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 at (4Sunahar S. Muramoto K. Tenma K. Kamiya H. Toxicon. 1987; 25: 211-219Crossref PubMed Scopus (44) Google Scholar). of the were of the and water was used of Bc-III was using to and Toxicon. PubMed Scopus Google Scholar). human Nav1.1, and in with 10% were obtained by with the corresponding S. S. S. PubMed Scopus Google and of and were as described M.A. 2000; Google Scholar, M.A. PubMed Scopus Google Scholar). was from the described in et al. 2001; PubMed Scopus Google was using a human from from human and and was using a human from from a from human were obtained of a containing the kind gift from of and extracellular 5 10 and 5 The 10 10 and 10 of the toxins were dissolved in the extracellular the were tetrodotoxin was used at nm Nav1.1, and and the were from to the The which has a tetrodotoxin higher showed at the The extracellular were a linear with an of that was the under and were recorded at room temperature by means of the as described L. M. Scopus Google with a of and were for each was to the to of the The was was by means of a in which a was from a of to a range of from (or to (or in or for by a to (or The peak current during the were to the of the first and against the of the The data were using two and a at the current the of the was to the obtained in from was using a which with from a of to (or for (or that of the channels. was by an at and a to (or The in were and in the and Inc.) were used during data and Bc-III and AFT-II and chromatography of B. caissarum venom six peaks the and was further purified by reverse-phase high performance liquid chromatography to the toxin Bc-III analysis showed that the mass of Bc-III was mass (5Malpezzi E.L.A. Freitas J.C. Muramoto K. Kamiya H. Toxicon. 1993; 31: 853-864Crossref PubMed Scopus (51) Google and that the determination of 25 N-terminal amino acid by Edman degradation confirmed the of the toxin Bc-III 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 of AFT-II was by mass and mass was also confirmed mass The of Bc-III in was to that for AFT-II (4Sunahar S. Muramoto K. Tenma K. Kamiya H. Toxicon. 1987; 25: 211-219Crossref PubMed Scopus (44) Google acid sequence of ATX-II, and AFT-II compared with sea anemone toxins numbers to in a new of of ATX-II on and under and all of the were performed at using the and during at was the is well known that the action of sea anemone is to the we used capable of these properties and compared at the same under the and during toxin The action of ATX-II on the channel was the used to the for the data and the of the action of toxin showed in first that the is in at a with a fraction of the of channels on each We this and the data on the that each Na+ current is the of two and a the of the component was and the two were low or During toxin was a in the component in some on the toxin an in a persistent We also that the of of the was not different whether we used a or that the recorded in the presence of the toxin were the of two of those from channels and those from channels and thus to is in at and nm a at in the presence of 5 nm toxin was into a and component The and in these is in for different which that the contribution of and channels in to the toxin this we investigated the from at at the by the of the toxin and toxin can be that was any of the component during of the of the of from in the The of the component at the various was as a of the and compared with the as in can be that the toxin induced a of the component in with the inactivation. of the component at an (from to this was not investigated The of AFT-II and Bc-III on effects of the toxins AFT-II and Bc-III on are in the six the data are divided the two for AFT-II and Bc-III The 3, A and B) the data with the data at various of the in a The 3, and the data for the from from one are for the toxin and the component The 3, and the data for the of of the and component under and at the The the from a of the under and at the data suggest that the action of AFT-II is to that of ATX-II and that Bc-III has a for this type of Interestingly, the obtained with these two new toxins differ from those obtained with ATX-II, as was a persistent component by were used to the data obtained with the three toxins on the Na+ in the different the Nav1.1, Nav1.2, Nav1.3, and Nav1.6 sodium channel genes as described in the The effects of ATX-II, AFT-II, and Bc-III on of on the Nav1.1 channel are in the nine the data are divided in three for ATX-II AFT-II and Bc-III The first the of different toxin from a The the data for the from from one in and the The the data for the of in the and and The the from a for the under and at the used the in the The of the component in not