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J. Biol. Chem., Vol. 283, Issue 2, 988-997, January 11, 2008

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The D-Diastereomer of ShK Toxin Selectively Blocks Voltage-gated K⁺ Channels and Inhibits T Lymphocyte Proliferation^*

Christine Beeton¹, Brian J. Smith¹, Jennifer K. Sabo, George Crossley^¶, Daniel Nugent^¶, Ilya Khaytin^¶, Victor Chi, K. George Chandy, Michael W. Pennington^¶, and Raymond S. Norton²

From the Department of Physiology & Biophysics, University of California, California, Irvine, 92697-4560, the Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia, and ^¶Bachem Bioscience, Incorporated, King of Prussia, Pennsylvania 19406

Received for publication, July 23, 2007 , and in revised form, October 31, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which are crucial in the activation of human effector memory T cells (T_EM); selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by T_EM cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. The critical motif on the toxin for potassium channel blockade consists of neighboring lysine and tyrosine residues. Because this motif is sufficient for activity, an ShK analogue was designed based on D-amino acids. D-Allo-ShK has a structure essentially identical with that of ShK and is resistant to proteolysis. It blocked Kv1.3 with K_d 36 nM (2,800-fold lower affinity than ShK), was 2-fold selective for Kv1.3 over Kv1.1, and was inactive against other K⁺ channels tested. D-Allo-ShK inhibited human T_EM cell proliferation at 100-fold higher concentration than ShK. Its circulating half-life was only slightly longer than that of ShK, implying that renal clearance is the major determinant of its plasma levels. D-Allo-ShK did not bind to the closed state of the channel, unlike ShK. Models of D-allo-ShK bound to Kv1.3 show that it can block the pore as effectively as ShK but makes different interactions with the vestibule, some of which are less favorable than for native ShK. The finding that an all-D analogue of a polypeptide toxin retains biological activity and selectivity is highly unusual. Being resistant to proteolysis and nonantigenic, this analogue should be useful in K⁺ channel studies; all-D analogues with improved Kv1.3 potency and specificity may have therapeutic advantages.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Sea anemones contain a family of polypeptide toxins that block potassium channels, the first representative of which to be isolated and characterized was ShK toxin, from Stichodactyla helianthus (1, 2). Its solution structure, determined by NMR spectroscopy (3, 4), consists of two short -helices encompassing residues 14-19 and 21-24, and an N terminus with an extended conformation up to residue 8, followed by a pair of interlocking turns that resembles a 3₁₀-helix. It contains no β-sheet and is thus distinct from the /β fold found in scorpion K⁺ channel blockers such as charybdotoxin (5) and margatoxin (6) but is similar to BgK toxin (7).

The surface of ShK involved in binding to voltage-activated (Kv) channels has been mapped using alanine scanning and selected toxin analogues (8, 9). Two residues, Lys²² and Tyr²³, are crucial for activity. Other residues in ShK also contribute to its affinity and selectivity for K⁺ channels, including Ile⁷, Arg¹¹, Ser²⁰, and Phe²⁷ (9) and His¹⁹ and Arg²⁴ (10), although the role of His¹⁹ may be at least partly structural (4).

ShK blocks K⁺ channels by binding to a shallow vestibule at the outer entrance to the ion conduction pathway and occluding the entrance to the pore. Mutational strategies similar to those employed for the scorpion toxins agitoxin and kaliotoxin (11, 12) were utilized to understand the molecular basis for channel blockade by ShK, although the availability of the KcsA crystal structure (13) allowed a more reliable model of the pore vestibule region of Kv1.3 to be constructed. ShK was docked initially with a crude model using restrained molecular dynamics simulations guided by data from mutant cycle analyses (14). In this configuration, Lys²² of ShK projected into the ion conduction pathway, and Arg¹¹ of ShK lay in the vicinity of His⁴⁰⁴ in one Kv1.3 subunit. Subsequently, the channel model was refined (15, 16), and ShK was docked using a larger number of restraints from complementary mutational analyses.

ShK blocks not only Kv1.3 (K_d = 11 pM) but also Kv1.1 (K_d = 16 pM), Kv1.6 (K_d = 165 pM) (14), and Kv3.2 (17, 18). More selective analogues have been created, such as ShK-Dap²², in which the critical Lys²² was replaced by the shorter, positively charged, non-natural residue 1,3-diaminopropionic acid (Dap) (14), ShK-F6CA, a fluorescein-labeled analogue of ShK (19), and ShK(L5), in which a Tyr(P) residue is attached through a hydrophilic linker to Arg¹ (17).

