Novel Structural Determinants of μ-Conotoxin (GIIIB) Block in Rat Skeletal Muscle (μ1) Na+ Channels
2000; Elsevier BV; Volume: 275; Issue: 36 Linguagem: Inglês
10.1074/jbc.m909719199
ISSN1083-351X
AutoresRonald A. Li, Irene L. Ennis, Patricio Vélez, Gordon F. Tomaselli, Eduardo Marbán,
Tópico(s)Nicotinic Acetylcholine Receptors Study
Resumoμ-Conotoxin (μ-CTX) specifically occludes the pore of voltage-dependent Na+ channels. In the rat skeletal muscle Na+ channel (μ1), we examined the contribution of charged residues between the P loops and S6 in all four domains to μ-CTX block. Conversion of the negatively charged domain II (DII) residues Asp-762 and Glu-765 to cysteine increased the IC50 for μ-CTX block by ∼100-fold (wild-type = 22.3 ± 7.0 nm; D762C = 2558 ± 250 nm; E765C = 2020 ± 379 nm). Restoration or reversal of charge by external modification of the cysteine-substituted channels with methanethiosulfonate reagents (methanethiosulfonate ethylsulfonate (MTSES) and methanethiosulfonate ethylammonium (MTSEA)) did not affect μ-CTX block (D762C: IC50, MTSEA+ = 2165.1 ± 250 nm; IC50, MTSES− = 2753.5 ± 456.9 nm; E765C: IC50, MTSEA+ = 2200.1 ± 550.3 nm; IC50, MTSES− = 3248.1 ± 2011.9 nm) compared with their unmodified counterparts. In contrast, the charge-conserving mutations D762E (IC50 = 21.9 ± 4.3 nm) and E765D (IC50 = 22.0 ± 7.0 nm) preserved wild-type blocking behavior, whereas the charge reversal mutants D762K (IC50 = 4139.9 ± 687.9 nm) and E765K (IC50 = 4202.7 ± 1088.0 nm) destabilized μ-CTX block even further, suggesting a prominent electrostatic component of the interactions between these DII residues and μ-CTX. Kinetic analysis of μ-CTX block reveals that the changes in toxin sensitivity are largely due to accelerated toxin dissociation (k off) rates with little changes in association (k on) rates. We conclude that the acidic residues at positions 762 and 765 are key determinants of μ-CTX block, primarily by virtue of their negative charge. The inability of the bulky MTSES or MTSEA side chain to modify μ-CTX sensitivity places steric constraints on the sites of toxin interaction. μ-Conotoxin (μ-CTX) specifically occludes the pore of voltage-dependent Na+ channels. In the rat skeletal muscle Na+ channel (μ1), we examined the contribution of charged residues between the P loops and S6 in all four domains to μ-CTX block. Conversion of the negatively charged domain II (DII) residues Asp-762 and Glu-765 to cysteine increased the IC50 for μ-CTX block by ∼100-fold (wild-type = 22.3 ± 7.0 nm; D762C = 2558 ± 250 nm; E765C = 2020 ± 379 nm). Restoration or reversal of charge by external modification of the cysteine-substituted channels with methanethiosulfonate reagents (methanethiosulfonate ethylsulfonate (MTSES) and methanethiosulfonate ethylammonium (MTSEA)) did not affect μ-CTX block (D762C: IC50, MTSEA+ = 2165.1 ± 250 nm; IC50, MTSES− = 2753.5 ± 456.9 nm; E765C: IC50, MTSEA+ = 2200.1 ± 550.3 nm; IC50, MTSES− = 3248.1 ± 2011.9 nm) compared with their unmodified counterparts. In contrast, the charge-conserving mutations D762E (IC50 = 21.9 ± 4.3 nm) and E765D (IC50 = 22.0 ± 7.0 nm) preserved wild-type blocking behavior, whereas the charge reversal mutants D762K (IC50 = 4139.9 ± 687.9 nm) and E765K (IC50 = 4202.7 ± 1088.0 nm) destabilized μ-CTX block even further, suggesting a prominent electrostatic component of the interactions between these DII residues and μ-CTX. Kinetic analysis of μ-CTX block reveals that the changes in toxin sensitivity are largely due to accelerated toxin dissociation (k off) rates with little changes in association (k on) rates. We conclude that the acidic residues at positions 762 and 765 are key determinants of μ-CTX block, primarily by virtue of their negative charge. The inability of the bulky MTSES or MTSEA side chain to modify μ-CTX sensitivity places steric constraints on the sites of toxin interaction. μ-conotoxin tetrodotoxin saxitoxin P loop S6 wild-type methanethiosulfonate methanethiosulfonate ethylammonium methanethiosulfonate ethylsulfonate μ-Conotoxin (μ-CTX)1is a receptor site I sodium channel blocker (1Catterall W.A. Science. 