Title: Binding Specificity of Sea Anemone Toxins to Nav 1.1-1.6 Sodium Channels
Abstract: Sea anemones are an important source of various biologically active peptides, and it is known that ATX-II from Anemonia sulcata slows sodium current inactivation. Using six different sodium channel genes (from Nav1.1 to Nav1.6), we investigated the differential selectivity of the toxins AFT-II (purified from Anthopleura fuscoviridis) and Bc-III (purified from Bunodosoma caissarum) and compared their effects with those recorded in the presence of ATX-II. Interestingly, ATX-II and AFT-II differ by only one amino acid (L36A) and Bc-III has 70% similarity. The three toxins induced a low voltage-activated persistent component primarily in the Nav1.3 and Nav1.6 channels. An analysis showed that the 18 dose-response curves only partially fit the hypothesized binding of Lys-37 (sea anemone toxin Anthopleurin B) to the Asp (or Glu) residue of the extracellular IV/S3-S4 loop in cardiac (or nervous) Na+ channels, thus suggesting the substantial contribution of some nearby amino acids that are different in the various channels. As these channels are atypically expressed in mammalian tissues, the data not only suggest that the toxicity is highly dependent on the channel type but also that these toxins and their various physiological effects should be considered prototype models for the design of new and specific pharmacological tools. Sea anemones are an important source of various biologically active peptides, and it is known that ATX-II from Anemonia sulcata slows sodium current inactivation. Using six different sodium channel genes (from Nav1.1 to Nav1.6), we investigated the differential selectivity of the toxins AFT-II (purified from Anthopleura fuscoviridis) and Bc-III (purified from Bunodosoma caissarum) and compared their effects with those recorded in the presence of ATX-II. Interestingly, ATX-II and AFT-II differ by only one amino acid (L36A) and Bc-III has 70% similarity. The three toxins induced a low voltage-activated persistent component primarily in the Nav1.3 and Nav1.6 channels. An analysis showed that the 18 dose-response curves only partially fit the hypothesized binding of Lys-37 (sea anemone toxin Anthopleurin B) to the Asp (or Glu) residue of the extracellular IV/S3-S4 loop in cardiac (or nervous) Na+ channels, thus suggesting the substantial contribution of some nearby amino acids that are different in the various channels. As these channels are atypically expressed in mammalian tissues, the data not only suggest that the toxicity is highly dependent on the channel type but also that these toxins and their various physiological effects should be considered prototype models for the design of new and specific pharmacological tools. As voltage-gated Na+ channels are responsible for the conduction of electrical impulses in most excitable tissues in the majority of animals (with the exception of nematodes), they have become important targets for the toxins of venomous animals from sea anemones to mollusks, scorpions, spiders, and even fishes. The peptides found in these venoms constitute various tools by means of which physiologists and pharmacologists can study the structure/function relationships of channel proteins in detail (1Cestèle S. Catteral W.A. Biochimie (Paris). 2000; 82: 883-892Crossref PubMed Scopus (595) Google Scholar). The α-subunits of voltage-gated Na+ channels (Nav1.x) have been divided into at least nine subtypes based on their tetrodotoxin binding, tissue expression, and amino acid sequences (2Catteral W.A. Chandy K.G. Gutman G.A. The IUPHAR Compendium of Voltage-gated Ion Channels. IUPHAR Media, Leeds, United Kingdom2002Google Scholar). Interestingly, the genes for four isoforms (Nav1.1, 1The abbreviations used are: Nav1.1, α-subunits of voltage-gated Na+ channel isoform; ApB, Anthopleurin B. 1The abbreviations used are: Nav1.1, α-subunits of voltage-gated Na+ channel isoform; ApB, Anthopleurin B. Nav1.2, Nav1.3, and Nav1.7) are located in human chromosome 2. Other genes (Nav1.5, Nav1.8, and Nav1.9) are located on chromosome 3, and Nav1.4 and Nav1.6 are located on chromosomes 17 and 12, respectively (2Catteral W.A. Chandy K.G. Gutman G.A. The