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Pharmacological Profiling of Orthochirus scrobiculosus Toxin 1 Analogs with a Trimmed N-Terminal Domain

Laboratoire Cellpep S.A., Marseille, France; Centre National de la Recherche Scientifique Formation de Recherche en Evolution 2738, Marseille, France (S.M., J.-M.S.); Universität Ulm, Ulm, Germany (G.T., V.V., S.G.); Department of Medical Pharmacology and Toxicology, University of California, Davis, Davis, California (D.H., H.W.); College of Life Sciences, Wuhan University, Wuhan, China (Y.W.); AFMB, Centre National de la Recherche Scientifique UPR 9039, Marseille, France (H.D.); Institut National de la Santé et de la Recherche Médicale U607, Département Réponse Dynamique et Cellulaire, Grenoble, France; CEA, Grenoble, France; and Université Joseph Fourier, Grenoble, France (M.D.W.)

Received for publication July 25, 2005.

Accepted for publication October 18, 2005.

	Abstract

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OSK1, a toxin from the venom of the Asian scorpion Orthochirus scrobiculosus, is a 38-residue peptide cross-linked by three disulfide bridges. A structural analog of OSK1, [Lys¹⁶,Asp²⁰]-OSK1, was found previously to be one of the most potent blockers of the voltage-gated K⁺ channel Kv1.3 hitherto characterized. Here, we demonstrate that progressive trimming of the N-terminal domain of [Lys¹⁶,Asp²⁰]-OSK1 results in marked changes in its pharmacological profile, in terms of both K⁺ channel affinity and selectivity. Whereas the affinity to Kv1.1 and Kv1.3 did not change significantly, the affinity to Kv1.2 and K_Ca3.1 was drastically reduced with the truncations. It is surprising that a striking gain in potency was observed for Kv3.2. In contrast, a truncation of the C-terminal domain, expected to partially disrupt the toxin -sheet structure, resulted in a significant decrease or a complete loss of activity on all channel types tested. These data highlight the value of structure-function studies on the extended N-terminal domain of [Lys¹⁶,Asp²⁰]-OSK1 to identify new analogs with unique pharmacological properties.

View larger version (30K): [in this window] [in a new window]   Fig. 1. Amino acid sequences and chemical syntheses of OSK1, AOSK1, and analogs thereof. a, the amino acid sequences (one-letter code) of OSK1, AOSK1, and AOSK1 analogs were aligned according to the positions of their half-cystine residues (numbered from 1 to 6). The positions of half-cystine residues are highlighted in gray boxes. Mutated residues in AOSK1 (Lys16 and Asp20) and related analogs are shown in boldface type. The positions of OSK1 secondary structures (an N-terminal extended domain located before the first half-cystine residue, an -helix, and a two-stranded -sheet structure) are indicated by solid lines above the toxin amino acid sequence (1). The 3-D solution structure of OSK1 can be found in the Protein Data Bank (code 1SCO [PDB] ). b, analytical C18 reverse-phase HPLC elution profiles of folded/oxidized OSK1, AOSK1, and its N- or C-terminal truncated analogs after their purification steps. c, deduced and experimental relative molecular masses (M+H)+ of synthetic OSK1, AOSK1, and its analogs.

	Materials and Methods

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Materials. Fmoc-L-amino acids, 4-hydroxymethylphenyloxy resin, and reagents used for peptide synthesis were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). Solvents were analytical grade products from SDS (Witham, Essex, UK). L929, B82, and MEL cells stably expressing rat Kv1.2, mouse Kv1.3, human Kv1.5, and mouse Kv3.1 have been described previously (Grissmer et al., 1994). Cells lacking leukocyte tyrosine kinase (LTK cells) expressing human Kv1.4 were obtained from Professor Michael Tamkun (Colorado State University, Fort Collins, CO), Chinese hamster ovary cells expressing mouse Kv1.7 were from Vertex Pharmaceuticals (San Diego, CA). Human embryonic kidney (HEK) 293 cells expressing human K_Ca1.1 were from Dr. Andrew Tinker (Centre for Clinical Pharmacology, University College London, London, UK); HEK 293 cells expressing human Kv11.1 (HERG) channel were from Professor Craig T. January (Department of Medicine, University of Wisconsin, Madison, WI); and HEK 293 cells expressing human K_Ca2.1 and K_Ca3.1 were from Dr. Khaled Houamed (University of Chicago, Chicago, IL).