1988; 242: 50-61Crossref PubMed Scopus (949) Google Scholar) isolated from the sea snail Conus geographus (2Cruz L.J. Gray W.R. Olivera B.M. Zeikus R.D. Kerr L. Yoshikami D. Moczydlowski E. J. Biol. Chem. 1985; 260: 9280-9288Abstract Full Text PDF PubMed Google Scholar, 3Gray W.R. Olivera B.M. Cruz L.J. Annu. Rev. Biochem. 1988; 57: 665-700Crossref PubMed Scopus (221) Google Scholar, 4Olivera B.M. Rivier J. Clark C. Ramilo C.A. Corpuz G.P. Abogadie F.C. Mena E.E. Woodward S.R. Hilliyard D.R. Cruz L.J. Science. 1990; 249: 257-263Crossref PubMed Scopus (514) Google Scholar) that specifically inhibits Na+ flux in skeletal muscle and eel Na+ channels with high affinity by physically occluding the channel pore (2Cruz L.J. Gray W.R. Olivera B.M. Zeikus R.D. Kerr L. Yoshikami D. Moczydlowski E. J. Biol. Chem. 1985; 260: 9280-9288Abstract Full Text PDF PubMed Google Scholar, 5Moczydlowski E. Olivera B.M. Gray W.R. Strichartz G.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5321-5325Crossref PubMed Scopus (143) Google Scholar, 6Yanagawa Y. Abe T. Satake M. Neurosci. Lett. 1986; 64: 7-12Crossref PubMed Scopus (19) Google Scholar, 7Chen L.-Q. Chahine M. Kallen R.G. Barchi R.L. Horn R. FEBS Lett. 1992; 309: 253-257Crossref PubMed Scopus (77) Google Scholar, 8Dudley Jr., S.C. Hannes T. Lipkind G. Fozzard H.A. Biophys. J. 1995; 69: 1657-1665Abstract Full Text PDF PubMed Scopus (100) Google Scholar, 9Li R.A. Tsushima R.G. Kallen R. Backx P.H. Biophys. J. 1997; 73: 1874-1884Abstract Full Text PDF PubMed Scopus (37) Google Scholar). μ-CTXs are peptides consisting of 22 amino acid residues with six cysteines that form three internal disulfide bonds, imparting extreme rigidity to the toxin molecule (2Cruz L.J. Gray W.R. Olivera B.M. Zeikus R.D. Kerr L. Yoshikami D. Moczydlowski E. J. Biol. Chem. 1985; 260: 9280-9288Abstract Full Text PDF PubMed Google Scholar, 3Gray W.R. Olivera B.M. Cruz L.J. Annu. Rev. Biochem. 1988; 57: 665-700Crossref PubMed Scopus (221) Google Scholar, 4Olivera B.M. Rivier J. Clark C. Ramilo C.A. Corpuz G.P. Abogadie F.C. Mena E.E. Woodward S.R. Hilliyard D.R. Cruz L.J. Science. 1990; 249: 257-263Crossref PubMed Scopus (514) Google Scholar, 5Moczydlowski E. Olivera B.M. Gray W.R. Strichartz G.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5321-5325Crossref PubMed Scopus (143) Google Scholar, 6Yanagawa Y. Abe T. Satake M. Neurosci. Lett. 1986; 64: 7-12Crossref PubMed Scopus (19) Google Scholar, 7Chen L.-Q. Chahine M. Kallen R.G. Barchi R.L. Horn R. FEBS Lett. 1992; 309: 253-257Crossref PubMed Scopus (77) Google Scholar, 10Nakamura H. Kobayashi J. Ohizumi Y. Hirata Y. Experientia (Basel ). 1983; 39: 590-591Crossref PubMed Scopus (38) Google Scholar, 11Hidaka Y. Sato K. Nakamura H. Ohizumi Y. Kobayashi J. Shimonishi Y. FEBS Lett. 1990; 264: 29-32Crossref PubMed Scopus (29) Google Scholar). The toxin contains a number of charged residues, including several positively charged amino acids that have been shown to be critical for its biological activity (12Sato K. Ishida Y. Wakamatsu K. Kato R. Honda H. Ohizumi Y. Nakamura H. Ohya M. Lancelin J.M. Kohda D. Inagaki F. J. Biol. Chem. 1991; 266: 16989-16991Abstract Full Text PDF PubMed Google Scholar, 13Becker S. Prusak-Sochazewski E. Zamponi G. Beck-Sickinger A.G. Gordon R.D. French R.J. Biochemistry. 