Dependence of μ-Conotoxin Block of Sodium Channels on Ionic Strength but Not on the Permeating [Na+]
2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês
10.1074/jbc.m301039200
ISSN1083-351X
AutoresRonald A. Li, Kwokyin Hui, Robert J. French, Kazuki Sato, Charles A. Henrikson, Gordon F. Tomaselli, Eduardo Marbán,
Tópico(s)Neuroscience and Neuropharmacology Research
Resumoμ-Conotoxins (μ-CTXs) are Na+ channel-blocking, 22-amino acid peptides produced by the sea snail Conus geographus. Although K+ channel pore-blocking toxins show specific interactions with permeant ions and strong dependence on the ionic strength (μ), no such dependence has been reported for μ-CTX and Na+ channels. Such properties would offer insight into the binding and blocking mechanism of μ-CTX as well as functional and structural properties of the Na+ channel pore. Here we studied the effects of μ and permeant ion concentration ([Na+]) on μ-CTX block of rat skeletal muscle (μ1, Nav1.4) Na+ channels. μ-CTX sensitivity of wild-type and E758Q channels increased significantly (by ∼20-fold) when μ was lowered by substituting external Na+ with equimolar sucrose (from 140 to 35 mm Na+); however, toxin block was unaltered (p > 0.05) when μ was maintained by replacement of [Na+] with N-methyl-d-glucamine (NMG+), suggesting that the enhanced sensitivity at low μ was not due to reduction in [Na+]. Single-channel recordings identified the association rate constant, k on, as the primary determinant of the changes in affinity (k on increased 40- and 333-fold for μ-CTX D2N/R13Q and D12N/R13Q, respectively, when symmetric 200 mm Na+ was reduced to 50 mm). In contrast, dissociation rates changed 0.05) when μ was maintained by replacement of [Na+] with N-methyl-d-glucamine (NMG+), suggesting that the enhanced sensitivity at low μ was not due to reduction in [Na+]. Single-channel recordings identified the association rate constant, k on, as the primary determinant of the changes in affinity (k on increased 40- and 333-fold for μ-CTX D2N/R13Q and D12N/R13Q, respectively, when symmetric 200 mm Na+ was reduced to 50 mm). In contrast, dissociation rates changed <2-fold for the same derivatives under the same conditions. Experiments with additional μ-CTX derivatives identified toxin residues Arg-1, Arg-13, and Lys-16 as important contributors to the sensitivity to external μ. Taken together, our findings indicate that μ-CTX block of Na+ channels depends critically on μ but not specifically on [Na+], contrasting with the known behavior of pore-blocking K+ channel toxins. These findings suggest that different degrees of ion interaction, underlying the fundamental conduction mechanisms of Na+ and K+ channels, are mirrored in ion interactions with pore-blocking toxins. μ-Conotoxins (μ-CTXs) 1The abbreviations used are: μ-CTX, μ-conotoxin; ChTX, charybdotoxin; GFP, green fluorescent protein; NMG, N-methyl-d-glucamine; MOPS, 4-morpholinepropanesulfonic acid; WT, wild-type; D, domain; μ, ionic strength.1The abbreviations used are: μ-CTX, μ-conotoxin; ChTX, charybdotoxin; GFP, green fluorescent protein; NMG, N-methyl-d-glucamine; MOPS, 4-morpholinepropanesulfonic acid; WT, wild-type; D, domain; μ, ionic strength. are 22-amino acid peptides produced by the sea snail Conus geographus with well defined three-dimensional structures (1Lancelin J.M. Kohda D. Tate S. Yanagawa Y. Abe T. Satake M. Inagaki F. Biochemistry. 1991; 30: 6908-6916Crossref PubMed Scopus (89) Google Scholar, 2Sato K. Ishida Y. Wakamatsu K. Kato R. Honda H. Ohizumi Y. Nakamura H. Ohya M. 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The selective binding of μ-CTX to the channel pore depends not only on the intimate physical fit of the toxin molecule to its receptor surface but also on electrostatic interactions among numerous charged toxin and channel residues (2Sato 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, 3Wakamatsu 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, 8Becker S. Prusak-Sochaczewski E. Zamponi G. Beck-Sickinger A.G. Gordon R.D. French R.J. Biochemistry. 1992; 31: 8229-8238Crossref PubMed Scopus (100) Google Scholar, 9Chahine M. Chen L.Q. Fotouhi N. Walsky R. Fry D. Santarelli V. Horn R. Kallen R.G. Receptors Channels. 1995; 3: 161-174PubMed Google Scholar, 10Chang N.S. French R.J. Lipkind G.M. Fozzard H.A. Dudley Jr., S. Biochemistry. 