Correspondence to: Gordon F. Tomaselli, Department of Medicine, Division of Cardiology, The Johns Hopkins University School of Medicine, 844 Ross Building, Baltimore, MD 21205. Fax:(410) 955-7953 E-mail:gtomasel{at}jhmi.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The permeation pathway of the Na channel is formed by asymmetric loops (P segments) contributed by each of the four domains of the protein. In contrast to the analogous region of K channels, previously we (Yamagishi, T., M. Janecki, E. Marban, and G. Tomaselli. 1997. Biophys. J. 73:195204) have shown that the P segments do not span the selectivity region, that is, they are accessible only from the extracellular surface. The portion of the P-segment NH2-terminal to the selectivity region is referred to as SS1. To explore further the topology and functional role of the SS1 region, 40 amino acids NH2-terminal to the selectivity ring (10 in each of the P segments) of the rat skeletal muscle Na channel were substituted by cysteine and expressed in tsA-201 cells. Selected mutants in each domain could be blocked with high affinity by externally applied Cd2+ and were resistant to tetrodotoxin as compared with the wild-type channel. None of the externally applied sulfhydryl-specific methanethiosulfonate reagents modified the current through any of the mutant channels. Both R395C and R750C altered ionic selectivity, producing significant increases in K+ and NH4+ currents. The pattern of side chain accessibility is consistent with a pore helix like that observed in the crystal structure of the bacterial K channel, KcsA. Structure prediction of the Na channel using the program PHDhtm suggests an helix in the SS1 region of each domain channel. We conclude that each of the P segments undergoes a hairpin turn in the permeation pathway, such that amino acids on both sides of the putative selectivity filter line the outer mouth of the pore. Evolutionary conservation of the pore helix motif from bacterial K channels to mammalian Na channels identifies this structure as a critical feature in the architecture of ion selective pores.
Key Words: pore helix, cysteine mutagenesis, tetrodotoxin, methanethiosulfonates, cadmium
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In voltage-gated ion channels the pore-lining (P)* segments in each of four internally homologous domains (Na and Ca channels) or subunits (K channels) have been proposed to form hairpins that partially traverse the membrane and together create an ion-selective pore ( helices or ß strands (
|
As might be predicted by the sequence divergence of the P segments in each domain of the Na channel, the contribution of each of the segments to the formation of the pore is not equivalent (
The crystal structure of the pore of a bacterial K channel (KcsA) exhibits several features predicted to be conserved across the superfamily of voltage-gated ion channels ( helix-turn-coil motif described in the KcsA structure (
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutagenesis and Channel Expression
Mutagenesis of the expression plasmid containing the rat skeletal muscle (µ1) subunit of the Na channel was performed as described previously (
subunit cDNA (
subunit, 1 µg of the ß1 subunit, and 0.3 µg of the green fluorescent protein cDNA-containing plasmids per 35-mm dish.
Electrophysiology and Data Analysis
Transfected cells were identified by epifluorescence microscopy and membrane currents were recorded with the whole-cell configuration of patch clamp ( tip resistances. Currents were recorded using a patch-clamp amplifier (model Axopatch 200A; Axon Instruments) interfaced to a personal computer. Cell capacitance was calculated by integrating the area under an uncompensated capacity transient elicited by a 20-mV hyperpolarizing test pulse from a holding potential of -80 mV. Series resistance was then compensated as much as possible without ringing, typically 7090%. Given the average series resistance of our electrodes, the maximal uncompensated voltage error was <|6 mV| for the largest currents studied. Whole-cell currents were recorded in a bath solution containing (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.4. The pipette solution was (in mM): 35 NaCl, 105 CsF, 1 MgCl2, 10 HEPES, and 1 EGTA, pH 7.2. Doseresponse curves for block of the mutant channels by extracellular Cd2+ were determined by adding the chloride salt of the blocker to the bath at concentrations between 1 µM and 5 mM, and TTX was added at concentrations between 1 nM and 10 µM, depending on the sensitivity of the particular mutant. Full current-voltage relationships were determined at each Cd2+ or TTX concentration. The half-blocking concentration (IC50) was determined by a least-squares fit (Levenberg-Marquardt algorithm; Microcal Origin) of the data to the function: I/Io = 1/{1 + ([blocker]/IC50)n}, where I and Io are the currents in the presence and absence of blocker, respectively. In nearly all cases, block by Cd2+ and TTX was reversible, when the current amplitude did not return to >90% of control channel run down could not be excluded and the experiments were not included in the analysis.