Production of AOSK1 Analogs by Solid-Phase Peptide Synthesis. AOSK1 was obtained as described previously (Mouhat et al., 2004b). The AOSK1 analogs ([¹]-AOSK1, [^1-4]-AOSK1, [^1-6]-AOSK1, [^1-7]-AOSK1, [^36-38]-AOSK1, and [¹, Thr²]-AOSK1) were produced by chemical synthesis using a peptide synthesizer (model 433A; Applied Biosystems, Foster City, CA). Peptide chains were assembled stepwise on 0.25 mmol 4-hydroxymethylphenyloxy resin (1% cross-linked; 0.65 mmol of amino group per gram) using 1 mmol Fmoc-L-amino acid derivatives (Merrifield, 1986). Side chain-protecting groups for trifunctional residues were trityl for cysteine, asparagine, histidine, and glutamine; t-butyl for serine, threonine, tyrosine, aspartate, and glutamate; 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl for arginine; and t-butyloxycarbonyl for lysine. N-amino groups were deprotected by successively treating with 18 and 20% (v/v) piperidine/N-methylpyrrolidone for 3 and 8 min, respectively. After three washes with N-methylpyrrolidone, the Fmoc-amino acid derivatives were coupled (20 min) as their hydroxybenzotriazole active esters in N-methylpyrrolidone (4-fold excess). After peptides were assembled, and removal of N-terminal Fmoc groups, the peptide resins (approximately 1.5 g) were treated under stirring for 3 h at 25°C with mixtures of TFA/H₂O/thioanisole/ethanedithiol (73:11:11:5, v/v) in the presence of crystalline phenol (2.1 g) in final volumes of 30 ml per gram of peptide resins. The peptide mixtures were filtered, precipitated, and washed twice with cold diethyloxide. The crude peptides were pelleted by centrifugation (3200g for 8 min). They were then dissolved in H₂O and freeze-dried. Reduced AOSK1 analogs were solubilized at a concentration of approximately 0.6 mM in 0.2 M Tris-HCl buffer, pH 8.3, for oxidative folding (40-120 h depending on the peptide, 20°C). The folded/oxidized peptides were purified to homogeneity by reverse-phase high-performance liquid chromatography (HPLC) (C₁₈ Aquapore ODS, 20 µm, 250 x 10 mm; PerkinElmer) by means of a 60-min linear gradient of 0.10% (v/v) TFA/H₂O (buffer A) with 0 to 40% of 0.08% (v/v) TFA/acetonitrile (buffer B) at a flow rate of 6 ml/min ( = 230 nm). The purity and identity of each peptide were assessed by 1) analytical C₁₈ reverse-phase HPLC (C₁₈ Lichrospher 5 µm, 4 x 200 mm; Merck, Darmstadt, Germany) using a 40-min linear gradient of buffer A with 0 to 60% of buffer B at a flow rate of 1 ml/min; 2) amino acid analysis after peptide acidolysis [6 M HCl/2% (w/v) phenol, 20 h, 120°C, N₂ atmosphere]; and 3) molecular mass determination by matrix-assisted laser-desorption ionization-time-of-flight spectrometry.

Conformational Analyses of AOSK1 Analogs by One-Dimensional ¹H NMR. AOSK1 analogs were dissolved in a 9/1 mixture of H₂O/D₂O (v/v) at final concentrations of 50 µM. All ¹H NMR measurements were performed on a Bruker DRX 500 spectrometer equipped with an HCN probe (Bruker, Newark, DE), and self-shielded triple axis gradients were used. Experiments were performed at 300 K.

Lethality of AOSK1 Analogs in Mice. The peptides were evaluated for toxicity in vivo by determining the LD₅₀ value after intracerebroventricular injection into 20-g C57/BL6 mice (approved by the French ethics committee; animal testing agreement number 13.231 delivered by the Direction Départementale des Services Vétérinaires des Bouches-du-Rhône, Préfecture des Bouches-du-Rhône, France). Groups of six to eight mice per dose were injected with 5 µl of peptide solution containing 0.1% (w/v) bovine serum albumin and 0.9% (w/v) NaCl.