IUPHAR Compendium of Voltage-gated Ion Channels. IUPHAR Media, Leeds, United Kingdom2002Google Scholar). During their evolution, different animals have developed a set of cysteine-rich peptides capable of binding different extracellular sites of the channel protein. A fundamental question concerning the mechanism of action of these toxins is whether they act at a common receptor site in Na+ channels when exerting their different pharmacological effects or at distinct receptor sites in different Nav channels subtypes whose particular properties lead to these pharmacological differences (1Cestèle S. Catteral W.A. Biochimie (Paris). 2000; 82: 883-892Crossref PubMed Scopus (595) Google Scholar). The aim of this study was to investigate the putative ability of some peptides to distinguish the different Na+ channels subtypes (from Nav1.1 to Nav1.6) either by differing biophysical effects or by differing potency or both. We used three sea anemone toxins, which have a range of sequence similarity, that were purified from different animals: the well known Anemonia sulcata ATX-II peptide (3Béress L. Béress R. Wunderer G. FEBS Lett. 1975; 50: 311-314Crossref PubMed Google Scholar); the Anthopleura fuscoviridis AFT-II peptide (4Sunahar S. Muramoto K. Tenma K. Kamiya H. Toxicon. 1987; 25: 211-219Crossref PubMed Scopus (44) Google Scholar); and the Bunodosoma caissarum Bc-III peptide (5Malpezzi E.L.A. Freitas J.C. Muramoto K. Kamiya H. Toxicon. 1993; 31: 853-864Crossref PubMed Scopus (51) Google Scholar). They are all 47-48-aa-long and have three disulfide bonds; however, interestingly, two peptides (ATX-II and AFT-II) differ by only one amino acid, whereas Bc-III has only a 70% identity. To the best of our knowledge, this is the first report of the characterization of sea anemone toxins against such a wide range of sodium channel subtypes. Animal and Venom Collection—30 specimens of B. caissarum were collected during periods of low tide-free diving at different rocky shores of the São Sebastião Channel on the northern coast of São Paulo, Brazil. The animals were transported live and starved in an aquarium for 24 h to eliminate any gastrovascular contents. The venom was obtained directly by means of nematocyst discharge as described by Malpezzi et al. (5Malpezzi E.L.A. Freitas J.C. Muramoto K. Kamiya H. Toxicon. 1993; 31: 853-864Crossref PubMed Scopus (51) Google Scholar). The protein content was estimated by means of absorbance at 280 nm and the BCA method (Pierce) following the manufacturer's instructions. Purification Procedures—The B. caissarum venom was fractionated in a Sephadex G-50 column (1.9 × 131 cm, Amersham Biosciences), equilibrated, and eluted with 0.1 m ammonium acetate (pH 7.0) at room temperature (5Malpezzi E.L.A. Freitas J.C. Muramoto K. Kamiya H. Toxicon. 1993; 31: 853-864Crossref PubMed Scopus (51) Google Scholar, 6Lanio M.E. Morera V. Alvarez C. Tejuca M. Gómez T. Pazos F. Besada V. Martínez D. Huerta V. Padrón G. Chávez M.A. Toxicon. 2001; 39: 187-194Crossref PubMed Scopus (113) Google Scholar). At each run, ∼1.0-2.0 g of the lyophilized material (corresponding to ∼200 mg of protein content) was dissolved in 15 ml of the same buffer and injected into the column. Fractions of 10 ml volume were collected using an automatic Fraction Collector Frac-100 (Amersham Biosciences), absorbance (280 nm) was monitored by a Spectra/Chrom Flow Thru UV monitor/controller (Spectrum), and the elution profile was recorded by a Spectra/Chrom 1 channel recorder (Spectrum). Each corresponding peak was pooled and lyophilized, and the neurotoxic fraction (peak IIIa) was further purified by reverse-phase high performance liquid chromatography. After being diluted in Milli-Q water (Millipore Inc.), the same fraction was injected into an Ultrasphere octadecylsilyl column (4.6 × 150 mm, 5 μm, Beckman Inc.) coupled to a Shimadzu high performance liquid chromatography purification system consisting of a UV-visible detector (SPD-10A VP), pumps (LC-10AD VP), and a system controller (SCL-10A VP, Shimadzu Corp., Tokyo, Japan). The peaks were obtained using a linear gradient of 5-50% buffer B containing 90% CH3CN and 10% buffer A (0.1% trifluoroacetic acid in H2O) over 25 min. The