on the toxin or the the range During at and AFT-II and Bc-III induced that were persistent but not in the and that not when at persistent a in with the and component and 131 in a new The of ATX-II, AFT-II, and Bc-III on of to those for Nav1.1 are in 5 for channels. the action of ATX-II was at that of AFT-II and Bc-III at high ATX-II and Bc-III to a in peak with to the of AFT-II on peak was of the of in a persistent component was induced that was not at The of ATX-II, AFT-II, and Bc-III on were also performed for the Nav1.3 channel The three toxins were only at higher As for Nav1.2, only AFT-II induced a in peak current are of this ATX-II to a of the The of ATX-II, AFT-II, and Bc-III on of the three toxins on the Nav1.4 channel are in The under was but this following the of the the component for Nav1.4 are the the subtypes is that ATX-II and AFT-II induced a of the that was the most and that the were of any persistent Bc-III a The of ATX-II, AFT-II, and Bc-III on those for the channels, the obtained for Nav1.6 channels are to those found for Nav1.3 channels the of a persistent component during a of and the of the that these effects are of the toxins but are to the of the specific channel The effects of ATX-II on peak were whereas AFT-II and Bc-III induced only of the on the (with the exception of the of the component not as a of toxin first three of can be that Bc-III the but Nav1.4 is an exception and was to the same by all three The same also the the of the curves for the and the The of the component was (with the exception of Bc-III on whereas the of were positive that Nav1.6 was by the three and the effects of the three toxins on the various Na+ channels, we the of the and in to of the data were for the of the component in the and to the peak in each The dose-response curves (ATX-II AFT-II and Bc-III are in We were to the when the toxin potency was was toxin were not with some and the that this to a of the curves that this is to in the of the channel as a of binding to sites those responsible for the inactivation. that some dose-response curves were not to fit the thus only an is all of the the of the are in for the component and for the data for the in the in a new data for the in the in a new As in ATX-II is highly at Nav1.1 and with an of nm AFT-II, which is only a amino acid has only for these subtypes and has for Nav1.4 and of and AFT-II and ATX-II at nm) and at Nav1.6 Bc-III has the of all of the toxins with the effects being on Nav1.1 and of the following was ATX-II, Nav1.4 Nav1.6 AFT-II, Nav1.4 Nav1.6 and The as the in the was obtained by AFT-II in the Nav1.3 against in Nav1.1 and in with for the The toxins also induced in this component in (3Béress L. Béress R. Wunderer G. FEBS Lett. 1975; 50: 311-314Crossref PubMed Google of ATX-II have that it is specific for cardiac or voltage-gated Na+ channels, but specific was known at that ATX-II the of the cardiac action by the contribution of the Na+ current to the G. M. M. Google and has been used by a of to investigate ability to persistent sodium S. T. C. 2000; PubMed Scopus Google Scholar, M. S. G. Scopus Google Scholar). of the different effects of sea anemone peptides on sodium channels have been M. PubMed Scopus Google Scholar, C. T. L. L. Alvarez 2001; PubMed Scopus Google Scholar, PubMed Scopus Google and various have been used to dose-response have the in the of the and have the of the current the of or the in the is the of and channels, we used the first for the and persistent D. PubMed Scopus Google Scholar). The in Na+ channel induced by Bc-III and AFT-II have been and we compared their effects on six Na+ channel subtypes. have highly but differing amino acid such that differences in the effects of any toxin should be directly to these sequence this sequence it should also be that the of Na+ channels is with the it has been that the of the in α-subunits not the T. Catteral W.A. Scopus Google Scholar). it is known that is expressed in human in this M. F. FEBS Lett. 2000; PubMed Scopus Google Scholar). in our we not investigate the of the of the by the of at the IV/S3-S4 of have that sea anemone toxins the purified from that with a acid residue in on the extracellular loop of the of the channel J.C. T. Catteral W.A. PubMed Scopus Google Scholar). that this amino acid has an in and channels (Nav1.5, and also has a in in Nav1.4 and The found that the the of ATX-II from to nm and that the the (from to they not the of but suggest that the of or to ATX-II and thus a for the of the channel and the of the and sequences of channels in the and extracellular loop of the in a new As these in Nav1.4 and our data of and nm in Nav1.4 and in with nm in suggest that is the which has only the first in in