1992; 31: 8229-8238Crossref PubMed Scopus (100) Google Scholar, 14Chahine M. Chen L.-Q. Fotouhi N. Walsky R. Fry D. Horn R. Kallen R.G. Receptors Channels. 1995; 3: 164-174Google Scholar). At physiological pH, the toxin carries a net charge of +6 or +7, respectively, for the GIIIA and GIIIB subtypes. The three-dimensional structure of μ-CTX resembles a tetragonal bipyramid with axial distances of approximately 25 and 20 Å (15Lancelin J.M. Knoda D. Tate S. Yanagawa Y. Abe T. Satake M. Inagaki F. Biochemistry. 1991; 30: 6908-6916Crossref PubMed Scopus (89) Google Scholar, 16Wakamatsu K. Kohda D. Hatanaka H. Lancelin J.M. Ishida Y. Oya M. Nakamura H. Inagaki F. Sato K. Biochemistry. 1992; 31: 12577-12584Crossref PubMed Scopus (90) Google Scholar, 17Hill J.M. Alewood P.F. Craik D.J. Biochemistry. 1996; 35: 8824-8835Crossref PubMed Scopus (96) Google Scholar). Despite the wealth of information regarding the three-dimensional structure of μ-CTX, the molecular configuration and components of its molecular receptor in the Na+ channel are much less certain. In contrast to other site I Na+ channel blockers such as tetrodotoxin (TTX) and saxitoxin (STX), μ-CTX is believed to bind more superficially in the channel pore. The docking site of μ-CTX is comprised of numerous toxin-channel interactions of varying affinities (7Chen L.-Q. Chahine M. Kallen R.G. Barchi R.L. Horn R. FEBS Lett. 1992; 309: 253-257Crossref PubMed Scopus (77) Google Scholar, 13Becker S. Prusak-Sochazewski E. Zamponi G. Beck-Sickinger A.G. Gordon R.D. French R.J. Biochemistry. 1992; 31: 8229-8238Crossref PubMed Scopus (100) Google Scholar). Mutagenesis experiments have identified a number of pore residues that are critical for μ-CTX binding to the channel (8Dudley Jr., S.C. Hannes T. Lipkind G. Fozzard H.A. Biophys. J. 1995; 69: 1657-1665Abstract Full Text PDF PubMed Scopus (100) Google Scholar,9Li R.A. Tsushima R.G. Kallen R. Backx P.H. Biophys. J. 1997; 73: 1874-1884Abstract Full Text PDF PubMed Scopus (37) Google Scholar). However, the characterization of the toxin footprint on the external vestibule of the channel is incomplete. In the present study, we attempted to identify novel residues that are responsible for interacting with μ-CTX. Within the P loop S6 (P-S6) linkers, located on the N-terminal side of the selectivity filter region, there are a large number of charged residues (in particular, the DIII linker contains five consecutive charged residues in a row) (Fig. 1). Several of the charged residues in P-S6 linkers significantly influence the permeation phenotype of the channel (18Li R.A. Velez P. Tomaselli G.F. Marbán E. J. Gen. Physiol. 2000; 115: 81-92Crossref PubMed Scopus (32) Google Scholar). Therefore, it seems likely that these residues also participate in μ-CTX binding to the channel (by optimizing its orientation via electrostatic interactions with the toxin). To test this hypothesis, we individually neutralized all charged residues in the P-S6 linkers and assessed μ-CTX block of the mutant channels. We identified two negatively charged residues (Asp-762 and Glu-765) in domain II of rat skeletal muscle Na+ channel that, when neutralized to cysteine, increased the IC50 for μ-CTX block by ∼100-fold. The molecular basis of the change in affinity was further investigated by manipulations of the charge at these positions using sulfhydryl-specific methanethiosulfonate (MTS) reagents, charge-altering mutations, and kinetic analysis of toxin block in both WT and mutant channels. A preliminary report has appeared (19Li, R. A., Velez, P., (abstr.) Tomaselli, G. F., and Marbán, E. (2000) Biophys. J.78, 87Google Scholar). Mutagenesis was performed on the rat skeletal muscle (μ1) sodium channel α-subunit (20Trimmer 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. Goodman R.H. Agnew W.S. Mandel G. Neuron. 