Susceptibilities to extracellular methanethiosulfonate ethylammonium (MTSEA), methanethiosulfonate ethyltrimethylammonium (MTSET), methanethiosulfonate ethylsulfonate (MTSES; Toronto Research Chemicals) modification was determined by bath application of saturating concentrations of each reagent (2.5 mM MTSEA, 1 mM MTSET, and 5 mM MTSES). MTS modification of the current was verified by the irreversibility of the change in the current amplitude. In no case was there an irreversible change in current amplitude after exposure to MTS reagents.
Single-channel recording was performed in those mutant channels that exhibited sensitivity to block by Cd2+ at the whole-cell level. The cell-attached configuration of the patch clamp was used (
Pooled data are presented as means ± SD with at least three determinations of the IC50 for block by Cd2+ and TTX with the exceptions noted as follows. The wild-type IC50 for Cd2+ block was 2.5 ± 0.2 mM. The mutant channels S387C, W388C, L393C, F742C, H743C, S744C, F749C, G1224C, G1516C, and C1521A exhibited no difference in the IC50 compared with the wild-type channel in a single determination that was confirmed by the absence of 50% block of the peak current by 0.5 mM Cd2+ in at least two additional cells. The wild-type IC50 for TTX block was 17 ± 5 nM. The mutant channels S387C, W388C, A389C, F390C, L391C, F742C, H743C, S744C, S1229C, L1230C, Q1232C, G1516C, N1517C, S1518C, C1521A, F1523C, E1524C, and I1525C exhibited no difference in the IC50 compared with the wild-type channel in a single determination that was confirmed by the greater 50% block of the peak current by 50 nM Cd2+ in at least two additional cells. Statistical comparisons were made using an analysis of variance (ANOVA) with P < 0.05 considered to be significant.
Molecular Modeling
Each homologous domain of the Na channel was evaluated by the program PHDhtm (http://www.embl-heidelberg.de/predictprotein), which is a neural network system that predicts the locations of transmembrane helices in integral membrane proteins (
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Modeling Predicts Pore Helices in the Na Channel
We tested the SS1 region of the Na channel P segments for helical content using PHDhtm, a neural network based program that detects transmembrane helices with 95% accuracy (
helices involving the italicized residues in the KcsA pore and the P segments of each Na channel domain (Fig 1). Based on the crystal structure, the second of the two vicinal tryptophans (W68), E71 and T72 in KcsA, are oriented with their side chains accessible to the channel pore (
1C subunit of the Ca channel (unpublished data). Several motifs in the putative helical regions of the P segments of the Na channel are conserved from the KcsA pore helix: a threonine residue one to two positions NH2-terminal to the selectivity filter (except domain II), and conserved aromatic and charged residues in the pore helix. We sought to experimentally test the hypothesis that the regions proximal to the selectivity filter in the Na channel are helical.