1998; 37: 4407-4419Crossref PubMed Scopus (90) Google Scholar, 11Dudley Jr., S.C. Todt H. Lipkind G. Fozzard H.A. Biophys. J. 1995; 69: 1657-1665Abstract Full Text PDF PubMed Scopus (100) Google Scholar, 12Li R.A. Tsushima R.G. Kallen R.G. Backx P.H. Biophys. J. 1997; 73: 1874-1884Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 13Li R.A. Velez P. Chiamvimonvat N. Tomaselli G.F. Marban E. J. Gen. Physiol. 1999; 115: 81-92Crossref Scopus (32) Google Scholar, 14Li R.A. Ennis I.L. Velez P. Tomaselli G.F. Marban E. J. Biol. Chem. 2000; 275: 27551-27558Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 15Li R.A. Ennis I.I. French R.J. Dudley Jr., S.C. Tomaselli G.F. Marban E. J. Biol. Chem. 2001; 276: 11072-11077Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 16Li R.A. Ennis I.L. Tomaselli G.F. French R.J. Marban E. Biochemistry. 2001; 40: 6002-6008Crossref PubMed Scopus (14) Google Scholar, 17Li R.A. Sato K. Kodama K. Kohno T. Xue T. Tomaselli G.F. Marban E. FEBS Lett. 2002; 511: 159-164Crossref PubMed Scopus (16) Google Scholar). Although the steric components of interactions are closely related to the pore and toxin geometries and thus are relatively short range, electrostatic interactions are longer range and should depend not only on the immediate ionic environment but also the proximity of complementary, interacting charges. Indeed, K+ channel pore-blocking toxins such as agitoxin and charybdotoxin (ChTX) are known to block their target pores with strong dependence on both the ionic strength and the permeant ion concentration (18Anderson C.S. MacKinnon R. Smith C. Miller C. J. Gen. Physiol. 1988; 91: 317-333Crossref PubMed Scopus (212) Google Scholar, 19MacKinnon R. Reinhart P.H. White M.M. Neuron. 1988; 1: 997-1001Abstract Full Text PDF PubMed Scopus (111) Google Scholar, 20Miller C. Biochemistry. 1990; 29: 5320-5325Crossref PubMed Scopus (31) Google Scholar, 21Park C.S. Miller C. Neuron. 1992; 9: 307-313Abstract Full Text PDF PubMed Scopus (170) Google Scholar, 22Stocker M. Miller C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9509-9513Crossref PubMed Scopus (86) Google Scholar, 23Ranganathan R. Lewis J.H. MacKinnon R. Neuron. 1996; 16: 131-139Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). However, the importance of these factors for Na+ channel block by μ-CTX has not been systematically explored. Does μ-CTX block the Na+ channel pore with comparable ion dependence as do its K+ channel pore-blocking counterparts? What are the molecular bases underlying such similarities (or differences) in the biological actions of these Na+ and K+ channel pore-blocking toxins? Answers to these questions will not only provide insights into the mechanisms underlying the activities of these specific toxin blockers but also, by inference, into the functional and structural properties of ion channel pores.We studied the effects of Na+ concentration ([Na+]) and ionic strength (μ) on μ-CTX block of toxin-sensitive rat skeletal muscle (μ1 or Nav1.4) Na+ channels. Unlike the action of K+ channel toxins, we found that μ-CTX block of Na+ channels depends on ionic strength but not specifically on the Na+ concentration. Using synthetic μ-CTX derivatives, we further identified toxin residues Arg-1, Arg-13 and Lys-18 as particularly important effectors of the dependence on ionic strength. These results offer novel insights into the mechanism by which μ-CTX interacts with the Na+ channel pore. A preliminary report of this work has appeared previously (24Li R.A. Henrikson C. French R.J. Hui K. Sato K. Tomaselli G.F. Marban E. Biophys. J. 2002; 82: 85aGoogle Scholar).MATERIALS AND METHODSSite-directed Mutagenesis and Heterologous Expression—The gene encoding the Nav1.4 sodium channel α-subunit (25Trimmer 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. Sigworth F.J. Goodman R.H. Agnew W.S. Mandel G. Neuron. 1989; 3: 33-49Abstract Full Text PDF PubMed Scopus (485) Google Scholar) was cloned into the pGFP-IRES vector with an internal ribosomal entry site interposed between it and the GFP reporter gene, enabling translation of two independent proteins (GFP and Nav1.4) from a single plasmid. Mutagenesis was performed in pGFP-IRES using PCR with overlapping mutagenic primers. All mutations were made in duplicate and confirmed by sequencing. Na+ channel constructs were transfected into tsA-201 cells (26Margolskee R. McHendry-Rinde B. Horn R. BioTechniques. 