flow rate was 1.0 ml/min, and UV was monitored at 214 nm. Pure Bc-III was obtained by means of repurification using the same column under 28% buffer B and at a higher flow rate (1.2 ml/min). The Bc-III and AFT-II mass spectra were obtained using a Q-TOF mass spectrometer (Micromass) in Qq-orthogonal time-of-flight configuration. An Ettan matrix-assisted laser desorption ionization time-of-flight/Pro mass spectrometer (Amersham Biosciences) was also used with α-cyano-4-hydroxycinnamic acid as the matrix. Both machines were operated in positive mode. The amino acid sequence of Bc-III was confirmed by means of N-terminal determination using automatic Edman degradation and a PPSQ/230 gas-phase sequencer (Shimadzu Corp.). A search for peptide homology/similarity and multiple sequence alignment was performed using the BLAST (www.ncbi.nlm.nih.gov) and Swiss-Prot databases (www.expasy.ch/sprot/sprot-top.html). The accession numbers are P01528 for ATX-II, P10454 for AFT-II, and A37435 for Bc-III. ATX-II was obtained from Sigma, and AFT-II was a kind gift from Dr. Koji Muramoto (Department of Biological Science at Tohoku University) (4Sunahar S. Muramoto K. Tenma K. Kamiya H. Toxicon. 1987; 25: 211-219Crossref PubMed Scopus (44) Google Scholar). All of the chemicals were of the highest purity available, and double-distilled water was used throughout. LD50Determination—The LD50 of Bc-III was determined intraperitoneally using white BALB/C mice (Mus musculus) according to Meier and Theakston (7Meier J. Theakston R.D.G. Toxicon. 1986; 24: 395-401Crossref PubMed Scopus (177) Google Scholar). Cell Culture—Human embryonic kidney 293 cell lines stably expressing human Nav1.1, 1.2, 1.3, 1.5, and 1.6 (cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum) were obtained by transfection with the corresponding pCIN5 expression vector (8Rees S. Coote J. Stables J. Goodson S. Harris S. Lee M.G. BioTechniques. 1996; 20: 102-110Crossref PubMed Scopus (262) Google Scholar) and selection of G418-resistant clones. pCIN5-hNaV1.3 and 1.6 were generated as described previously (9Chen Y.H. Dale T.J. Romanos M.A. Whitaker W.R. Xie X.M. Clare J.J. Eur. J. Neurosci. 2000; 12: 4281-4289PubMed Google Scholar, 10Burbidge S.A. Dale T.J. Powell A.J. Whitaker W.R. Xie X.M. Romanos M.A. Clare J.J. Mol. Brain Res. 2002; 103: 80-90Crossref PubMed Scopus (71) Google Scholar). pCIN5-hNaV1.2 was generated from the hNaV1.2 cDNA clone described in Xie et al. (11Xie X.M. Dale T.J. John H.L. Cater T.C. Peakman T.C. Clare J.J. Pfluegers Arch. Eur. J. Physiol. 2001; 441: 425-433Crossref PubMed Scopus (61) Google Scholar), pCIN5-hNaV1.1 was generated using a human NaV1.1 cDNA assembled from partial clones isolated from human adult cerebellum and medulla cDNA libraries, and pCIN5-hNaV1.5 was generated using a human NaV1.5 cDNA assembled from partial clones isolated from a cDNA library made from human heart. 2J. J. Clare, unpublished data. Nav1.4-expressing cells were obtained after transient transfection of a plasmid containing the hNav1.4 construct (a kind gift from Prof. Diana Conti-Camerino, University of Bari). Solutions and Drugs—The standard extracellular solution contained (in mm) 130 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES-NaOH, and 5 d-glucose, pH 7.4. The standard pipette solution contained (in mm) 130 K+-aspartate, 10 NaCl, 2 MgCl2, 10 EGTA-KOH, and 10 HEPES-KOH, pH 7.3. Known quantities of the toxins were dissolved in the extracellular solution immediately before the experiments. When contaminating potassium currents were seen, tetrodotoxin (Sigma) was used at 100 nm (on Nav1.1, 1.2, 1.3, 1.4, and 1.6 currents) and the resulting traces were subtracted from control traces to obtain the tetrodotoxin-sensitive currents. The Nav1.5 clone, which has a tetrodotoxin ID50 much higher than 100 nm, never showed significant potassium currents at the test potentials. The extracellular solutions were delivered through a 9-hole (0.6-mm) remote-controlled linear positioner with an average response time of 2-3 s that was placed near the cell under study. Patch Clamp Recordings and Data Analysis—The currents were recorded at room temperature by means of the MultiClamp 700A (Axon Instruments) as described previously (12Faravelli L. Arcangeli A. Olivotto M. Wanke E. J. Physiol. (Lond.). 