Cell Cultures and Transfections. B82, MEL, L929, and HEK cells stably expressing the above-mentioned voltage- and Ca²⁺-activated K⁺ channels and COS-7 cells used for transfection were maintained in Dulbecco's modified Eagle's medium with Earle's salts (Invitrogen, Carlsbad, CA), and 10% heat-inactivated fetal calf serum (PAA Laboratories GmbH, Linz, Austria) as described previously (Grissmer et al., 1994). Mouse Kv1.1 in a GFPIre vector (Invitrogen), human Kv1.2 in a pcDNA3/Hygro vector (Invitrogen), and rat Kv3.2 in a pcDNA3 vector (Protinac GmbH, Hamburg, Germany) were transfected into COS-7 cells either alone or with a green fluorescent protein-expressing construct using FuGene6 Transfection Reagent (Roche Diagnostics, Mannheim, Germany) according to the recommended protocol. Currents were recorded 1 to 3 days later in green fluorescent protein-positive cells.

Electrophysiology. Electrophysiological experiments were carried out at 22 to 24°C using the patch-clamp whole-cell recording mode (Hamill et al., 1981; Rauer and Grissmer, 1996). Cells were bathed with mammalian Ringer's solution: 160 mM NaCl, 4.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, and 10 mM HEPES, pH 7.4 with NaOH, with an osmolarity of 290 to 320 mOsM. When AOSK1 analogs were applied, 0.1% bovine serum albumin was added to the Ringer's solution. A syringe-driven perfusion device was used to exchange the external recording bath solution. Two internal pipette solutions were used: one for measuring voltage-gated K⁺ currents that contained 155 mM KF, 2 mM MgCl₂, 10 mM HEPES, and 10 mM EGTA, pH 7.2 with KOH, with an osmolarity of 290 to 320 mOsM; the other was for measuring Ca²⁺-activated K⁺ currents that contained 135 mM potassium aspartate, 8.7 mM CaCl₂, 2 mM MgCl₂, 10 mM EGTA, and 10 mM HEPES, pH 7.2 with KOH, with an osmolarity of 290 to 320 mOsM. A free [Ca²⁺]_i value of 1 µM was calculated. All currents through voltage-gated K⁺ channels were elicited by 200-ms depolarizing voltage steps from -80 to +40 mV. Potassium currents through K_Ca1.1, K_Ca2.1, and K_Ca3.1 were elicited by 1 µM internal [Ca²⁺]_i and 200-ms voltage ramps from -120 to +40 mV. Electrodes were pulled from glass capillaries (Science Products, GmbH, Hofheim, Germany) and fire-polished to resistances of 2.5 to 5 M. Membrane currents were measured with an EPC-9 or EPC-10 patch-clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany) interfaced to a computer running acquisition and analysis software (Pulse and PulseFit). When voltage-gated K⁺ currents were measured, the capacitive and leak currents were subtracted using a P/10 procedure. Series resistance compensation (>80%) was used for currents greater than 2 nA. The holding potential was -80 mV in all experiments. Data analyses were performed with IgorPro (WaveMetrics, Lake Oswego, OR), and IC₅₀ values were deduced by fitting a modified Hill equation to the data (I_toxin/I_control = 1/[1 + ([AOSK1 analog]/IC₅₀)], where I is the peak current (for voltage-gated K⁺ channels) or the slope of the ramp current (for Ca²⁺-activated K⁺ channels) to the normalized data points obtained with at least four different AOSK1 analog concentrations.