also higher for ATX-II. channel is to it has the in of the in thus suggesting a further of toxin Interestingly, which is by ATX-II has the of the but the is the same amino acid as it is in with the potency of ATX-II in Nav1.3 be by the that amino acids can the The of a to a residue is which is with the that a of should a for ATX-II J.C. T. Catteral W.A. PubMed Scopus Google Scholar). AFT-II the Nav1.4 and channels and has effects on the channels. the amino acid ATX-II and these the that the in is for the effects of ATX-II on channels. Bc-III has a different amino acid sequence from that of ATX-II and AFT-II in the of and which it is ATX-II. is that a report F. H. PubMed Google has that sea anemone (from Bunodosoma and toxins have a common of in their we the by et al. S. B. M. PubMed Scopus Google the fundamental and the of and of (corresponding to the positive of and of should be in the loop the first and the and the and the of the by H. M. K. PubMed Scopus Google Scholar). is located the amino acids and of ATX-II, and it be that this positive a in binding the toxin to the of the IV/S3-S4 in the positive in this suggest in Bc-III new toxin has effects on all of the investigated channels. the and which are toxins to have been to the of the cardiac action with the of and respectively C. T. L. L. Alvarez 2001; PubMed Scopus Google Scholar). in the these become and and have only one to be important for and Bc-III has the same in and can be considered to and et al. F. FEBS Lett. PubMed Scopus Google a potency of which is different from that of the presence of the or the different the it is our as the majority of the the important of the loop of are also is by the that Nav1.6 has in and and amino acids in ATX-II and AFT-II have only thus suggesting that with of the channel are different with to on the by the of the that either or Asp at the of the IV/S3-S4 loop is important for sea anemone toxin binding has been further investigated by PubMed Google Scholar, PubMed Scopus Google Scholar, S. B. M. PubMed Scopus Google the toxin The of of purified from Anthopleura sea anemone that and are for PubMed Google Scholar, PubMed Scopus Google but it has been by et al. PubMed Scopus Google that the Lys-37 of with the of (corresponding to the of They found that the a in toxin a toxin a in the type channel and a in the channel ATX-II and AFT-II, the corresponding is in and by a well that is common to all of the The following amino acids is also a in ATX-II but is to an in AFT-II only the two AFT-II and are in this and our the of this hypothesized We found that AFT-II and ATX-II are on of in with data and the of the of the it is not at all confirmed in ATX-II is AFT-II on the first two channels but AFT-II on the only the two toxins is the and Nav1.1 and are also in that it is to a to at least in and not to that the amino acids of the toxin be for the Nav1.4 is only different from in this has a residue in of the in all of the channels, we to that the action of ATX-II and AFT-II be we found that AFT-II is ATX-II of which that the amino acid which is in Nav1.4 of is important for binding or that are in and data to the of in the same amino acid is also in Nav1.6 by an as in but AFT-II and ATX-II are in this channel the the amino acid in is a of the in all of the channels. we are to that the amino acids should be it is confirmed by the dose-response data in which is to to During our we found that the toxins only a which has a by a persistent component at of to the two effects can be are on this and channels are to be in this AFT-II was capable of this to a and Nav1.3 and Nav1.6 with a that the amino acid in of the toxin can a that to the persistent The that ATX-II is in in Nav1.3 AFT-II be to ability to the persistent component in the effects should suggest an that the toxins not the Nav1.3 channel in the same as ATX-II in Nav1.1 and of of have to the of a of mammalian sodium channel subtypes and to the of an of peptide toxins by animals that their effects these channels. The study of these is a to that their and should lead to an of the and mechanism of action of the channels and the toxins study is to the effects of the toxins on channel a and of the were The physiological of this with to and of and cardiac action to be we are to the of these We Dr. of the of São Paulo, for the amino acid sequences and Dr. C. V. of the Paulo, for with the purification We are to Dr. M. of the C. for suggesting the of and Dr. Koji Muramoto of the of Biological at for with We also and for the and G. for S. are at the of the of the of São R. and R. R. C. are of at the of and of the of of sea anemone toxins to Nav sodium channels. from differences in the IV/S3-S4 of Biological 10 of the should be to