1993; 15: 906-911PubMed Google Scholar), which constitutively express t-antigen to boost the level of channel expression, using LipofectAMINE Plus (Invitrogen, Gaithersburg, MD) according to the manufacturer's protocol. Briefly, plasmid DNA encoding the WT or mutant α subunit (1 μg/60-mm dish) was added to the cells with LipofectAMINE, followed by incubation at 37 °C in a humidified atmosphere of 95% O2-5% CO2 for 48–72 h before electrical recordings.Synthesis of Point-mutated μ-CTX GIIIA—μ-CTX derivatives were created as previously described (2Sato 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, 8Becker S. Prusak-Sochaczewski E. Zamponi G. Beck-Sickinger A.G. Gordon R.D. French R.J. Biochemistry. 1992; 31: 8229-8238Crossref PubMed Scopus (100) Google Scholar, 10Chang N.S. French R.J. Lipkind G.M. Fozzard H.A. Dudley Jr., S. Biochemistry. 1998; 37: 4407-4419Crossref PubMed Scopus (90) Google Scholar). Briefly, peptides were synthesized using N-(9-fluorenyl)methoxycarbonyl chemistry and were high-performance liquid chromatography-purified. Peptide composition was verified by quantitative amino acid analysis and/or mass spectroscopy. One-dimensional proton NMR spectra of a number of synthesized toxins were compared with those of the native toxin to test for proper folding.Electrophysiology—Macroscopic currents were recorded using the whole-cell patch clamp technique (27Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflugers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15093) Google Scholar) at room temperature. Transfected cells were identified by their green epifluorescence. Pipette electrodes had final tip resistances of 1–3 MΩ. The bath solution contained (in mm): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, with pH adjusted to 7.4 with NaOH. Designated concentrations of toxin were added to the bath as indicated. External NaCl was substituted by either sucrose or N-methyl-d-glucamine (NMG) as noted. Osmolalities (in mosmol-kg–1 H2O) of the solutions were: 140 Na, 313; 70 Na/105 sucrose, 269; 35 Na/105 sucrose, 259; 140 NMG, 295; the internal (pipette) solution contained (in mm): 35 NaCl, 105 CsF, 1 MgCl2, 10 HEPES, 1 EGTA, with pH adjusted to 7.2 with CsOH (286 mosmol-kg–1 H2O).Single-channel recordings were performed with bilayer-incorporated Na+ channels incubated with 50 μm batrachotoxin as previously described (8Becker S. Prusak-Sochaczewski E. Zamponi G. Beck-Sickinger A.G. Gordon R.D. French R.J. Biochemistry. 1992; 31: 8229-8238Crossref PubMed Scopus (100) Google Scholar). Briefly, bilayers were formed from a 4:1 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (Avanti Polar-Lipids) solution dissolved in decane (Fisher Scientific). The decane-lipid mixture was painted on a 100- to 200-μm hole in a plastic partition between the two compartments of the experimental chamber. Sodium channels were incorporated into the bilayers from surface membrane vesicles from rat skeletal muscle. Sodium channel-containing plasmalemmal vesicles were isolated as described before (Becker et al. (8Becker S. Prusak-Sochaczewski E. Zamponi G. Beck-Sickinger A.G. Gordon R.D. French R.J. Biochemistry. 1992; 31: 8229-8238Crossref PubMed Scopus (100) Google Scholar)), sonicated, and incubated with 50 μm batrachotoxin in a 0.3 m sucrose, 20 mm HEPES solution (pH7.4), and kept at –20 °C for at least 1 day prior to use to inhibit channel inactivation. 1–5 μl of incubated vesicles (∼2.5 mg of protein/ml) were pipetted into one well of the bilayer chamber. Bath solutions were symmetric and initially contained (in mm): 50 or 200 NaCl, 10 MOPS, 0.1 EDTA, with pH adjusted to 7.0 using 10 n NaOH. The salt concentration was increased by addition of an identically buffered 2.3 m NaCl solution to both sides of the bilayer. Data were filtered at 200 Hz and digitized at 1 kHz. Blocking activities of μ-CTX derivatives D2N/R13Q, D12N/R13Q, and R13W were assayed for their dependence on ionic strength. Comments on the choice of these derivatives are given under "Results."Data Analysis and Statistics—Ionic strength (μ) was calculated using the following equation, μ=12Σcizi2(Eq. 1) where c i is the concentration of ion species i and z i is its valence.Toxin was superfused continuously during the whole-cell experiments. Equilibrium half-blocking concentrations (IC50) for toxins were determined from the following binding isotherm, I/IO=(1-R)/{1+([toxin]/IC50)}+R(Eq. 2) where IC50 is the half-blocking concentration, R is the residual current or sub-conductance