1989; 3: 33-49Abstract Full Text PDF PubMed Scopus (485) Google Scholar) gene cloned into pGFP-IRES vector (21Johns D.C. Nuss H.B. Marban E. J. Biol. Chem. 1997; 272: 31598-31603Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) using polymerase chain reaction with overlapping mutagenic primers as described previously (22Yamagishi T. Janecki M. Marban E. Tomaselli G.F. Biophys. J. 1997; 73: 195-204Abstract Full Text PDF PubMed Scopus (52) Google Scholar). The desired mutations were confirmed by DNA sequencing. WT and mutant channels were expressed in TSA-201 cells (a transformed HEK 293 cell line stably expressing the SV40 T antigen) using a LipofectAMINE Plus transfection kit (Life Technologies, Inc., Gaithersburg, MD). DNA encoding the WT or mutant α-subunit (1 μg/60-mm dish) was added to the cells with LipofectAMINE. 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. Electrophysiological recordings were performed using the whole-cell patch clamp technique (23Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pfluegers Arch. Eur. J. Physiol. 1981; 391: 85-100Crossref PubMed Scopus (15138) Google Scholar) with an integrating amplifier (Axopatch 200A, Axon Instruments). Transfected cells were identified by epifluorescence microscopy. Pipettes were fire-polished with final tip resistances of 1–3 MΩ. Recordings were performed at room temperature in a bath solution containing (in mm): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH adjusted to 7.4 with NaOH. Designated amounts of μ-CTX (GIIIB), or MTS reagents (methanethiosulfonate ethylsulfonate (MTSES) and methanethiosulfonate ethylammonium (MTSEA), Toronto Research Chemicals, Ontario, Canada) were added to the bath when required. The internal recording solution contained (in mm): 35 NaCl, 105 CsF, 1 MgCl2, 10 HEPES, 1 EGTA, pH adjusted to 7.2 with CsOH. All chemicals were purchased from Sigma unless otherwise specified. μ-CTX (GIIIB) was continuously applied by bath superfusion during the experiment before washing out. Flow rate was maintained at 10 to 20 ml/min (bath volume was 150 μl). Washout began only after the peak currents had reached a steady-state level. Raw current records were analyzed using customized software. Mutant channels were screened for changes in μ-CTX block affinity by determining the degree of current reduction by the toxin at doses of 30 and (or) 100 nm. Full dose-response relationships were obtained for wild-type and for the D762C and E765C mutant channels. Half-blocking concentrations (IC50) for μ-CTX were determined by least-square fits of the dose-response data to a binding isotherm of the form: I/I0=1/[1+([blocker]/IC50)]Equation 1 where IC50 is the half-blocking concentration,I 0 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 blocker, respectively. For kinetic analysis of μ-CTX block, the time course of the change of peak sodium currents elicited by depolarization to −10 mV from a holding potential of −100 mV during toxin wash-in or wash-out was fit to a single exponential function using a nonlinear least-squares method for estimation of the time constants τon and τoff. The pseudo first order association rate constant (k on) and the first order dissociation rate constant (k off) were calculated from these time constants using the following relationships. koff=1/τoffEquation 2 kon=((1/τon)−(1/τoff))/[μCTX]Equation 3 The kinetically derived equilibrium constant (K D) was determined from the equation, KD=koff/konEquation 4 Data reported are mean ± S.E. Statistical significance was determined using a paired Student's t test at the 