All human T lymphocytes express two types of K⁺ channels, Kv1.3 and KCa3.1, which play crucial roles in human T cell activation (20-22). The number of channels expressed by a given cell depends on its state of activation and differentiation (23). Kv1.3 channels dominate in terminally differentiated effector memory (T_EM) cells, and Kv1.3 blockers inhibit the activation of these cells, whereas KCa3.1 blockers are ineffective (23, 24). Naïve and central memory (T_CM) cells are less sensitive to Kv1.3 blockade because they rapidly up-regulate KCa3.1 channels upon activation (23). Kv1.3 blockers might therefore constitute valuable new therapeutics for the treatment of autoimmune diseases mediated by T_EM cells, such as multiple sclerosis, rheumatoid arthritis, and type 1 diabetes mellitus (23, 24). Indeed, ShK and its analogue ShK(L5) potently inhibit proliferation and cytokine production by disease-associated autoreactive T cells from patients with autoimmune diseases (23, 24). ShK, ShK-Dap²² (25), and ShK(L5) (17, 24) have been shown to prevent and treat adoptive transfer experimental autoimmune encephalomyelitis in rats, an animal model for multiple sclerosis, treat pristane-induced arthritis in rats, a model for rheumatoid arthritis, and suppress delayed-type hypersensitivity caused by skin-homing T_EM cells. ShK has a short half-life in vivo ( 30 min) (25) as a result of proteolytic degradation and/or rapid renal clearance. As a means of dissecting the relative importance of these processes and of possibly lengthening the half-life of ShK in vivo, an analogue resistant to proteolysis would be valuable. A strategy to achieve this is offered by the minimalist nature of the crucial functional motif of the toxin. The dyad of a lysine and neighboring aromatic residue (7) is conserved across toxins from several species, including anemone, scorpion, snake, and cone shell (26). The potent activity conferred by these two residues in the context of a wide variety of toxins suggests a high level of tolerance in the scaffold that presents them on the toxin surface. Therefore, a mirror image of ShK toxin in which all amino acids have the enantiomeric (mirror image) configuration but that maintains the spatial arrangement of these critical residues might also bind to and block the channel, while being completely resistant to proteolysis as a result of its inability to be recognized by endogenous proteases. In this paper we describe the synthesis, structural characterization, channel binding, and in vivo evaluation of an analogue of ShK in which all residues have the D-configuration at C. The side chain configurations were unchanged, so this analogue should be described as D-allo-ShK, although it is only for Thr and Ile that the side chain configurations differ from the exact mirror image (because each contains a chiral center). We therefore refer to it as a diastereomer of ShK toxin rather than an enantiomer.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Peptide Synthesis—Fmoc³ D-amino acids (Bachem Feinchemikalien) included D-Arg(Pmc), D-Asp(OtBu), D-Cys-(Trt), D-Gln(Trt), D-His(Trt), D-Lys(Boc), D-Ser(tBu), and D-Thr(tBu). All other non-side chain protected derivatives used for synthesis were also in the D-configuration except for Gly, which is achiral. Stepwise assembly was carried out on an Applied Biosystems 431A peptide synthesizer at the 0.25-mmol scale using Fmoc-Ramage^TM-amide-resin. Residues 34-22 were single coupled. At this point, half of the resin was removed to effect better mixing. The remainder of the peptide sequence was double coupled to the remaining resin aliquot. All of the couplings were mediated by dicyclohexylcarbodiimide in the presence of 2 eq of 1-hydroxybenzotriazole. Following final removal of the Fmoc group, the peptide resin (2.42 g) was cleaved from the resin and simultaneously deprotected using reagent K (27) for 2 h at room temperature. Following cleavage, the peptide was filtered to remove the spent resin beads and precipitated with ice-cold diethyl ether. The peptide was collected on a fine filter funnel, washed with ice-cold ether, and finally extracted with 20% AcOH in H₂O. The peptide extract was subsequently diluted into 2 liters of H₂O, the pH adjusted to 8.0 with NH₄OH and allowed to air oxidize at room temperature for 36 h. Following oxidation of the disulfide bonds, the peptide solution was acidified to pH 2.5 and pumped onto a Rainin Dynamax C₁₈ column (5.0 x 30 cm). The sample was eluted with a linear gradient from 5 to 30% acetonitrile into H₂O containing 0.1% trifluoroacetic acid. The resulting fractions were analyzed using two analytical RP-HPLC systems: trifluoroacetic acid and triethylammonium phosphate (28). Pure fractions were pooled and lyophilized. Upon lyophilization, 120 mg of D-allo-ShK toxin amide was obtained, representing a theoretical yield of 21% (from the starting resin).