	Results

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Syntheses of AOSK1 Analogs. A comparison of the amino acid sequences of OSK1, AOSK1, and its analogs is provided in Fig. 1a. AOSK1 is an OSK1 analog with two mutations in positions 16 (acidic Glu¹⁶ replaced by basic lysine) and 20 (basic Lys²⁰ replaced by acidic aspartic acid). Although the net global charge of AOSK1 is the same as that of OSK1, the distribution of charged amino acid residues is different between the two compounds. A previous study demonstrated that AOSK1 is a 5-fold more potent blocker of Kv1.3 than is OSK1 (Mouhat et al., 2004b). AOSK1 is also more selective for Kv1.3 than OSK1. We therefore used it as a template for the rationale design of new structural analogs in which we trimmed either the N or C terminus. In both cases, the number and relative positioning of all six half-cystine residues forming the three-disulfide bridges were preserved. The standard structural motif for the / scaffold in most scorpion toxins can be described by X_nCX_nCX₃CX_n-(G/A/S)XCX_nCXCX_n (where X represents an unspecified amino acid residue and n a variable number of residues). Boldface X_n at N- and C-terminal extremities represent the residues that were selectively trimmed in the design of AOSK1 analogs. Using this approach, four N-terminal truncated AOSK1 analogs ([¹]-AOSK1, [^1-4]-AOSK1, [^1-6]-AOSK1, and [^1-7]-AOSK1) were chemically produced that shortened the N-terminal extended domain but kept the first AOSK1 half-cystine residue intact. We further synthesized an analog in which we truncated the first N-terminal amino acid residue (Gly¹) and mutated the second amino acid residue of AOSK1 from Val² to threonine ([¹,Thr²]-AOSK1). This point mutation was selected to produce an AOSK1 analog possessing an N-terminal domain (i.e., TIINVK) that is identical with those of margatoxin and noxiustoxin, two scorpion toxins that potently block Kv1.3 (Possani et al., 1999; Rodriguez de la Vega et al., 2003). In addition, a C-terminal-truncated AOSK1 analog ([^36-38]-AOSK1) was synthesized to evaluate the importance of this domain. The latter truncates half of the second strand (amino acid sequence 32-38) of the -sheet structure, and we therefore expected it to show a reduced potency because of the reported importance of the -sheet structure for Kv channel blockage (Regaya et al., 2004). All of the peptides were assembled by solid-phase synthesis using stepwise Fmoc/t-butyl chemistry, as described previously (Merrifield, 1986). After assembly, crude reduced peptides were obtained in yields ranging from 70 to 80%. The peptides were folded/oxidized under standard alkaline conditions and purified by preparative C₁₈ reverse-phase HPLC. Homogeneity of the purified peptides was >99%, as assessed by analytical HPLC (Fig. 1b). Mass spectrometry analyses of the peptides using the matrix-assisted laser-desorption ionization-time-of-flight technique gave experimental molecular masses similar to the deduced molecular masses (Fig. 1c), validating the identity of the desired products. All peptides were also characterized and quantified by amino acid analyses after acidolysis. A complete Edman sequencing was performed for some of them to further validate the final products (data not shown). The yield of peptide synthesis ranged between 1 and 5%.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Structural analysis of AOSK1 peptides by 1H NMR. One-dimensional 1H NMR spectra of AOSK1 and its truncated analogs. The spectrum of OSK1 is shown for comparison. Only the representative amide proton regions are shown. The overall distribution of resonance frequencies suggests that all peptides have similar global folds.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Lethal activities of AOSK1 and its analogs in vivo in mice. LD50 values are provided in micrograms of peptide per kilogram of mouse. The LD50 value of OSK1 is indicated for comparison.

Comparison of Lethal Activities between AOSK1 and Its Analogs in Mice. The synthetic peptides were compared for their lethal activities by intracerebroventricular injection in mice (Fig. 3). All peptides were lethal in mice with characteristic symptoms of K⁺ channel-acting toxins, such as tremor, convulsions, and spasmic paralysis followed by death. The following order of in vivo lethality was observed (from the most to the least potent peptide): OSK1 > AOSK1 > [¹]-AOSK1 = [¹,Thr²]-AOSK1 > [^1-4]-AOSK1 > [^1-6]-AOSK1 > [^1-7]-AOSK1 >> [^36-38]-AOSK1. The greatest difference in lethality between AOSK1 and [^1-7]-AOSK1, its N-terminal most truncated analog, is 6-fold. A good correlation seems to exist between the size of the N-terminal truncation of AOSK1 and the resulting lethality of the AOSK1 peptides. This progressive decrease in lethality is believed to be associated with a decline in pharmacological potency toward vital K⁺-channel subtype(s). In contrast, removal of the last three C-terminal AOSK1 residues (Thr³⁶, Pro³⁷, and Lys³⁸) dramatically reduced toxicity and increased the LD₅₀ value 16-fold compared with AOSK1. Accordingly, a previous structural characterization of OSK1 (Jaravine et al., 1997) revealed that Thr³⁶ side chain and Lys³⁸ main chain mobility are important for the docking of OSK1 to K⁺ channels.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Block of K+ channel subtypes by AOSK1, N-terminal-truncated [1-7]-AOSK1, and C-terminal-truncated [36-38]-AOSK1. a, representative current traces in the absence (control) and presence (from left to right) of AOSK1, [1-7]-AOSK1 or [36-38]-AOSK1. The concentration of applied peptide is provided on the figure. From top to bottom: representative Kv1.1, Kv1.2, Kv1.3, and KCa3.1 potassium currents. b, average normalized current inhibition by various concentrations of AOSK1 (), [1-7]-AOSK1 (), or [36-38]-AOSK1 () for each channel type (Kv1.1, Kv1.2, Kv1.3, and KCa3.1 channels, as indicated). Each data point represents the mean value ± S.D. of four experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Impact of N- and C-terminal truncations on blocking potencies of AOSK1 toward Kv1.1, Kv1.2, and Kv1.3 channels. AOSK1 versus its analogs: factors of increase in IC50 values for Kv1.1 (top), Kv1.2 (middle), and Kv1.3 channels (bottom). For each of these channel subtypes, the IC50 value of AOSK1 was normalized to 1 for the sake of comparison. The same scale was used for all three-channel types, illustrating the more drastic effects of truncations on Kv1.2 current blockage.