Accessibility Pattern to Cd 2+, MTS Reagents, and TTX
Fig 1 shows the positions in the Na channel pore studied here. The rat µ1 channel P segment sequences are aligned with the bacterial inward-rectifier channel (KcsA). The residues that form the turret and pore helix of the KcsA channel are designated (
The permeation phenotype of each expressing mutant channel was characterized by determination of the IC50 for block by Cd2+ and TTX, accessibility to modification by MTS reagents, and peak current size with cationic substitutions. Fig 2 shows the sensitivity to externally applied Cd2+ of wild-type and mutant Na channels. Eight mutants had enhanced sensitivity to block by Cd2+ compared with the wild-type channel: F390C and R395C in domain I; R750C, L746C, and F745C in domain II; Y1227C and L1228C in domain III; and I1519C in domain IV (Fig 2 A). It is notable that the mutated residues in the same P segments that exhibit enhanced sensitivity to Cd2+ are either adjacent to one another or separated by three to four amino acids. The easy reversibility of Cd2+ block and the relatively low, micromolar affinity of sensitive channels suggests that single cysteine residues participate in the divalent cation block.
|
Mutations in the pore of the Na channel also alter sensitivity to block by guanidinium toxins. The toxin molecule is much larger than Cd2+, with more points of contact with amino acids in the channel pore. The IC50 for TTX of the wild-type µ1 channel expressed in tsA201 cells is 17 ± 0.5 nM, comparable to that in the oocyte expression system (
MTS reagents selectively modify free cysteinyl side chains in an aqueous environment. External application of saturating concentrations of MTS reagents produced no significant effect on the current through any of the mutant channels; data from R395C are summarized in Fig 3. To test the MTS accessibility from the inside of the channel, MTSEA or MTSET was applied to the cytoplasmic face of F394C, R395C, and L396C (domain I) and I751C and R750C (domain II). Internal MTS reagents had no effect on the mutant channels (unpublished data).
|
The lack of effect of MTS reagents may result from an inability of the reagents to form a mixed thiol. Alternatively, the cysteinyl may be modified, but the adduct does not block the permeation pathway and, therefore, does not affect current amplitude. To distinguish between these possibilities, we measured sensitivity to Cd2+ block before and after application of MTS reagents. Fig 3 shows representative experiments for R395C. Application of 1 mM Cd2+ blocks 60% of the current; the subsequent administration of MTSEA, MTSET, and MTSES does not alter the current amplitude nor does it modify the sensitivity to subsequent applications of Cd2+ (
Single-channel Analysis
The voltage dependence of blockade of the unitary current by external Cd2+ was used to determine the fractional electrical distance of divalent cation binding. The fractional electrical distance for Cd2+ block of cysteine mutants provides the relative location of the substituted thiol in the channel pore. Fig 4 (top) shows unitary Na+ currents for R395C and R750C, Cd2+-sensitive mutants with robust whole-cell expression, in the presence and absence of Cd2+. In the absence of Cd2+, the single-channel conductances of R395C and R750C differed from the wild-type (50 ± 0.3 pS in 140 mM [Na+]o) by <30%. In the presence of Cd2+, block is rapid and, therefore, appears as a reduction in the single-channel current amplitude. The voltage dependence of block is evident from the change in single-channel current through the mutant channels in the presence and absence of Cd2+ over a wide voltage range (Fig 4, middle). In each case, the reduction in unitary current is more prominent at negative voltages. By plotting the ratio of the blocked and unblocked unitary currents and assuming a single-site model for Cd2+ binding (
|
Ion Selectivity of the Mutant Na Channels
Several residues in both the third (K1237) and fourth domain (G1530, W1531, and D1532) P segments of the Na channel influence ionic selectivity (
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The P segments contain the major determinants of permeation in voltage-gated ion channels. In the Na channel, the DEKA ring is a central, but not the only element that mediates conductance and selectivity (
The role of the residues NH2-terminal to the DEKA ring (often referred to as the SS1 region) in the formation of the outer pore mouth is unknown. In the KcsA channel, the analogous region forms an -helical structure referred to as the pore helix. Side chain accessibility to the aqueous phase of SS1 residues was gauged by a combination of cysteine substitution mutagenesis, and sensitivity to block or modification by hydrophilic, thiol-specific modifying reagents. The probes used provide complementary structural information. Alterations in external Cd2+ block sensitivity and MTS modification of the current imply side chain accessibility in the outer mouth of the pore. Modest changes in TTX block may be associated with global changes in pore structure with retained specific channeltoxin interaction sites. Dramatic changes in toxin affinity are more likely to result if a specific channeltoxin interaction is disrupted (
In each domain, at least one substituted thiol side chain is accessible to the extracellular pore. In domain I, the side chains of both F390C and R395C (separated by four residues) are accessible. In domain II, F745C, L746C, and R750C exhibit side chain accessibility. In domain III, Y1227C and L1228C have enhanced sensitivity to Cd2+ block, whereas G1226C and L1231C have an indeterminate phenotype since they do not express functional channels. Similarly, in domain IV, the mutant I1519C exhibits side chain accessibility to Cd2+ and is three residues NH2-terminal from the nonfunctional mutant L1522C. In the context of previous mutagenesis data in adjacent regions of the P segments ( helices.