1996; 496: 13-23Crossref Scopus (91) Google Scholar) with a pipette resistance of ∼1.5-2.2 megaohms. Cell capacitance and series resistance errors were carefully compensated for (85-90%) before each voltage clamp protocol was run to reduce the voltage errors to <5% of the protocol pulse. The P/N leak procedure was routinely used. Voltage-dependent steady-state inactivation was determined by means of a double-pulse protocol in which a conditioning pulse was applied from a holding potential of -80 mV to a range of potentials from -110 (or -130) to -10 (or -30) in 10- or 15-mV increments for 450 ms immediately followed by a test pulse to -10 (or -20) mV. The peak current amplitudes during the tests were normalized to the amplitude of the first pulse and plotted against the potential of the conditioning pulse. The data were fitted using two exponential terms and a constant value beginning at the current peak. When necessary, the time constant of the fast inactivating term was fixed to the average fast time constant obtained in control curves. Recovery from inactivation was determined using a three-pulse protocol, which began with conditioning depolarization from a holding potential of -120 to -10 (or -20) mV for 60 (or 100) ms that inactivated >95% of the channels. This was followed by an increasing recovery time interval at -80 mV and a test depolarization to -10 (or -20) mV. The time intervals in successive episodes were 2, 5, 10, 20, and 50 ms (see details in the figure legends). pClamp 8.2 (Axon Instruments) and Origin 7 (Microcal Inc.) software were routinely used during data acquisition and analysis. Bc-III and AFT-II Purification, Amino Acid Sequence, and LD50—Gel-filtration chromatography of B. caissarum venom yielded six main peaks (I-VI). Peak IIIa (Fig. 1A) had the greatest neurotoxicity and was further purified by reverse-phase high performance liquid chromatography steps, leading to the pure toxin Bc-III (Fig. 1B). Mass spectrometry analysis showed that the molecular mass of Bc-III was 4.976 atomic mass units (4.973) (5Malpezzi E.L.A. Freitas J.C. Muramoto K. Kamiya H. Toxicon. 1993; 31: 853-864Crossref PubMed Scopus (51) Google Scholar) and that the determination of 25 N-terminal amino acid residues (GVACRCDSDGPTSRGNTLTGTLWLT) by automated Edman degradation confirmed the identity of the major toxin Bc-III (Table I) reported by Malpezzi et al. (5Malpezzi E.L.A. Freitas J.C. Muramoto K. Kamiya H. Toxicon. 1993; 31: 853-864Crossref PubMed Scopus (51) Google Scholar). The high purity of AFT-II was determined by mass spectrometry, and its molecular mass was also confirmed (4.941 atomic mass units). The intraperitoneal LD50 of Bc-III in mice was 600 μg/kg, very similar to that reported for AFT-II (450 μg/kg) (4Sunahar S. Muramoto K. Tenma K. Kamiya H. Toxicon. 1987; 25: 211-219Crossref PubMed Scopus (44) Google Scholar).Table IAmino acid sequence of ATX-II, Bc-III, and AFT-II compared with other similar sea anemone toxins (the numbers refer to ATX-II)View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab Basic Properties of Biophysical Effects of ATX-II on Nav1.5 and Data Analysis—As explained under "Materials and Methods," all of the experiments were performed at fixed drug concentrations using the currents elicited before and during drug perfusion (for 25-40 s) at successively increasing concentrations. Recovery was always checked after the experiment. It is well known that the main action of sea anemone neurotoxins is to slow the inactivation process. Therefore, we mainly used protocols (double pulse, see Fig. 2A, inset) capable of deriving these properties and compared traces at the same test potential under the control conditions and during toxin perfusion. The action of ATX-II on the Nav1.5 channel was the starting point used to establish the protocols for recording the data and analyzing the results. Observation of the action of increasing toxin concentrations clearly showed in first approximation that the drug is in equilibrium at a fixed concentration with a fraction of the total number of channels on each cell. We checked this result several times