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Cn11, the first example of a scorpion toxin that is a true blocker of Na⁺ currents in crayfish neurons

Martha E. Ramirez-Dominguez¹, Timoteo Olamendi-Portugal¹, Ubaldo Garcia², Consuelo Garcia¹, Hugo Arechiga³ and Lourival D. Possani^1,*

¹ Department of Molecular Recognition and Structural Biology, Biotechnology Institute, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Cuernavaca 62210, México,
² Department of Physiology, Biophysics and Neurosciences, Centro de Investigación y de Estudios Avanzados del I.P.N. México DF 07000 and
³ Division of Graduate Studies and Research, Medical School, Universidad Nacional Autónoma de México, Ciudad Universitaria, México DF 04510

View larger version (20K): [in a new window]   Fig. 1. Purification of toxin Cn11. Sub-fraction II-5 (1 mg), obtained by previous separation of soluble venom of the scorpion Centruroides noxius, was applied to an HPLC C4 reverse-phase column (Vydac, Hisperia, CA, USA) and eluted with a linear gradient of solvent A (0.12 % trifluoroacetic acid in water) to 60 % solvent B (0.1 % trifluoacetic acid in acetonitrile) over 60 min. Component 4 (140 µg) was further separated by HPLC using a C18 reverse-phase column (Vydac) by application of 25 % solvent B (inset). The asterisk indicates the elution position of the pure toxin.

View larger version (8K): [in a new window]   Fig. 2. Complete amino acid sequence of Cn11. The N-terminal amino acid sequence up to residue 20 (underlined –d) was obtained by direct sequencing of a sample of native peptide, whose sequence was confirmed up to residue 34 with a reduced and carboxymethylated sample (underlined –RCM). Four additional sub-peptides obtained by enzymatic cleavage of alkylated toxin, followed by HPLC separation (as in Fig. 1, data not shown), were sequenced. They are under-labelled Tryp1 (positions 39–52) and Tryp2 (positions 55–62), for trypsin digestion and AspN1 (positions 20–44) and AspN2 (positions 51–61), for aspartic-N endopeptidase digestion. The last residue in position 63 was determined by mass spectrometry (under-labelled ms).

View larger version (14K): [in a new window]   Fig. 3. Blockade of Na+ currents by Cn11 in crayfish neurons. (A) After 3 min of establishing the whole-cell configuration, inward Na+ currents were elicited by test depolarizations to 0 mV from –60 mV, each for 15 s. The first three traces were averaged and taken as the control value in the absence of toxin. Subsequent traces were obtained during perfusion of the preparation with 1 µmol l–1 Cn11. The traces show how the current decreases over time. (B) Cn11 blocks the current without affecting its kinetics, as shown by superimposition of the time course of control Na+ currents and those in the presence of toxin (scaled up fourfold), which blocked 75 % of the control current (labelled Im 4x).

View larger version (21K): [in a new window]   Fig. 4. Time course of the blockade of Na+ currents by Cn11 and determination of Km. (A) Under control conditions, the current amplitude decreases steadily by approximately 10 % during the first 160 s, and this is taken as a normal rundown (open circles). Subsequent curves were obtained after superfusion with the toxin at various concentrations: filled squares, 50 nmol l–1; open squares, 100 nmol l–1; open diamonds, 200 nmol l–1; filled diamonds, 500 nmol l–1. Normalized current (Im) was obtained as I/Im. After 160 s, the preparation was superfused with the external solution. The current amplitude decreased in a concentration-dependent manner, with no apparent recovery. (B) The percentage of Na+ current blockade plotted against Cn11 concentration, 70 s after application of the toxin, corrected for 5 % rundown. The points were fitted to a Boltzman’s equation, giving a Km of approximately 320 nmol l–1.

View larger version (20K): [in a new window]   Fig. 5. The blockade of the Na+ current by Cn11 is not voltage-dependent. Current/voltage relationships were obtained under control conditions (open circles) and 3 min after the application of 1 µmol l–1 (filled squares) or 3 µmol l–1 (open squares) Cn11. The inward currents were elicited by depolarizing from a holding potential of –60 mV to +50 mV, in increments of 10 mV, with pulses of 10 ms duration. Currents start at approximately –40 mV and reach a maximum value of approximately 0 mV. I/Im, normalized Na+ current.

View larger version (30K): [in a new window]   Fig. 6. Pairwise comparison of identity between Cn11 and the other 10 representative groups of scorpion toxin specific for Na+ channels. The amino acid sequence of the 10 representative examples of each sub-group of Na+ channel scorpion toxin was compared with the amino acid sequence of Cn11 (the eleventh group proposed here). AaHII is toxin II from Androctonus australis Hector, the prototype of an -scorpion toxin; CssII is toxin II from Centruroides sufussus sufussus, the prototype of a ß-scorpion toxin; Tsgamma is toxin gamma from Tityus serrulatus; LqhIT2 is insect toxin 2 from Leiurus quinquestriatus hebraeus; LqqIV is toxin IV from Leiurus quinquestriatus quinquestriatus; LqqIII is toxin III from the same scorpion; AaHIT4, toxin 4, is an insect toxin from Androctonus australis Hector; CsEv3 is variant 3 from Centruroides sculpturatus Ewing; Cn10, toxin 10, is an insect toxin from Centruroides noxius Hoffmann; AaHIT is an insect toxin from Androctonus australis Hector; and Cn11 is from this study (data from Possani et al., 1999a, 2000, 2001). Gaps (dashes) were introduced to enhance similarities. Cysteine residues are in bold type. The right-hand column indicates the percentage identity of the pairwise comparison.