5% level. Representative records shown in the figures were single current sweeps. All cysteine mutants expressed functional channels except D1248C, R1250C, K1252C, and E1259C, which did not express measurable currents in 5–10 rounds of transfection. We first screened for changes in susceptibility to current blockade by μ-CTX of these mutants relative to WT channels. Fig. 2 summarizes the half-blocking concentrations (IC50) for μ-CTX block of the cysteine-substituted channels. Most cysteine replacements did not significantly alter μ-CTX sensitivity compared with WT channels (p > 0.05). However, neutralization of the negative charges at positions 762 and 765 in domain II decreased toxin affinity: D762C (IC50 = 2558 ± 250 nm,n = 8) and E765C (IC50 = 2020 ± 379 nm, n = 5) channels were ∼100-fold less sensitive to μ-CTX block than was WT (IC50 = 22.3 ± 7.0 nm, n = 4) (p < 0.05). Fig. 3 compares μ-CTX block of WT, D762C, and E765C channels in more detail. Fig. 3 A shows typical Na+ currents recorded in the absence and presence of μ-CTX. Application of 100 nm μ-CTX to WT channels blocked peak sodium current (I Na) to 22.3 ± 4.0% (n = 9) of the control level. However, even 1 μm μ-CTX produced significantly (p < 0.05) less block of D762C and E765C channels and reduced the peak currents to 71.8 ± 1.7% (n = 8) and 65.2 ± 6.2% (n = 5), respectively. Fig. 3 B shows the corresponding complete dose-response curves for μ-CTX block of these channels. The binding curves of both D762C and E765C channels were shifted to the right relative to WT. All data (both WT and mutant channels) were well-fitted with a binding equation assuming a Hill coefficient of one (i.e. no cooperativity), consistent with μ-CTX blocking the Na+ channel pore in a 1:1 stoichiometry (2Cruz L.J. Gray W.R. Olivera B.M. Zeikus R.D. Kerr L. Yoshikami D. Moczydlowski E. J. Biol. Chem. 1985; 260: 9280-9288Abstract Full Text PDF PubMed Google Scholar, 5Moczydlowski E. Olivera B.M. Gray W.R. Strichartz G.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5321-5325Crossref PubMed Scopus (143) Google Scholar, 6Yanagawa Y. Abe T. Satake M. Neurosci. Lett. 1986; 64: 7-12Crossref PubMed Scopus (19) Google Scholar, 24Stephan M.M. Potts J.F. Agnew W.S. J. Membr. Biol. 1994; 137: 1-8Crossref PubMed Scopus (53) Google Scholar). As anticipated, when these DII charges were neutralized by replacement with glutamine, the resulting double mutant D762Q/E765Q was also resistant to μ-CTX block. Application of 1 μm μ-CTX incompletely blocked the peak current of this channel to 28.5 ± 2.6% (n = 3) (Fig.3 C). We have recently shown that the P-S6 residues Asp-762 and Glu-765 are accessible from the extracellular surface of the channel; therefore, it is possible that these residues contribute to μ-CTX binding to the Na+ channel by electrostatically interacting with the toxin (19Li, R. A., Velez, P., (abstr.) Tomaselli, G. F., and Marbán, E. (2000) Biophys. J.78, 87Google Scholar). To test this hypothesis, we examined the effects of charge restoration and reversal at these positions by determining the changes in μ-CTX affinity of D762C and E765C channels after MTSES and MTSEA modification, respectively. MTSES and MTSEA are sulfhydryl-specific modifiers that respectively attach a negatively and positively charged adduct to a free cysteinyl (9Li R.A. Tsushima R.G. Kallen R. Backx P.H. Biophys. J. 1997; 73: 1874-1884Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 25Akabas M.H. Stauffer D.A. Xu M. Karlin A. Science. 1992; 258: 307-310Crossref PubMed Scopus (595) Google Scholar, 26Akabas M.H. Kauffmann C. Archdeacon P. Karlin A. Neuron. 