Peptide Analysis—Synthetic peptide samples were hydrolyzed in 6 N HCl at 110 °C for 22 h in vacuo. Amino acid analysis was performed on a Beckman 126AA System Gold amino acid analyzer. Matrix-assisted laser desorption ionization time-of-flight mass spectroscopic analysis was performed on a Kratos Kompact mass spectrometer using -cyano-4-hydroxycinnamic acid as a matrix. Amino acid analysis of the purified D-allo-ShK showed the following average amino acid ratios: Asx (1) 0.97, Thr (4) 3.89, Ser (4) 4.04, Glx (1) 0.98, Pro (1) 0.93, Gly (1) 1.02, Ala (1) 1.00, Met (1) 0.88, Ile (2) 1.78, Leu (1) 1.01, Tyr (1) 0.99, Phe (2) 2.00, Lys (4) 3.99, His (1) 0.95, Arg (4) 3.89, and Cys (<6) 5.26.

NMR Spectroscopy—The spectra were recorded on a sample of D-allo-ShK in 95% H₂O, 5% ²H₂O at pH 4.9. Two-dimensional homonuclear total correlation spectra with a spin-lock time of 60 ms and double quantum filtered correlation NMR spectra were acquired at 500 MHz on a Bruker AMX-500 spectrometer. A two-dimensional NOESY spectrum with a mixing time of 200 ms was also acquired on a Bruker AMX-500 spectrometer. NOESY spectra for ShK were acquired at 500 and 600 MHz as described previously (3, 4); the 600-MHz NOESY was used in the comparison with D-allo-ShK. Water was suppressed using the WATERGATE pulse sequence (29). All of the spectra were collected at 20 °C unless otherwise stated and were referenced to an impurity peak at 0.15 ppm or to the water resonance.

Diffusion measurements were performed using a pulsed field gradient longitudinal eddy current delay pulse sequence (30, 31) as implemented by Yao et al. (32). The spectra were processed using XWINNMR (Version 3.5, Bruker Biospin) and analyzed using XEASY (Version 1.3.13) (33). Structural figures were prepared using VMD (34).

Proteolytic Digestion—The stability of D-allo-ShK to proteolytic digestion was investigated under the same conditions used to determine the disulfide bridges of ShK toxin (35). D-Allo-ShK (15 µg) was dissolved in 0.05 M HEPES, pH 6.5, containing 10 mM CaCl₂ (30 µl) and trypsin, chymotrypsin, or a mixture of trypsin and chymotrypsin (enzyme:substrate1:50, w/w, 30 °C, 6 h). The digestion was terminated by acidification with 10% aqueous trifluoroacetic acid (3 µl), the solution was centrifuged (13,000 x g, 5 min), and the supernatant was analyzed directly by RP-HPLC.

Modeling and Docking—An initial model of D-allo-ShK was created by inverting the structure of ShK derived by NMR (3) (Protein Data Base code 1ROO, structure 1) and correcting the side chains of threonine and isoleucine residues for the appropriate stereochemistry. Both the D-allo model and NMR-derived structures were subjected to molecular dynamics (MD) simulation using the GROMACS (v3.3.1) package of programs (36). All of the simulations consisted of an initial minimization of water molecules followed by 100 ps of MD with the peptide fixed. Following positional restraints MD, the restraints on the peptide were removed, and MD continued for a further 10 ns.