|
Helical wheel representations of the SS1 regions demonstrate that the accessible or indeterminate side chains lie on the same face of the predicted helices with the exception of G1226 (Fig 6). The secondary structure of amino acid residues on the NH2-terminal side of DEKA are distinct from the random coil structure predicted by mutagenesis of amino acids on the COOH-terminal side of the ring (
The detailed pattern of accessibility differs for each of the residues that alter channel interaction with the structural probes. The mutations R395C (domain I), F745C, R750C (domain II), and L1228C (domain III) increase the affinity for Cd2+ block and are resistant to TTX. However, external application of MTS reagents does not alter the current through these mutant channels. R395C and R750C, but not F745C or l1228C, alter cation selectivity of the channel (Fig 5). R395 and R750 are at homologous positions by sequence alignment in domains I and II, respectively. It is possible that they form a charge ring in the external pore that alters permeation. However, R395C and R750C do not effect permeation by a simple electrostatic mechanism. Neutralization of positive charges in the external pore would be expected to increase channel conductance; neither mutant increases conductance compared with wild type, in fact, R750C is 30% smaller than R395C or wild type (Fig 4). A simple electrostatic mechanism cannot explain the change in ion selectivity observed with these two mutations (Fig 5). Furthermore, an electrostatic impediment to TTX binding is inconsistent with the data: a reduction in the net positive charge of the pore, all else being equal, should increase TTX sensitivity. Instead, these two charge-reducing mutations decrease the channel sensitivity to block by TTX, perhaps by changing the interaction of other toxin channel contact sites (
Another mutation that alters both Cd2+ and TTX sensitivity is replacement of aromatic phenylalanine in domain II (F745C). Both mutations in domain II that alter Cd2+ sensitivity (F745C and R750C) decrease TTX affinity by nearly two orders of magnitude. If F745 and R750 participate in the formation of a pore helix, the cysteine substitutions at these positions disrupt an important interaction between the channel and toxin. F745C produces no change in channel selectivity and is not modified by MTS reagents. Similarly, L1228C increases affinity for Cd2+ block and makes the channel resistant to TTX block. The magnitude of the destabilization of TTX block is similar to that exhibited by the domain I mutant R395C and less than that of the domain II mutants F745C and R750C. The remainder of the cysteine mutations, Y1227C (domain III) and I1519C (domain IV) increase Cd2+ affinity without affecting toxin block or MTS binding.
The IC50 for Cd2+ block of all of the mutants is not affected by prior addition of MTS reagents. This suggests that the cysteine mutants rather than being modified but not altering current are, instead, not modified by MTS reagents. These data imply that some parts of the P segments in the external pore are not accessible to modification by external MTS reagents, which require a reaction sphere of 6 A.