and consequently analyzed the data on the assumption that each Na+ current trace is the sum of two exponential decaying components and a steady-state component. Under control conditions, the amplitude of the fast component was generally large and the other two components were normally very low or negligible. During toxin action, there was a large increase in the slow component and, in some cases (depending on the toxin type), an additional increase in a persistent component. We also systematically observed that the quality of fitting of the toxin-induced currents was not different whether we used a free or fixed (control value) fast time constant. This strongly suggests that the currents recorded in the presence of the toxin were always the sum of two types of currents: those deriving from toxin-bound channels (modified) and those deriving from toxin-free channels (not modified and thus equivalent to control channels). This is shown in Fig. 2, A-C, at 0, 10, and 200 nm [ATX-II]. In Fig. 2D, a single trace (continuous line) elicited at -20 mV in the presence of 5 nm toxin was dissected into a fast (○) and slow component (▵). The relative increase and decrease in these components is plotted in Fig. 2E for different concentrations (n = 4), which shows that the contribution of bound and unbound channels consistently changes in relation to the toxin concentration. Following this finding, we investigated the recovery from inactivation at -80 mV (see protocol below Fig. 2F) at the maximal concentration by superimposing the traces of the control (line), toxin (line), and toxin control (open circle). It can be seen that there was hardly any recovery of the slow component during times of the order of the time constants of recovery from inactivation in the control traces. The normalized voltage dependence of the slow component amplitudes at the various concentrations was plotted as a function of the preconditioning level and compared with the classical steady-state inactivation as shown in Fig. 2G. It can be observed that the toxin induced a net leftward shift of the slow component in comparison with the control inactivation. Moreover, comparison of the slow component at increasing concentrations indicates an increased time constant (from 14.5 to 33.2 ms, see Fig. 2G, inset), although this observation was not investigated further. The Effect of AFT-II and Bc-III on Nav1.5—The effects of the toxins AFT-II and Bc-III on Nav1.5 are shown in Fig. 3. In the six panels, the data are divided along the two columns for AFT-II (n = 4) and Bc-III (n = 4). The top row (Fig. 3, A and B) shows the control data (line) with the data observed at various concentrations (different symbols of the superimposed traces in a representative cell). The second row (Fig. 3, C and D) shows the data for the recovery from inactivation (traces from one representative cell are superimposed for the control (line), toxin (line), and the toxin-sensitive component (open squares). The third row (Fig. 3, E and F) shows the data for the voltage dependence of inactivation of the slow (○) and steady-state component (▵) under control conditions (▪) and at the highest concentration used. The insets show the superimposed recordings from a representative cell of the complete inactivation protocol under control conditions and at the highest concentration. These data suggest that the action of AFT-II is similar to that of ATX-II and that Bc-III has a lower affinity for this type of channel. Interestingly, the results obtained with these two new toxins differ from those obtained with ATX-II, insofar, as there was a small supplementary persistent component characterized by right-shifted inactivation curves. Similar procedures were used to analyze the data obtained with the three toxins acting on the Na+ currents in the different cell lines expressing the Nav1.1, Nav1.2, Nav1.3, Nav1.4, and Nav1.6 sodium channel genes as described in the subsequent paragraphs. The effects of ATX-II, AFT-II, and Bc-III on Nav1.1—The results of experiments on cells expressing the Nav1.1 channel are shown in Fig. 4. In the nine panels, the data are divided in three columns for ATX-II (n = 3), AFT-II (n = 4), and Bc-III (n = 4). The first