MD simulations of both diastereomers of ShK were performed using the OPLS-aa force field (37). Ionizable residues were assumed to be in their standard state at neutral pH. Each peptide was placed in a 50 x 50 x 50 ų water box with no pressure coupling. The total charge on the system was made neutral by replacing water molecules with chloride ions using the Genion program. Peptide, water, and ions were coupled separately to a thermal bath at 300 K using a Berendsen thermostat (38) applied with a coupling time of 0.1 ps. All of the simulations were performed with a single nonbonded cut-off of 10 Å, applying a neighbor list update frequency of 10 steps (20 fs). The particle mesh Ewald method was used to account for long range electrostatics, applying a grid width of 1.2 Å, and a fourth-order spline interpolation. Bond lengths were constrained using the LINCS algorithm (39). All of the simulations consisted of an initial minimization of water molecules followed by 100 ps of MD with the peptide fixed. Following positional restraints MD, the restraints on the peptide were removed, and MD continued for a further 10 ns.

Comparative models of the trans-membrane region (only) of the murine Kv1.3 channel were constructed using the x-ray structure of the K⁺ channel from Streptomyces lividans (KcsA, Protein Data Base code 1BL8 [PDB] ) as a template. The MODELLER (6v2) program (40) was used to create nine models based on the sequence alignment shown in supplemental Table S2. mKv1.3 has good sequence similarity with KcsA over the entire pore domain (32% identity), whereas there is 91% sequence identity with Kv1.2. Despite the greater sequence similarity with Kv1.2, the structure of KcsA was chosen as the template for model building because the structure of the two loops comprising the extracellular face of KcsA, the site of toxin binding, has been well characterized (41), whereas those in the more closely related channel, Kv1.2, are disordered in the electron density (42), and are therefore less suitable for model building.

Complexes of the D-allo and L forms of ShK with mKv1.3 were modeled using the ZDOCK program (43). This program uses a fast Fourier transform to explore all of the possible binding modes of the two proteins; docking was restricted to residues on the extracellular surface of the channel to ensure the exclusion of unphysical binding predictions. The interaction is evaluated using shape complementarity, desolvation energy, and electrostatics.

Models of each form of the toxin were extracted at 1-ns intervals during the MD simulation. Including the initial model, we considered 11 models of each form of the toxin. Each model of the toxin was docked with one of the nine models of the channel; thus, we considered all 99 possible combinations of toxin with channel for both D-allo and L forms of the toxin. The top 2,000 scoring predictions from each combination were then refined using the RDOCK program (44), in which the binding interface was refined using molecular mechanics minimization. The final docking predictions from all 198,000 complexes (for both forms of the toxin) were ranked according to the RDOCK scoring function.

The highest ranked complexes of both D-allo and L forms of ShK with the channel were subjected to a short 100-ps MD simulation to permit further relaxation of the atoms at the interface. The extracellular face of the complex was capped in a sphere of water molecules, with the molecules at the surface of the sphere fixed at their originally minimized positions (to prevent evaporation). Atoms of residues of the channel more than 8 Å from the ShK peptide were held fixed during these MD calculations. The structures of the complexes were minimized without restraints at the completion of the MD simulation.

The buried surface areas were calculated from the difference in surface areas of channel and toxin from the complex. The surface areas were calculated using the NACCESS program (45).

Cells and Cell Lines—L929, B82, and MEL cells stably expressing mKv1.1, rKv1.2, mKv1.3, and hKv1.5 have been described previously (46) and were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum, 4 mM L-glutamine, 1 mM sodium pyruvate, and 500 µg/ml G418 (Calbiochem). LTK cells expressing hKv1.4 were obtained from M. Tamkun (University of Colorado, Boulder, CO), CHL cells expressing mKv1.7 were from Vertex Pharmaceutical Inc (San Diego, CA), and HEK293 cells stably expressing hKCa3.1 were a kind gift from Dr. Khaled Houamed (Chicago, IL). PAS T cells, a major histocompatibility complex class II-restricted myelin basic protein-specific encephalitogenic CD4⁺ rat T cell line (47), were a kind gift from Dr. Evelyne Béraud (Marseille, France). Mononuclear cells were isolated from Lewis rat (Harlan-Sprague-Dawley, Indianapolis, IN) spleens using Histopaque-1083^TM gradients (Sigma).

Electrophysiology—The cells were studied in the whole-cell configuration of the patch clamp technique. The holding potential in all experiments was -80 mV. Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, and Kv1.7 currents were recorded in normal Ringer solution with a calcium-free pipette solution containing 145 mM KF, 10 mM HEPES, 10 mM EGTA, 2 mM MgCl₂, pH 7.2, 300 mOsm, as described previously (46). KCa3.1 currents were recorded as described previously (48). The K_d values were determined from dose-response curves shown using Microcal Origin software.