1994; 13: 919-927Abstract Full Text PDF PubMed Scopus (357) Google Scholar, 27Akabas M.H. Kauffmann C. Cook T.A. Archdecon P. J. Biol. Chem. 1994; 269: 14865-14868Abstract Full Text PDF PubMed Google Scholar). Figs. 4 and5 demonstrate the effects of MTS modifications on μ-CTX block of D762C and E765C channels, respectively. Representative raw currents through D762C and E765C channels recorded in the absence and presence of μ-CTX are shown (Figs. 4 A and 5 A). In contrast to the predictions of a simple electrostatic interaction, neither MTSES nor MTSEA application altered the toxin sensitivities of D762C channels compared with unmodified channels. Clearly, restoration of a negative charge by MTSES was insufficient to restore the sensitivity of either D762C and E765C mutant channels, and charge reversal by MTSEA also failed to further destabilize toxin block. However, MTS modification does not simply alter side-chain charge; instead, a mixed disulfide is formed, adding a bulky charged adduct. Thus, the failure of charged MTS reagents to restore or further reduce μ-CTX affinity does not rule out an electrostatic contribution to the toxin-channel interaction.Figure 5Effects of charge reversal and restoration at position 765 by MTS agents or point mutations. A, representative Na+ current tracings of D765C, MTSEA-modified E765C, MTSES-modified E765C, E765K, and E765D channels elicited by depolarization to −10 mV from a holding potential of −100 mV (−150 mV for MTSEA-modified E765C) in the absence (solid line) and presence (broken line) of μ-CTX as indicated by arrows. Control peak currents were normalized.B, bar graphs summarizing the half-blocking concentrations (IC50) for μ-CTX of the same channels shown inA. Similar to D762C, neither MTSEA (2200.1 ± 550.3 nm, n = 2) nor MTSES (3248.1 ± 2011.9 nm, n = 2) modification affected μ-CTX sensitivity of E765C (2025 ± 450 nm,n = 5). Wild-type μ-CTX sensitivity was restored in E765D (22.0 ± 7.0 nm, n = 4) channels but was further destabilized in E765K (4202.7 ± 1888.0 nm, n = 3) channels. The data presented are the mean ± S.E.View Large Image Figure ViewerDownload (PPT) To determine whether the negative charges at positions 762 and 765 are indeed critical for toxin-channel interactions, we introduced the point mutations D762E and E765D for charge conservation and D762K and E765K for charge reversal (Figs. 4and 5). Unlike MTSES modification of the cysteine mutants, the charge-conserving mutants D762E (IC50 = 21.9 ± 4.3,n = 6) and E765D (IC50 = 22.0 ± 7.0 nm, n = 4) have wild-type sensitivity to μ-CTX block. Conversely, the D762K (IC50 = 4139.9 ± 687.9 nm, n = 4) and E765K (IC50 = 4202.7 ± 1088.0 nm,n = 3) mutations further destabilized toxin affinity compared with their cysteine-substituted counterparts. Taken together with the MTS experiments, our observations suggest that not only do the negative charges at positions 762 and 765 play a crucial role in determining the μ-CTX sensitivity of the wild-type channel but also the local structural environment in which the residues reside is important for optimal toxin-channel interactions. Furthermore, when the mutations D762K and E765K were combined together, the double charge-reversed mutant (i.e. D762K/E765K) displayed an additional decrease in toxin block when compared with the single mutations alone. Addition of 3 μm μ-CTX blocked peak Na+ current merely to 87.8 ± 3.8% (n= 4) of the control value. This observation suggests that both negative charges participate simultaneously in setting the wild-type blocking affinity. To further characterize the toxin-channel interactions, we measured the time course of onset and offset of μ-CTX block. Fig. 6 shows the time course of the development of and recovery from μ-CTX block of WT, D762C, and E765C channels. Single-exponential fits of these