row (Fig. 4, A-C) shows the effect of different toxin concentrations (superimposed traces from a representative cell). The second row (Fig. 4, D-F) shows the data for the recovery from inactivation (superimposed traces from one representative cell in control, toxin, and the toxin-sensitive component). The third row (Fig. 4, G-I) shows the data for the voltage dependence of inactivation in the control (▪) and slow (○) and steady-state components (▵). The insets show the superimposed recordings from a representative cell for the complete inactivation protocol under control conditions and at the highest concentration used (note the break in the time axis). The average time constant of the slow component (shown in Table II) did not depend on the toxin concentration or the preconditioning voltage (in the range -110/-50 mV). During preconditioning at -35 and -20 mV, AFT-II and Bc-III induced currents that were almost persistent but not present in the control and that did not inactivate when tested at -20 mV. These persistent components had a right-shifted voltage-dependent curve in comparison with the control and slow component behavior.Table IIInactivation and time constantsτslow (msec)Voltage-dependent inactivationShift (Slow) (mV)Shift (SS) (mV)ATX-IIAFT-IIBc-IIIATX-IIAFT-IIBc-IIIATX-IIAFT-IIBc-IIINav 1.17 ± 1.113 ± 1.316 ± 2.1n.a.n.a.6 ± 0.49 ± 0.615 ± 1.530 ± 2.8Nav 1.212 ± 0.912.7 ± 1.113 ± 1.5n.a.n.a.n.a.15.2 ± 2.230 ± 313.2 ± 1Nav 1.37.8 ± 0.611 ± 1.217 ± 2.1−7 ± 1n.a.n.a.22 ± 3.534 ± 6.113 ± 2.6Nav 1.46.3 ± 0.84.5 ± 0.68.5 ± 1.6−6.1 ± 1−6.4 ± 0.9n.a.n.a.n.a.15.2 ± 2.5Nav 1.5Var18 ± 131 ± 1.8−11 ± 1n.a.n.a.n.a.30 ± 2.325 ± 1.8Nav 1.69.8 ± 0.616 ± 1.311 ± 0.9−8 ± 0.4−5 ± 0.5n.a.30 ± 2.940 ± 5.638 ± 7 Open table in a new tab The Effects of ATX-II, AFT-II, and Bc-III on Nav1.2—The results of experiments similar to those reported for Nav1.1 are shown in Fig. 5 for Nav1.2 channels. Once again, the action of ATX-II (n = 3) was seen at much lower concentrations than that of AFT-II and Bc-III (n = 4). Although at high concentrations ATX-II and Bc-III often led to a slight increase in peak currents with respect to control, the effect of AFT-II on peak currents was more marked (increase of the order of 60 ± 5.4% (n = 3)) and, in addition, a large persistent component was induced that was not inactivated at -20 mV (Fig. 5H, insets). The Effects of ATX-II, AFT-II, and Bc-III on Nav1.3—Similar experiments were also performed for the Nav1.3 channel (see Fig. 6). The three toxins were effective only at relatively higher concentrations. As noted for Nav1.2, only AFT-II induced a large increase in peak current (see Fig. 6H, insets). Persistent currents are typical of this channel. Only ATX-II led to a negatively shifted inactivation curve of the slow component. The Effects of ATX-II, AFT-II, and Bc-III on Nav1.4—The effect of the three toxins on the Nav1.4 channel are shown in Fig. 7. The inactivation time constant under control conditions was 0.5 ± 0.15 ms (n = 7), but this increased ∼10-fold following the application of the toxins. Nevertheless, the slow component time constants for Nav1.4 are the lowest among the subtypes tested (see Table II). It is worth noting that both ATX-II and AFT-II induced a non-parallel leftward shift of the inactivation curve that was more pronounced toward the most negative region and that the recordings were devoid of any persistent component. Only Bc-III generated a small steady-state component. The Effects of ATX-II, AFT-II, and Bc-III on Nav1.6—Unlike those observed for the Nav1.5 channels, the results obtained for Nav1.6 channels (see Fig. 8) are similar to those found for Nav1.3 channels including the appearance of a persistent component during activation, a remarkable right shift of voltage-dependent inactivation and the leftward shift of the slow component. This finding suggests that these effects are less characteristic of the toxins themselves but are probably more related to the structure of the specific channel subtype. The effects of ATX-II on peak currents were considerable, whereas AFT-II and Bc-III induced only minor increases. Comparative Effects of the Inactivation Process on the Whole NavSeries—In general (with the