[³H]Thymidine Incorporation Assays—Rat splenocytes seeded at 2 x 10⁵ cells/well in RPMI culture medium in flat-bottomed 96-well plates (final volume, 200 µl) were preincubated with increasing concentrations of ShK or D-allo-ShK for 30 min and then stimulated with 2 µg/ml concanavalin A for 48 h. PAS T cells (2 x 10⁴ cells/well) were stimulated in the presence of 2 x 10⁶ irradiated (2500 rad) Lewis rat thymocytes as antigen presenting cells with 10 µg/ml myelin basic protein isolated from guinea pig spinal cords as described previously (49). [³H]Thymidine (1 µCi/well) was added for the last 16-18 h. The cells were harvested onto glass fiber filters, and radioactivity was measured in a β-scintillation counter.

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Analysis of D-allo-ShK. A, RP-HPLC (Vydac C18-silica 5 µm, 120 Å, 0.46 x 250 mm) analysis of crude oxidized D-allo-ShK following overnight oxidation. Gradient conditions were 5-50% B in 20 min at 1.5 ml/min (A buffer is 0.1% trifluoroacetic acid in water and B is acetonitrile containing 0.1% trifluoroacetic acid); detection is at 215 nm. The major product eluting at 10.5 min is the correctly folded peptide. B, RP-HPLC analysis of the purified product following preparative HPLC. Gradient conditions were identical to those used in A. C, matrix-assisted laser desorption ionization time-of-flight mass spectral analysis of the purified product using -cyano-4-hydroxycinnamic acid as the matrix. Theoretical mass is 4054.1 observed M+H = 4055.2. D, RP-HPLC profile of D-allo-ShK following6hof treatment with chymotrypsin and trypsin. Gradient conditions were identical with those used in A. E, amino acid sequence of ShK toxin showing location of three disulfide bridges.

To determine the circulating half-life of D-allo-ShK, known amounts of D-allo-ShK were added to Lewis rat serum, and the blocking activity on Kv1.3 channels was tested by patch clamp to establish a standard dose-response curve. Blood samples from rats were obtained from the saphenous vein (50) at various times after a single subcutaneous injection of 1 mg/kg D-allo-ShK in phosphate-buffered saline + 2% rat serum. In another series of experiments, the rats received daily subcutaneous injections of 1 mg/kg D-allo-ShK, and blood was drawn 24 h after each injection. The serum samples were tested for Kv1.3 blocking activity by patch clamp, and the levels of D-allo-ShK were determined from the standard curve as described (25).

For the DTH experiments, the rats were immunized with an emulsion of ovalbumin in complete Freund's adjuvant (Difco, Detroit, MI) (51). Seven days later, they received an injection of ovalbumin dissolved in saline in the pinna of one ear and saline in the other ear (52). The rats then received a subcutaneous injection of D-allo-ShK (1 mg/kg) or vehicle (phosphate-buffered saline + 2% rat serum). Ear swelling was measured 24 h later using a spring-loaded micrometer (Mitutoyo, Spokane, WA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Synthesis of D-Allo-ShK Amide—ShK amide consists of 35 residues with a C-terminal amide (1). Synthesis of D-allo-ShK with a C-terminal amide was initiated on Ramage amide resin using an automated protocol where the first 12 residues were single-coupled, and the remaining 22 residues were double-coupled. The biologically active form of ShK toxin requires proper folding of three disulfide bonds (53). Following cleavage and deprotection, 36 h was allowed for peptide folding and disulfide bond formation under alkaline conditions in the presence of air under the same conditions used for ShK amide (2); after 36 h there was no further change in the HPLC profile (Fig. 1). Folding proceeded smoothly to a unique, earlier eluting, major product with the same profile as ShK amide. The purified D-allo-ShK was homogeneous by analytical RP-HPLC in two different solvent systems (trifluoroacetic acid and triethylammonium phosphate) (Fig. 1). Matrix-assisted laser desorption ionization time-of-flight analysis yielded a (M+H) of 4054, consistent with the theoretical value following formation of three disulfide bonds.