The pattern of side chain accessibility in the pore helix region has been studied in a number of K channels. As in the case of the Na channel, and as predicted by the KcsA structure, hydrophobic residues at the NH2-terminal side of the pore helix appear to have their side chains exposed using Ag+ (
Summary
Our data demonstrate that the P segments on the NH2-terminal side of the DEKA residues of the Na channel participate in formation of the outer pore. The P segments do not span the selectivity filter; none of the cysteine substitutions are accessible from the internal mouth of the channel. The pattern of side chain accessibility is consistent with pore helices similar to that seen in the KcsA channel pore crystal structure and side chain accessibility in other K channels. Two of the mutations in domain II (F745C and R750C) that alter Cd2+ block dramatically reduce TTX sensitivity, and may be sites of direct toxinchannel interaction. The data are consistent with previous studies that demonstrate the importance of domains I and II in guanidinium toxin binding and block (
Our data and previous work ( helix-turn-coil motif (Fig 6, bottom). The precise location of the end of the helix and beginning of the turn are not defined; however, modeling predicts that 810 amino acids preceding the selectivity filter residue in each domain, have helical character. Overall, this region is hydrophobic, and helical packing is likely to be thermodynamically favorable for this water-accessible part of the protein. The preference for acidic residues to flank the NH2 terminus and basic residues the COOH terminus (
helices may extend beyond the region studied; however, in domains III and IV, this would mean the incorporation of one or more glycines into the helices.
The KcsA channel appears to be an evolutionary forerunner of the voltage-dependent family of K channels (
![]() |
Footnotes |
---|
* Abbreviations used in this paper: DEKA, Na channel pore resi-dues D400, E755, K1237, and A1529; MTS, methanethiosulfonate; MTSEA, methanethiosulfonate ethylammonium; MTSES, methanethiosulfonate ethylsulfonate; MTSET, methanethiosulfonate ethyltrimethylammonium; P, pore-lining; TTX, tetrodotoxin.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We thank Ailsa Mendez-Fitzwilliam for construction of the mutants.
This work was supported by the National Institutes of Health (R01 HL50411 and R01 HL52376). R.A. Li is supported by 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 January 2001
Revised: 14 May 2001
Accepted: 8 June 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Benitah, J.P., Tomaselli, G.F., and Marban, E. 1996. Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis. Proc. Natl. Acad. Sci. USA. 93:7392-7396
Benitah, J.P., Ranjan, R., Yamagishi, T., Janecki, M., Tomaselli, G.F., and Marban, E. 1997. Molecular motions within the pore of voltage-dependent sodium channels. Biophys. J. 73:603-613[Abstract].
Catterall, W.A. 1995. Structure and function of voltage-gated ion channels. Annu. Rev. Biochem. 64:493-531[Medline].
Chang, N.S., French, R.J., Lipkind, G.M., Fozzard, H.A., and Dudley, S., Jr. 1998. Predominant interactions between mu-conotoxin Arg-13 and the skeletal muscle Na+ channel localized by mutant cycle analysis. Biochemistry. 37:4407-4419[Medline].
Chen, S., Hartmann, H., and Kirsch, G. 1997. Cysteine mapping in the ion selectivity and toxin binding region of the cardiac Na+ channel pore. J. Membr. Biol. 155:11-25[Medline].
Chiamvimonvat, N., Perez-Garcia, M.T., Ranjan, R., Marban, E., and Tomaselli, G.F. 1996. Depth asymmetries of the pore-lining segments of the Na+ channel revealed by cysteine mutagenesis. Neuron. 16:1037-1047[Medline].
Colquhoun, D., and Sigworth, F.J. 1983. Fitting and statistical analysis of single-channel records. In Sakmann B., Neher E., eds. Single-channel Recording. New York, Plenum Press, 191-263.
Doyle, D.A., Cabral, J.M., Pfuetzner, R.A., Kuo, A., Gulbis, J.M., Cohen, S.L., Chait, B.T., and Mackinnon, R. 1998. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 280:69-77
Dudley, S., Jr., Todt, H., Lipkind, G., and Fozzard, H. 1995. A mu-conotoxin-insensitive Na+ channel mutant: possible localization of a binding site at the outer vestibule. Biophys. J. 69:1657-1665[Abstract].