exception of Nav1.5), the time constants of the toxin-induced slow inactivation component did not vary as a function of toxin concentration (see first three columns of Table II). It can be seen that Bc-III produced the relatively highest time constant values, but Nav1.4 is an exception and Nav1.2 was affected to the same extent by all three toxins. The same table also shows the shifts (on the voltage axis) of the voltage-dependent inactivation curves for both the slow and the steady-state components. The shift of the slow component was almost always negative (with the exception of Bc-III on Nav1.1), whereas the shifts of steady-state components were always positive (note that Nav1.6 was greatly affected by the three toxins). Dose-response Curves—To quantify and compare the effects of the three toxins on the various Na+ channels, we evaluated the amplitude of the slow and steady-state toxin-induced components in relation to drug concentrations. All of the data were corrected for the amplitude of the slow component in the control recordings (normally <7-8%) and normalized to the control peak amplitude in each cell. The resulting dose-response curves (ATX-II (▪), AFT-II (□), and Bc-III (○)) are shown in Fig. 9. We sometimes were unable to reach the maximal effect when the toxin potency was too low, because there was insufficient toxin material. Steady-state components were not observed with some subtypes, and the fact that this always led to a marked rightward shift of the inactivation curves suggests that this is probably due to changes in the biophysics of the channel as a result of binding to sites other than those responsible for slowing the inactivation. Simple visual inspection indicated that some dose-response curves were not sufficiently good to fit the classical logistic equation reasonably; thus only an approximate EC50 is given. For all of the other data, the results of the fitting procedure are shown in Table III for the slow component and Table IV for the steady-state component.Table IIIDose-response curve data for the fractional increase in the slow componentATX-IIAFT-IIBc-IIINav1.1A0.56 ± 0.020.67 ± 0.02Estimated ∼ 0.6EC506.01 ± 0.46390.55 ± 30.75Estimated ∼ 300p1.86 ± 0.092.46 ± 0.24n.a.Nav1.2A0.64 ± 0.021.42 ± 0.220.65 ± 0.08EC507.88 ± 0.481998 ± 519.31449.17 ± 216.47p3.33 ± 0.411.31 ± 0.112.86 ± 0.64Nav1.3A0.96 ± 0.091.91 ± 0.070.4 ± 0.02EC50759.22 ± 99.9459.36 ± 48.131458.42 ± 128.52p2.13 ± 0.371.85 ± 0.122.93 ± 0.51Nav1.4A1.15 ± 0.030.91 ± 0.041.26 ± 0.09EC50109.49 ± 7.0130.62 ± 3.46820.84 ± 144.31p1.41 ± 0.121.64 ± 0.231.16 ± 0.13Nav1.5A1 ± 0.031.14 ± 0.041.02 ± 0.04EC5049.05 ± 3.3162.5 ± 4.05307 ± 32.5p1.52 ± 0.122.5 ± 0.421.72 ± 0.21Nav1.6AEstimated ∼ 0.9Estimated ∼ 0.65Estimated ∼ 1.4EC50Estimated ∼ 180Estimated ∼ 300Estimated ∼ 900pn.a.n.a.n.a. Open table in a new tab Table IVDose-response curve data for the fractional increase in the steady-state componentATX-IIAFT-IIBc-IIINav1.2A0.17 ± 0.03n.a.n.a.EC5026.9 ± 16.47p1.1 ± 0.4Nav1.3An.a.0.64 ± 0.02n.a.EC50580.32 ± 51.7p1.48 ± 0.14Nav1.6A0.32 ± 0.020.61 ± 0.070.64 ± 0.08EC50368.72 ± 56.71301.9 ± 85.5683.3 ± 210.6p1.13 ± 0.121.33 ± 0.321.15 ± 0.24 Open table in a new tab As shown in Table III, ATX-II is highly potent at Nav1.1 and Nav1.2 with an EC50 of ∼7 nm (n = 6). In contrast AFT-II, which is only a single amino acid different, has only weak efficacy for these subtypes and has greatest efficacy for Nav1.4 and Nav1.5 (EC50 of ∼30 and 62 nm, respectively). AFT-II and ATX-II had similar efficacies at both Nav1.5 (∼55 nm) and at Nav1.6 (∼240 nm). Bc-III has the lowest efficacy of all of the toxins with the greatest effects being on Nav1.1 and Nav1.5 (EC50 values of ∼300 nm). In conclusion, the following EC50 rank order was found: ATX-II, Nav1.1-Nav1.2 ≪ Nav1.5 < Nav1.4 < Nav1.6 < Nav1.3; AFT-II, Nav1.4 < Nav1.5 < Nav1.6 < Nav1.3-Nav1.1 < Nav1.2; and Bc-III, Nav1.5-Nav1.1 < Nav1.4-Nav1.6 < Nav1.2-Nav1.3. The greatest efficacy (measured as the increase in the slow component) was obtained by AFT-II in the Nav1.3 channel, 1.91 (as against 0.67 in Nav1.1 and 1.42 in Nav1.2) with 0.6 for the steady-state component. The toxins also induced large increases in this component in Nav1.6. Previous stud