of an ion-binding site within the pore of mammalian sodium channels. Science 257:248-251[Medline].
Balser, J.R., Nuss, H.B., Chiamvirnonvat, N., Perez-Garcia, M.T., Marban, E., Tomaselli, G.F. 1996. External pore residue mediates slow inactivation in µl rat skeletal muscle sodium channels. J. Physiol. 494:431-442[Abstract].
Benitah, J.P., Tomaselli, G.F., Marban, E. 1996. Adjacent pore-lining residues within sodium channels identified by paired cysteine replacements. Proc. Natl. Acad. Sci. USA. 93:7392-7396
Benitah, J.P., Ranjan, R., Yamagishi, T., Janecki, M., Tomaselli, G.F., Marban, E. 1997. Molecular motions within the pore of voltage-dependent sodium channels. Biophys. J 73:603-613[Abstract].
Benitah, J.P., Chen, Z., Balser, J.R., Tomaselli, G.F., Marban, E. 1999. Molecular dynamics of the sodium channel pore vary with gating: interactions between P-segment motions and inactivation. J. Neurosci. 19:1577-1585
Catterall, W.A. 1988. Structure and function of voltage-sensitive ion channels. Science. 242:50-61[Medline].
Chiamvimonvat, N., Pérez-García, M.T., Tomaselli, G.F., Marban, E. 1996a. Control of ion flux and selectivity by negatively charged residues in the outer mouth of rat sodium channels. J. Physiol. 491:51-59[Abstract].
Chiamvimonvat, N., Perez-Garcia, M., Ranjan, R., Marban, E., Tomaselli, G.F. 1996b. Depth asymmetries of the pore-lining segments of the sodium channel revealed by cysteine mutagenesis. Neuron. 16:1037-1047[Medline].
Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J. 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch. 391:85-100[Medline].
Heinemann, S.H., Terlau, H., Stühmer, W., Imoto, K., Numa, S. 1992a. Calcium channel characteristics conferred on the sodium channel by single mutations. Nature. 356:441-443[Medline].
Heinemann, S.H., Terlau, H., Imoto, K. 1992b. Molecular basis for pharmacological differences between brain and cardiac sodium channels. Pflügers. Arch. 422:90-92.
Hille, B. 1992. Ionic Channels of Excitable Membranes. 2nd ed Sunderland, MA, Sinauer Associates Inc, pp. 195.
Holloway, S.F., Salgado, V.L., Wu, C.H., Narahashi, T. 1989. Kinetic properties of single sodium channels modified by fenvalerate in mouse neuroblastoma cells. Pflügers Arch. 414:613-621.
Johns, D.C., Nuss, H.B., Marban, E. 1997. Suppression of neuronal and cardiac transient outward currents by viral gene transfer of dominant-negative Kv4.2 constructs. J. Biol. Chem. 272:31598-31603
Kürz, L.L., Zühlke, R.D., Zhang, H.-J., Joho, R.H. 1995. Side-chain accessibilities in the pore of a K+ channel probed by sulfhydryl-specific reagents after cysteine-scanning mutagenesis. Biophys. J. 68:900-905[Abstract].
Lancelin, J.M., Knoda, D., Tate, S., Yanagawa, Y., Abe, T., Satake, M., Inagaki, F. 1991. Tertiary structure of conotoxin GIIIA in aqueous solution. Biochemistry. 30:6908-6916[Medline].
Li, R.A., Tsushima, R.G., Backx, P.H. 1997. Critical pore residues for µ-conotoxin binding to rat skeletal muscle Na+ channel. Biophys. J. 73:1874-1884[Abstract].
Li, R.A., Tsushima, R.G., Himmeldirk, K., Dime, D.S., Backx, P.H. 1999a. Local anesthetic anchoring to cardiac sodium channels. Implications into tissue-selective drug targeting. Circ. Res. 85:88-98
Li, R.A., Velez, P., Chiamvimonvat, N., Tomaselli, G.F., Marban, E. 1999b. Modeling of the Na+ channel pore based on mutagenesis, covalent modification and toxin sensitivity of charged residues in the outer vestibule. Circulation. 100:I-277.
Liu, Y., Jurman, M.E., Yellen, G. 1996. Dynamic rearrangement of the outer mouth of a K+ channel during gating. Neuron. 16:859-867[Medline].
Marbán, E., Yamagishi, T., Tomaselli, G.F. 1998. Structure and function of voltage-gated sodium channels. J. Physiol. 508:647-657
Mikala, G., Bahinski, A., Yatani, A., Tang, S., Schwartz, A. 1993. Differential contribution by conserved glutamate residues to an ion-selectivity site in the L-type Ca2+ channel pore. FEBS Lett. 335:265-269[Medline].
Noda, M., Suzuki, H., Numa, S., Stühmer, W. 1989. A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel-II. FEBS Lett. 259:213[Medline].
Pascual, J.M., Shieh, C.-C., Kirsch, G.E., Brown, A.M. 1995. K+ pore structure revealed by reporter cysteines at inner and outer surfaces. Neuron. 14:1055-1063[Medline].
Perez-Garcia, M.T., Chiamvimonvat, N., Marban, E., Tomaselli, G.F. 1996. Structure of the sodium channel pore revealed by serial cysteine mutagenesis. Proc. Natl. Acad. Sci. USA. 93:300-304
Pusch, M., Noda, M., Stühmer, W., Numa, S., Conti, F. 1991. Single point mutations of the sodium channel drastically reduce the pore permeability without preventing its gating. Eur. Biophys. J. 20:127-133[Medline].
Satin, J., Kyle, J.W., Chen, M., Bell, P., Cribbs, L.L., Fozzard, H.A., Rogart, R.B. 1992. A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties. Science. 256:1202-1205[Medline].
Sunami, A., Lipkind, G., Glaaser, I.W., Fozzard, H.A. 1999. Characterizing structural rearrangement of the sodium channel outer vestibule induced by S6 mutants. Biophys. J. 76:A8.
Terlau, H., Heinemann, S.H., Stühmer, W., Pusch, M., Conti, F., Imoto, K., Numa, S. 1991. Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II. FEBS Lett. 293:93-96[Medline].
Tomaselli, G.F., Nuss, H.B., Balser, J.R., Perez-Garcia, M.T., Kluge, K., Orias, D.W., Backx, P.H., Marban, E. 1995. A mutation in the pore of the sodium channel alters gating. Biophys. J. 68:1814-1827[Abstract].
Torchinsky, Y.M. 1981. Sulfur in Proteins. Oxford, UK, Pergamon Press, pp. 198.
Tsushima, R.G., Li, R.A., Backx, P.H. 1997a. Altered ionic selectivity of Na+ channel revealed by cysteine mutagenesis. J. Gen. Physiol. 109:1-13
Tsushima, R.G., Li, R.A., Backx, P.H. 1997b. P-loops of Na+ channels are highly flexible structures. J. Gen. Physiol. 110:59-72
Trimmer, J.S., Cooperman, S.S., Tomiko, S.A., Zhou, J., Crean, S.M., Boyle, M.B., Kallen, R.G., Sheng, Z., Barchi, R.L., Sigworth, F.J. et al. 1989. Primary structure and functional expression of a mammalian skeletal muscle sodium channel. Neuron. 3:33-49[Medline].
Woodhull, A.M. 1973. Ionic blockade of sodium channels in nerve. J. Gen. Physiol. 61:687-708
Yamagishi, T., Janecki, M., Marban, E., Tomaselli, G.F. 1997. Topology of the P segments in the sodium channel pore revealed by cysteine mutagenesis. Biophys. J. 73:195-204[Abstract].