data allow estimation of the time constants τon and τoff and, hence, the rate constants (i.e. k on and k off) for toxin binding and unbinding. Similar kinetic analysis was performed on the charge-conserved D762E and E765D and the charge-reversed D762K and E765K mutant channels. The rate constants for these channels are plotted in Fig. 7. All mutant channels but E765D significantly increased k off (3- to 50-fold, p < 0.05). The largest effects onk off were seen with the charge reversal mutants (50- and 30-fold increases for D762K and E765K channels, respectively,p < 0.05), whereas the charge-conserving D762E (3-fold, p < 0.05) and E765D (1.3-fold,p > 0.05) mutations modestly increasedk off. Despite the significant effects onk off, the same mutations had relatively minor effects on k on (2- to 5-fold decrease,p > 0.05) except for D762K channels, which displayed a 10-fold decrease in k on (p < 0.05). The kinetic equilibrium constants (K D) derived from these association and dissociation rate constants were quantitatively similar to and displayed similar trends as their corresponding equilibrium IC50 values (TableI).Figure 7Logarithmic plot of the dissociation rate constants (k off ) versusthe reciprocal of the association rate constants (k on ). The horizontal andvertical dotted lines, respectively, represent the levels of 1/k on and k off for the WT channels. Overall, channel mutations at positions 762 and 765 chiefly affected k off with relatively small effects onk on (see text for details).View Large Image Figure ViewerDownload (PPT)Table IThe effects of charge replacements at positions 762 and 765 on μ-CTX block of the Na + channelChannelk onk on, mutant/ k on, WTkoffk off, mutant/ k off, WTKDK d, mutant/ K d, WTIC50IC50, mutant/ IC50, WTm −1 s −1s −1nmnmWT μ1–21.0 ± 0.3 × 105 (7)1-aNumbers in parentheses, number of individual determinations.8.5 ± 1.4 × 10−4 (5)1.5 ± 0.8 × 10 (7)2.2 ± 0.7 × 10 (4)D762C5.5 ± 1.3 × 104 (5)0.62.2 ± 0.3 × 10−2 (5)1-bp < 0.05.26.27.0 ± 3.6 × 102 (5)1-bp < 0.05.45.62.6 ± 0.3 × 103 (8)1-bp < 0.05.114.7D762E2.1 ± 2.4 × 104 (7)0.22.9 ± 0.4 × 10−4 (5)1-bp < 0.05.3.43.6 ± 0.1 × 10 (5)2.32.2 ± 0.4 × 10 (6)0.98D762K1.4 ± 0.6 × 104 (8)1-bp < 0.05.0.14.0 ± 0.1 × 10−2 (8)1-bp < 0.05.47.23.8 ± 2.0 × 104 (5)1-bp < 0.05.2486.74.1 ± 0.7 × 103 (4)1-bp < 0.05.185.7E765C4.9 ± 1.0 × 104 (5)0.54.8 ± 0.9 × 10−3 (5)1-bp < 0.05.5.71.5 ± 0.6 × 102 (6)1-bp < 0.05.10.02.0 ± 0.5 × 103 (5)1-bp < 0.05.90.8E765D3.7 ± 0.8 × 104 (5)0.41.1 ± 0.3 × 10−3 (5)1.33.5 ± 0.9 × 10 (5)2.32.2 ± 0.7 × 10 (4)1.0E765K5.1 ± 1.1 × 104 (5)0.52.3 ± 0.1 × 10−2 (6)1-bp < 0.05.27.12.8 ± 2.2 × 103 (6)1-bp < 0.05.1844.2 ± 1.1 × 103 (3)1-bp < 0.05.188.51-a Numbers in parentheses, number of individual determinations.1-b p < 0.05. Open table in a new tab To further strengthen our hypothesis that Asp-762 and Glu-765 play electrostatic roles in μ-CTX binding to the channel, we also replaced these native negatively charged amino acids with alanine, valine, and leucine. These amino acids are neutral yet less reactive than cysteine and should therefore result in similar effects as with the cysteine mutants, with the additional benefit of enabling us to rule out any nonspecific effects that may arise from cysteine or lysine substitutions. Indeed, D762V, D762A, E765A, and E765L displayed results anticipated from electrostatic theory (TableII). In general, theirk off values were more affected than their values for k on. Similar to their cysteine and lysine counterparts, diverse position 762 mutations showed larger effects on toxin block than mutations at 765 suggesting Asp-762 may play a more critical role than Glu-765 in stabilizing the toxin-channel interactions.Table IIThe effects of