when channels are fully blocked, I O and I are the peak currents measured from a step depolarization to –10 mV from a holding potential of –100 mV before and after application of the toxin, respectively.For single-channel experiments, the unitary current amplitude was determined visually from the current records and was similar to Gaussian fits of the all-points amplitude histograms. Single-channel events were detected by the half-amplitude threshold technique using a minimum cutoff of 400 ms for blocked events. The kinetic parameters, k on and k off, were determined from mean unblocked and blocked times, 〈t u〉 and 〈t b〉, respectively, as follows: k on = 1/(〈t u〉[toxin]), and k off = 1/t b. The reported values of K d, k on and k off, were determined from estimates at multiple voltages interpolated to 0 mV. All data reported are mean ± S.E. Statistical significance was determined using a paired Student's t test at the 5% level.RESULTSEffects of External Na + Concentrations and Ionic Strength on μ-CTX Block of Na + Channels—We first studied the effects of ionic strength (μ) on μ-CTX block of WT Nav1.4 Na+ channels. Given the slow kinetics of toxin binding and dissociation (macroscopic time constants generally of many seconds), the current elicited by a brief test pulse (∼10 ms) from a holding potential of –100 mV reflects the fraction of channels that are not blocked by the toxin at the holding potential. Toxin-bound channels show subtle shifts of activation gating and perhaps of inactivation, which have previously been analyzed in detail, but these do not influence the assay of toxin binding by the protocol used here (28French R.J. Prusak-Sochaczewski E. Zamponi G.W. Becker S. Kularatna A.S. Horn R. Neuron. 1996; 16: 407-413Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 29French R.J. Horn R. Sáez R. L.a.J.C. From Ion Channels to Cell-to-Cell Conversations. Plenum Press, New York1997: 67-89Crossref Google Scholar)External Na+ was substituted by sucrose to reduce changes in the osmolarity while varying μ. Fig. 1 shows the effects of 140 mm Na+, 70 mm Na+/70 mm sucrose, and 35 mm Na+/105 mm sucrose on the half-blocking concentration (IC50) of μ-CTX on WT Nav1.4 channels. The toxin sensitivity increased ∼18-fold as μ was lowered over the range examined. The channel mutation E758Q is known to significantly reduce μ-CTX sensitivity (11Dudley Jr., S.C. Todt H. Lipkind G. Fozzard H.A. Biophys. J. 1995; 69: 1657-1665Abstract Full Text PDF PubMed Scopus (100) Google Scholar); despite the lower toxin sensitivity of E758Q channels, μ-CTX block also increased substantially (p < 0.05) when μ was lowered (Fig. 1). Indeed, the affinity of E758Q channels for μ-CTX increased by ∼28-fold when the external solution was changed from 140 mm Na+ to 35 mm Na+/105 mm sucrose.We next investigated whether the changes in toxin affinity were directly due to the lowering of μ, changes in the permeant concentration, or both. To distinguish among these possibilities, we studied μ-CTX block of E758Q channels by substituting external Na+ with NMG+ so as to maintain both the osmotic and ionic strengths while varying [Na+]. Fig. 2 summarizes these results. In contrast to the sucrose experiments, μ-CTX sensitivity was not altered when external [Na+] was lowered from 140, to 70, and to 35 mm (p > 0.05). These results indicate that the changes observed in Fig. 1 were entirely due to the effects of ionic strength. Therefore, unlike K+ channel toxins, μ-CTX block depends on ionic strength but not, in an obligatory manner, on permeant ion concentration.Fig. 2Effects of external Na + on μ-CTX block. A, representative Na+ currents through E758Q channels recorded in the absence and presence of μ-CTX with 140, 70, and 35 mm external Na+ in the bath solution. Unlike Fig. 1, external Na+ was substituted by NMG+ in these experiments to preserve both osmotic and ionic strengths. The same protocol as described in Fig. 1 was used to elicit currents. B, bar graphs summarizing the half-blocking concentrations (IC50) for μ-CTX block of E758Q channels with 140, 70, and 35 mm external [Na+] in the bath. Under these conditions, μ-CTX sensitivity did not appear to depend on external [Na+].View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effects of Ionic Strength on μ-CTX Block of Single Na + Channels—To measure directly the kinetic changes, we performed single-channel recordings in neutral lipid bilayers to