Favre, I., Moczydlowski, E., and Schild, L. 1995. Specificity for block by saxitoxin and divalent cations at a residue which determines sensitivity of sodium channel subtypes to guanidinium toxins. J. Gen. Physiol. 106:203-229[Abstract].
Favre, I., Moczydlowski, E., and Schild, L. 1996. On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel. Biophys. J. 71:3110-3125[Abstract].
Hamill, O.P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F.J. 1981. Improved patch-clamp techniques for high-resolution current recording form cells and cell-free membrane patches. Pflügers Arch. 391:85-100[Medline].
Heinemann, S., Terlau, H., Stuhmer, W., Imoto, K., and Numa, S. 1992. Calcium channel characteristics conferred on the sodium channel by single mutations. Nature. 356:441-443[Medline].
Heinemann, S.H., Schlief, T., Mori, Y., and Imoto, K. 1994. Molecular pore structure of voltage-gated sodium and calcium channels. Braz. J. Med. Biol. Res. 27:2781-2802[Medline].
Hidalgo, P., and MacKinnon, R. 1995. Revealing the architecture of a K+ channel pore through mutant cycles with a peptide inhibitor. Science. 268:307-310[Medline].
Hsu, K., Amzel, M., Tomaselli, G.F., and Marban, E. 1999. Prediction of pore helices in sodium and calcium channels. Biophys. J. 76:A82.
Isom, L.L., De Jongh, K.S., Patton, D.E., Reber, B.F., Offord, J., Charbonneau, H., Walsh, K., Goldin, A.L., and Catterall, W.A. 1992. Primary structure and functional expression of the beta 1 subunit of the rat brain sodium channel. Science. 256:839-842[Medline].
Kurz, L.L., Zuhlke, R.D., Zhang, H.J., and 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].
Li, R.A., Tsushima, R.G., Kallen, R.G., and Backx, P.H. 1997. Pore residues critical for mu-CTX binding to rat skeletal muscle Na+ channels revealed by cysteine mutagenesis. Biophys. J. 73:1874-1884[Abstract].
Li, R.A., Ennis, I.L., Velez, P., Tomaselli, G.F., and Marban, E. 2000a. Novel structural determinants of mu-conotoxin block in rat skeletal muscle Na+ channels. J. Biol. Chem. 275:27551-27558
Li, R.A., Velez, P., Chiamvimonvat, N., Tomaselli, G.F., and Marban, E. 2000b. Charged residues between the selectivity filter and S6 segments contribute to the permeation phenotype of the sodium channel. J. Gen. Physiol. 115:81-92
Lipkind, G., and Fozzard, H. 1994. A structural model of the tetrodotoxin and saxitoxin binding site of the Na+ channel. Biophys. J. 66:1-13[Abstract].
Lipkind, G.M., and Fozzard, H.A. 2000. KcsA crystal structure as framework for a molecular model of the Na(+) channel pore. Biochemistry. 39:8161-8170[Medline].
Lu, Q., and Miller, C. 1995. Silver as a probe of pore-forming residues in a potassium channel. Science. 268:304-307[Medline].
MacKinnon, R., Cohen, S.L., Kuo, A., Lee, A., and Chait, B.T. 1998. Structural conservation in prokaryotic and eukaryotic potassium channels. Science. 280:106-109
MacKinnon, R., and Yellen, G. 1990. Mutations affecting TEA blockade and ion permeation in voltage-activated K+ channels. Science. 250:276-279[Medline].
Marban, E., Yamagishi, T., and Tomaselli, G.F. 1998. Structure and function of voltage-gated sodium channel. J. Physiol. 508:647-657
Noda, M., Suzuki, H., Numa, S., and Stühmer, W. 1989. A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel II. FEBS Lett. 259:213-216[Medline].