We further evaluated [¹,Thr²]-AOSK1 because its N-terminal domain is identical with the Kv1.3-blocking scorpion toxins [-KTx2.1 (noxiustoxin) and -KTx2.2 (margatoxin)] (Tytgat et al., 1999; Rodriguez de la Vega et al., 2003). [¹,Thr²]-AOSK1 was as potent as [¹]-AOSK1 on Kv1.3, but its affinity further decreased by more than 3-fold for Kv1.2 compared with [¹]-AOSK1 to finally give a 34-fold difference in affinity for this channel compared with AOSK1. As such, [¹,Thr²]-AOSK1 has a better selectivity than [¹]-AOSK1 for Kv1.3. The N-terminal truncation strategy thus proves valuable for generating analogs with modified selectivity profiles and, possibly, with increased affinity toward specific channel subtype(s). Because AOSK1, but not OSK1, has a weak affinity for Kv3.2, we wondered whether any of the truncated AOSK1 analogs might be more potent Kv3.2 blockers than AOSK1. Figure 6 illustrates that two analogs, [¹]-AOSK1 and [¹,Thr²]-AOSK1, are able to block Kv3.2 currents more potently than AOSK1. The increases in affinity were 11- and 15-fold for [¹]-AOSK1 and [¹,Thr²]-AOSK1, respectively. Further truncations of AOSK1 had the opposite effect and resulted in a complete loss of activity on Kv3.2, which is in line with observations made for other voltagegated K⁺ channels (Fig. 4).

View larger version (17K): [in this window] [in a new window]   Fig. 6. Increased blocking potencies of [1]-AOSK1 and [1, Thr2]-AOSK1 on Kv3.2 channel. Representative current traces in the absence (control) and presence of OSK1, AOSK1, [1]-AOSK1, [1,Thr2]-AOSK1, [1-7]-AOSK1, or [36-38]-AOSK1. The concentration of applied peptide is provided for each. Note that OSK1 is inactive on Kv3.2 at micromolar concentration, contrary to AOSK1, and [1]-AOSK1 and [1,Thr2]-AOSK1 are more potent than AOSK1 on Kv3.2 channel.

Comparison for each peptide between IC₅₀ values on individual channels and LD₅₀ values indicates an interesting trend worth discussing. In brief, it seems that the two most important channels for lethality in mouse brain are Kv1.1 and Kv1.2, as noticed previously (Mouhat et al., 2004b). It also seems that pharmacological activity on Kv3.2 is not essential for lethal potency. A tendency for a greater implication of Kv1.1 over Kv1.2 in the peptide-induced lethal effects is also evidenced. However, it should be noted that lethality probably involves other uncharacterized channels making such comparisons difficult.

	Discussion

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The scorpion toxin OSK1 serves as a good lead for the design of new Kv1.3 channel blockers because of its potent action on this channel type, with an IC₅₀ value of 14 pM. OSK1's ability to also block other voltage-gated K⁺ channels such as Kv1.1 and Kv1.2 (in the low nanomolar concentration range) and one Ca²⁺-activated K⁺ channel (K_Ca3.1) with moderate activity (at high nanomolar concentration) means that the toxin displays an unusual wide range of channel targets. AOSK1 is an even more interesting lead for further derivatization because it is one of the most potent Kv1.3 channel blockers described so far (Middleton et al., 2003; Mouhat et al., 2004b). Compared with other published compounds acting on Kv1.3, AOSK1 displays specificity factors of 133 (over Kv1.1; Table 1) and 987 (over Kv1.2), which thus seems to be similar to ShK-Dap²² (Kalman et al., 1998) but better than ShK-F6CA (Beeton et al., 2003) or ShK(L5) (Beeton et al., 2005). In addition, AOSK1 displays an even larger pharmacological profile through its additional action on Kv3.2. Therefore, AOSK1 is, to our knowledge, the second Kv3.2 channel blocker reported hitherto, after the sea anemone toxin ShK acting in the low nanomolar concentration range (Yan et al., 2005). Here, a strategy of domain trimming was used for the first time to generate new pharmacological profiles.