study the effects of ionic strength on the blockade by the μ-CTX-based derivatives D2N/R13Q, D12N/R13Q, and R13W. This approach is advantageous for two reasons. First, it is much easier to symmetrically change solutions over a wide range of ionic strength than it is using single-channel patch recording. Second, the long steady-state recordings from batrachotoxin-modified channels enable toxin association and dissociation rate constants to be determined directly from the unblocked and blocked times in a single record, for toxin kinetics varying over a wide range. This avoids the need for continuous perfusion and step changes in concentration that are required to estimate the rate constants from macroscopic recordings and allows more detailed and precise kinetic measurements.The three toxin derivatives studied in these experiments were chosen in part because they have shorter blocked times, than the native toxin, thus facilitating kinetic analysis. Wild-type μ-CTX shows mean blocked times on the order of minutes, thereby limiting the number of blocking and unblocking events that can normally be collected in a single experiment. Two of the derivatives (D2N/R13Q and D12N/R13Q) have the same nominal net charge as wild-type μ-CTX (+6), but the charge is maintained by counter charge neutralizations at widely separated locations on the toxin (cf. Ref. 30Hui K. Lipkind G. Fozzard H.A. French R.J. J. Gen. Physiol. 2002; 119: 45-54Crossref PubMed Scopus (79) Google Scholar). R13W (+5) also shows relatively rapid blocking and unblocking kinetics. All of these derivatives cause incomplete block of single-channel currents and thus enabled us to monitor the efficacy of single-channel block by the bound toxin as a function of ionic strength, while simultaneously recording binding and unbinding kinetics.Fig. 3A shows representative traces of single Na+ channel activities recorded in the absence and presence of D2N/R13Q at a test voltage of +60 mV. It is evident from these records that at low ionic strength the mean blocked and unblocked times increased and decreased, respectively. As shown in Fig. 3C, the association rate (k on) decreased dramatically (∼40-fold for D2N/R13Q), accompanied by a modest, <2-fold increase in the dissociation rate constant (k off), as [Na+] was increased from 50 to 200 mm. These changes in blocking kinetics conferred significant increases in the kinetically derived K D with increasing ionic strength (Fig. 3D), consistent with the changes in steady-state IC50 of μ-CTX block of Nav1.4 channels observed at the whole-cell level when μ was changed (cf. Fig. 1).Fig. 3Block of single Na + channels by μ-CTX mutants D2N/R13Q, D12N/R13Q, and R13W under different ionic conditions. A, representative records of single-channel activities of bilayer-incorporated muscle Na+ channels measured in the absence (with 50 mm NaCl) or presence of 1 μm μ-CTX D2N/R13Q under different ionic conditions at +60 mV. μ = [Na+] in these experiments, because ionic strength was changed by varying [Na+]. B, the residual current (R), as a fraction of the unblocked current, varies with [Na+]. All three μCTX mutants produced more complete block of single channel current at low ionic strength. C, rate constants, k on and k off, determined from the inverse of the mean unblocked and blocked times, respectively, at different ionic conditions. The kinetic parameters were determined at multiple voltages and extrapolated to 0 mV, k = k 0 exp (±zδFE/RT), where the exponential is positive for k off and negative for k on. The values shown in the bar plots are k 0 at the indicated NaCl concentration. Clearly, k on displays a dramatic dependence, whereas k off is much less affected. D, the dissociation constant, K D, was determined as the ratio k off/k on at different ionic conditions. As with the rate constants, K D extrapolated to 0 mV is shown. The combined effect of the rate constants, particularly k on, lowers toxin affinity at higher [NaCl], consistent with the whole-cell observations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In addition to the kinetics, the blocked and unblocked unitary conductances were also affected. Notably, the fractional residual conductance (R) increased with the increase in μ (Fig. 3B). Similar trends were observed for single-channel block and binding kinetics shown by the two other derivatives, D12N/R13Q and R13W (Fig. 3, B–D). The different ranges over which the fractional residual current changed depend on both size and charge of the particular amino acids substituted into the toxin (see "Discussion" and Ref. 30Hui K. Lipkind G. Fozzard H.A. French R.J. J. Gen. Physiol. 