Pascual, J.M., Shieh, C.C., Kirsch, G.E., and Brown, A.M. 1995. K+ pore structure revealed by reporter cysteines at inner and outer surfaces. Neuron. 14:1055-1063[Medline].
Penzotti, J.L., Fozzard, H.A., Lipkind, G.M., and Dudley, S.C., Jr. 1998. Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule. Biophys. J. 75:2647-2657
Perez-Garcia, M., Chiamvimonvat, N., Ranjan, R., Balser, J.R., Tomaselli, G.F., and Marban, E. 1997. Mechanisms of sodium/calcium selectivity in sodium channels probed by cysteine mutagenesis and sulfhydryl modification. Biophys. J. 72:989-996[Abstract].
Perez-Garcia, M.T., Chiamvimonvat, N., Marban, E., and Tomaselli, G.F. 1996. Structure of the sodium channel pore revealed by serial cysteine mutagenesis. Proc. Natl. Acad. Sci. USA. 93:300-304
Phillipson, L.H., Malayev, A., Kuznetkov, A., Chang, C., and Nelson, D. 1993. Functional and biochemical characterization of the human potassium channel Kv1.5 with a transplanted carboxyl-terminal epitope in stable mammalian cell lines. Biochem. Biophys. Acta. 1153:112-121.
Ranganathan, R., Lewis, J.H., and MacKinnon, R. 1996. Spatial localization of the K+ channel selectivity filter by mutant cycle-based structure analysis. Neuron. 16:131-139[Medline].
Rost, B., Casadio, R., Fariselli, P., and Sander, C. 1995. Transmembrane helices predicted at 95% accuracy. Prot. Sci. 4:521-533
Satin, J., Kyle, J., Chen, M., Bell, P., Cribbs, L., Fozzard, H., and Rogart, R. 1992. A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties. Science. 256:1202-1205[Medline].
Strong, M., Chandy, K.G., and Gutman, G.A. 1993. Molecular evolution of voltage-sensitive ion channel genes: on the origins of electrical excitability. Mol. Biol. Evol. 10:221-242[Abstract].
Stuhmer, W. 1991. Structurefunction studies of voltage-gated ion channels. Annu. Rev. Biophys. Biophys. Chem. 20:65-78[Medline].
Sunami, A., Glaaser, I.W., and Fozzard, H.A. 2000. A critical residue for isoform difference in tetrodotoxin affinity is a molecular determinant of the external access path for local anesthetics in the cardiac sodium channel. Proc. Natl. Acad. Sci. USA. 97:2326-2331
Terlau, H., Heinemann, S., Stühmer, W., Pusch, M., Conti, F., Imoto, K., and Numa, S. 1991. Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II. FEBS Lett. 293:93-96[Medline].
Trimmer, J.S., Cooperman, S.S., Tomiko, S.A., Zhou, J.Y., Crean, S.M., Boyle, M.B., Kallen, R.G., Sheng, Z.H., Barchi, R.L., and Sigworth, F.J. et al. 1989. Primary structure and functional expression of a mammalian skeletal muscle sodium channel. Neuron 3:33-49[Medline].
Tsushima, R., Li, R., and Backx, P. 1997a. P-loop flexibility in Na+ channel pores revealed by single- and double-cysteine replacements. J. Gen. Physiol. 110:59-72
Tsushima, R., Li, R., and Backx, P.H. 1997b. Altered ionic selectivity of the sodium channel revealed by cysteine mutations within the pore. J. Gen. Physiol. 109:463-475
Yamagishi, T., Janecki, M., Marban, E., and Tomaselli, G. 1997. Topology of the P segments in the sodium channel pore revealed by cysteine mutagenesis. Biophys. J. 73:195-204[Abstract].
Yellen, G., Jurman, M.E., Abramson, T., and MacKinnon, R. 1991. Mutations affecting internal TEA blockade identify the probable pore-forming region of a K+ channel. Science. 251:939-942[Medline].