D762A, D762V, E765A, and E765L mutations on μ-CTX block of the Na + channelChannelk onk on, mutant/k on, WTk offk off, mutant/k off, WTK dK d, mutant/K d, WTm −1 s −1s −1nmD762A2.9 ± 0.4 × 104 (7) a,b0.36.5 ± 0.9 × 10−3 (9)2-bp < 0.05 when compared with WT channels.7.62.5 ± 0.4 × 102 (7)2-bp < 0.05 when compared with WT channels.16.7D762V1.5 ± 0.4 × 104 (9)2-bp < 0.05 when compared with WT channels.0.21.6 ± 0.2 × 10−22-bp < 0.05 when compared with WT channels.18.82.6 ± 1.1 × 103 (9)2-bp < 0.05 when compared with WT channels.173.3E765A1.6 ± 3.2 × 104 (8)2-bNumbers in parentheses, number of individual determinations.0.21.7 ± 0.2 × 10−32-bp < 0.05 when compared with WT channels.21.5 ± 0.4 × 102 (8)2-bp < 0.05 when compared with WT channels.10E765L6.0 ± 0.9 × 104 (7)0.63.6 ± 0.7 × 10−3 (7)2-bp < 0.05 when compared with WT channels.4.28.0 ± 2.6 × 10 (7)2-bp < 0.05 when compared with WT channels.5.32-a Numbers in parentheses, number of individual determinations.2-b p < 0.05 when compared with WT channels. Open table in a new tab Tetrodotoxin (TTX) is a guanidinium toxin of known three-dimensional structure and is another useful molecular probe of the Na+ channel pore structure. However, TTX is much smaller than μ-CTX (molecular weights ∼300 versus ∼2600). Our results indicate that none of the P-S6 cysteine mutants alter TTX block by more than a factor of 3; in the three mutant channels (D762C, E765C, and E1251C) where changes were evident, IC50 values were only modestly reduced (≤2-fold) (Table III). These observations contrast starkly with the >1000-fold difference in affinity observed with deep-pore mutations such as Y401C, E403C, and E758C (28Noda M. Suzuki H. Numa S. Stuhmer W. FEBS Lett. 1989; 259: 213-216Crossref PubMed Scopus (270) Google Scholar, 29Backx P. Yue D. Lawrence J. Marban E. Tomaselli G. Science. 1992; 257: 248-251Crossref PubMed Scopus (240) Google Scholar, 30Heinemann S.H. Terlau H. Imoto K. Pfluegers Arch. Eur. J. Physiol. 1992; 422: 90-92Crossref PubMed Scopus (97) Google Scholar, 31Satin J. Kyle J.W. Chen M. Bell P. Cribbs L.L. Fozzard H.A. Rogart R.B. Science. 1992; 256: 1202-1205Crossref PubMed Scopus (335) Google Scholar, 32Perez-Garcia M.T. Chiamvimonvat N. Marban E. Tomaselli G.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 300-304Crossref PubMed Scopus (107) Google Scholar).Table IIIHalf-blocking concentration (IC 50 ) for TTX block of cysteine- substituted Na + channel pore mutantsChannelIC50nmrSkM130.4 ± 3.6 (5)3-aNumbers in parentheses, number of individual determinations.R411C34.6 ± 9.7 (3)K415C24.3 ± 2.7 (4)D762C14.7 ± 3.1 (4)3-bp < 0.05.E765C16.1 ± 5.5 (3)3-bp < 0.05.D1249CN.E.3-cN.E., no expression.R1250CN.E.E1251C18.8 ± 3.3 (6)3-bp < 0.05.K1252CN.E.E1253C39.4 ± 13.3 (4)E1254C34.0 ± 10.9 (3)E1259CN.E.D1545C48.7 ± 15.4 (4)D1547C45.4 ± 11.7 (2)E1551C39.8 ± 1.8 (4)R1558C31.2 ± 4.9 (3)D1560C21.6 ± 3.5 (2)Values represent the mean ± S.E.3-a Numbers in parentheses, number of individual determinations.3-b p < 0.05.3-c N.E., no expression. Open table in a new tab Values represent the mean ± S.E. We have identified outer-pore charged residues in the domain II P-S6 linker of the μ1 Na+ channels that are critical for high affinity μ-CTX block. Most Na+ channel mutations studied so far showed only moderate effects (2- to 10-fold) on μ-CTX block. Among the most dramatic mutations known to affect μ-CTX block is the charge neutralization of the domain II pore residue Glu-758 by glutamine, which produces a ∼50-fold decrease in toxin affinity when expressed in Xenopus oocytes (8Dudley Jr., S.C. Hannes T. Lipkind G. Fozzard H.A. Biophys. J. 1995; 69: 1657-1665Abstract Full Text PDF PubMed Scopus
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