2002; 119: 45-54Crossref PubMed Scopus (79) Google Scholar).The key points to note here are the consistent increases, with decreasing ionic strength, in both the toxin binding affinity, and completeness of single channel block. We suggest that both changes are directly related to the decreased screening of various charges on the toxin and/or the channel.Arg-13 and Glu-758 Are Not Essential for Ionic Strength Dependence of Binding—Arg-13 is critical for high affinity, complete blockade of Na+ channels by μ-CTX (2Sato 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, 3Wakamatsu 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, 8Becker S. Prusak-Sochaczewski E. Zamponi G. Beck-Sickinger A.G. Gordon R.D. French R.J. Biochemistry. 1992; 31: 8229-8238Crossref PubMed Scopus (100) Google Scholar, 10Chang N.S. French R.J. Lipkind G.M. Fozzard H.A. Dudley Jr., S. Biochemistry. 1998; 37: 4407-4419Crossref PubMed Scopus (90) Google Scholar). We therefore created the μ-CTX derivative R13A by neutralizing the native basic toxin residue at position 13 by alanine substitution and studied the effects of ionic strength on its interactions with the pore. Under normal conditions (140 mm Na+), R13A, applied to WT Nav1.4 channels, displayed significantly reduced affinity and showed incomplete block of the whole-cell current even at high toxin concentrations (Fig. 4, A and B) consistent with previous single-channel observations (8Becker S. Prusak-Sochaczewski E. Zamponi G. Beck-Sickinger A.G. Gordon R.D. French R.J. Biochemistry. 1992; 31: 8229-8238Crossref PubMed Scopus (100) Google Scholar, 30Hui K. Lipkind G. Fozzard H.A. French R.J. J. Gen. Physiol. 2002; 119: 45-54Crossref PubMed Scopus (79) Google Scholar). However, the leftward shift of its dose-response curve in Fig. 4B upon lowering of μ indicates that the affinity of R13A also increased significantly (p < 0.05) in a manner qualitatively similar to WT μ-CTX block (cf. Fig. 1, and see Fig. 5 for quantification of this dependence on ionic strength). Consistent with our observations with D2N/R13Q, D12N/R13Q, and R13W block at the single-channel level, the asymptotic residual whole-cell current at high [R13A] block was also reduced at low ionic strength. Because Arg-13 is known to interact with the domain II (DII) pore residue Glu-758 (10Chang N.S. French R.J. Lipkind G.M. Fozzard H.A. Dudley Jr., S. Biochemistry. 1998; 37: 4407-4419Crossref PubMed Scopus (90) Google Scholar), we studied also the ionic strength dependence of R13A block of E758Q channels (Fig. 4, C and D). Indeed, the magnitude of changes in affinity and residual currents were similar to those seen with WT Nav1.4 channels.Fig. 4Effects of ionic strength on μ-CTX R13A block of WT and E758Q channels. Representative Na+ current traces through WT (A) and E758Q (C) channels blocked by μ-CTX R13A with 140 mm Na+ and 70 mm Na+/70 mm sucrose. Dose-response relationships of block of WT (B) and E758Q (D) channels by R13A peptide under different ionic conditions. Similar to the wild-type toxin, the blocking affinity of μ-CTX R13A increased as the ionic strength lowered. Furthermore, the residual current at high toxin concentrations also decreased as the ionic strength decreased.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Effects of ionic strength on block of E758Q channels by μ-CTX R1A, K8A, K9A, K11A, D12A, K16A, and R19A. A, representative Na+ currents through E758Q channels recorded in the absence and presence of μ-CTX R1A, K9A, K11A, D12A, R13A, and K16A as indicated under different ionic conditions. B, plot of IC50 values for block of WT and E758Q channels against ionic strength (in mm) by the same μ-CTX derivatives in A. δμ = δ[Na+] in these experiments. C, the slopes obtained from linear fits of data from panel B, which reflect the dependence, on ionic strength, of binding of the indicated peptides to E758Q, are plotted. The binding of R1A, R13A, and K16A was less dependent on ionic strength than WT μ-CTX and other derivatives. These observations suggest that the interactions of different charged toxin residues are differentially affected by extracellular ionic strength.View Large Image
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