Several conclusions can be drawn from the experimental data obtained with these AOSK1 analogs. First, trimming in the C-terminal region of AOSK1 is expected to alter its -sheet structure (Jaravine et al., 1997) and thereby reduces activity on all tested target channels. Second, trimming of the N-terminal region seems to be a more attractive strategy because the changes in activity differed greatly from one channel subtype to the other. Indeed, Kv1.1 and Kv1.3 channels were far less susceptible to decreases in analog affinity than was Kv1.2. It is interesting that a limited trimming of the AOSK1 N terminus turned out to be an interesting route for producing analogs with increased activity toward Kv3.2. We surmise that limited truncation may facilitate the access to the binding site of the peptides in the outer vestibule of the channel. As such, this indicates that some toxins can be inactive for a given channel type because of accessibility problems and perhaps not because they do not recognize a binding site on the channel. In future experiments, it will be interesting to combine various experimental strategies to obtain still more powerful toxin-derived drugs acting on specific voltage-gated and Ca²⁺-activated potassium channels. In particular, structural determination and docking simulations of truncated AOSK1 peptides on the various target K⁺ channels will be greatly helpful for this purpose. The challenge is to produce more selective AOSK1 analogs with high potencies on the Kv1.3 channel, with or without potent activity on K_Ca3.1 channel. In the end, such structure-function studies on AOSK1 may be useful for the production of potent immunosuppressive drugs. Combining N-terminal truncations with point mutations is clearly a powerful way to create new selective toxin-derived peptide blockers for Kv1.3, Kv1.1, K_Ca3.1, and Kv3.2 channels.

	Acknowledgements

 
We thank Dr. P. Mansuelle for Edman sequencing and amino acid analysis of AOSK1 peptides. Drs. B. De Rougé and E. Béraud are acknowledged for helpful discussions.

	Footnotes

 
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.017210.

ABBREVIATIONS: OSK1, toxin 1 from the scorpion Orthochirus scrobiculosus; AOSK1, [Lys¹⁶,Asp²⁰]-OSK1; [¹]-AOSK1, AOSK1 deleted of the amino acid residue at position 1; [^1-4]-AOSK1, AOSK1 deleted of the four N-terminal residues; [^1-6]-AOSK1, AOSK1 deleted of the six N-terminal residues; [^1-7]-AOSK1, AOSK1 deleted of the seven N-terminal residues; [^36-38]-AOSK1, AOSK1 deleted of the last three C-terminal residues; [¹,Thr²]-AOSK1, AOSK1 deleted of the amino acid residue at position 1 and mutated at position 2 by a threonine residue; Fmoc, N-fluoren-9-ylmethyloxycarbonyl; TFA, trifluoroacetic acid; HPLC, high-performance liquid chromatography; Kv, mammalian voltage-gated K⁺ channels (Kv1.1 to Kv1.7); K_Ca2.1 (also referred to as SK1), type-1 small-conductance Ca²⁺-activated K⁺ channel; K_Ca2.2 (also referred to as SK2), type 2 small-conductance Ca²⁺-activated K⁺ channel; K_Ca3.1 (also referred to as IK1), type 1 intermediate-conductance Ca²⁺-activated K⁺ channels; K_Ca1.1 (also referred to as BK), large-conductance Ca²⁺-activated K⁺ channel; Kv11.1 (also referred to as HERG), human ether-a-go-go-related K⁺ channel; HEK, human embryonic kidney.

Address correspondence to: Dr. Jean-Marc Sabatier, Centre National de la Recherche Scientifique Formation de Recherche en Evolution 2738, Boulevard Pierre Dramard, 13916 Marseille Cedex 20, France. E-mail: sabatier.jm{at}jean-roche.univ-mrs.fr

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