Susceptibility of D-Allo-ShK to Proteolysis—Neither trypsin nor chymotrypsin nor a mixture of both proteases had any effect on D-allo-ShK, as assessed by RP-HPLC (Fig. 1D). Cleavage at basic or aromatic residues was prevented by the D-stereochemistry at C. This result was not unexpected, because Milton et al. had shown previously that only an all-D enzyme was capable of cleaving an all-D peptide substrate, but it could not digest an all-L peptide substrate (54).

View larger version (40K): [in this window] [in a new window]   FIGURE 2. Pharmacology of D-allo-ShK. A, effect of ShK (top panels) and D-allo-ShK (bottom panels) on Kv1.3 (left panels) and Kv1.1 (right panels) currents in stably transfected cells. B, dose-dependent inhibition of Kv1.3 (open symbols) and Kv1.1 (closed symbols) by ShK (), ShK-amide (), and D-allo-ShK (). Each data point is the mean ± S.D. of three independent determinations. C, selectivity of D-allo-ShK for K+ channels.

A potentially more discriminating comparison of the two structures is afforded by a comparison of NOESY cross-peak intensities. This is compromised somewhat by the higher quality of the D-allo-ShK NOESY spectrum compared with that for ShK, but some differences are noted here. A d_N NOE between Thr⁶ and Ile⁷ is missing in D-allo-ShK, probably reflecting an alteration in the backbone in this region. A long range NOE from Ile⁴ to Arg²⁹ is also missing in D-allo-ShK, but two new NOEs between these residues are present. There are 20 additional NOEs that are observed only in the D-allo-ShK spectrum, including sequential medium range and long range NOEs; about half of these involve the side chains or backbone of Thr⁶, Ile⁷, Thr¹³, Thr³¹, and Thr³⁴, reflecting the different side chain conformations relative to the backbone. Nonetheless, the overall similarities of the chemical shifts and NOE patterns for the ShK and D-allo-ShK indicate that their conformations are essentially mirror images of one another and that the different stereochemistry of the Thr and Ile side chains in D-allo-ShK relative to the backbone does not cause significant perturbations beyond the resolution of the solution structures.

Our findings for ShK versus D-allo-ShK are consistent with previous studies of all-D analogues of other structured polypeptides. Crystals of racemic rubredoxin, prepared by independent chemical synthesis of the L- and D-enantiomers, were centrosymmetric, and the 2 Å resolution structures of the two forms were mirror images of one another (55). NMR spectra of the all-D and all-L versions of the plant trypsin inhibitor EETI-II were identical with each other (56).

K⁺ Channel Blocking Activity—We tested ShK, ShK-amide, and D-allo-ShK on Kv1.3 and Kv1.1 channels stably expressed in L929 cells. Fig. 2A shows the effects of ShK and D-allo-ShK on Kv1.3 (left panels) and Kv1.1 (left panels) currents elicited by 200-ms depolarizing pulses from a holding potential of -80 to 40 mV. Both peptides reversibly blocked Kv1.3 and Kv1.1 in a dose-dependent manner with Hill coefficients of 1 (Fig. 2B). Native ShK and ShK-amide blocked Kv1.3 with essentially identical affinities, and both displayed a 2-fold selectivity for Kv1.3 over Kv1.1 (ShK: K_d for Kv1.3 13 ± 4 pM and for Kv1.1 29 ± 3 pM; ShK-amide: K_d for Kv1.3 14 ± 3 pM and for Kv1.1 31 ± 4 pM), as expected (Fig. 2). D-Allo-ShK blocked Kv1.3 with a 2,800-fold lower affinity than ShK (K_d 36 ± 3 nM) but displayed the same 2-fold selectivity for Kv1.3 over Kv1.1 as ShK (K_d on Kv1.1 83 ± 9 nM) (Fig. 2). D-Allo-ShK had no effect on Kv1.2, Kv1.4, Kv1.5, Kv1.7, or KCa3.1 at concentrations up to 1 µM (Fig. 2C). The activation time constant (_n) and the inactivation time constant (_h) of the Kv1.3 current (_n = 6.2 ± 1.3 ms; n = 13; _{h =} 174 ± 6 ms) (57) were not altered by ShK (_n = 7.4 ± 1.6 ms; n = 6; _{h =} 161 ± 6 ms), and D-allo-ShK (_n = 5.1 ± 2 ms; n = 6; _{h =} 165 ± 27 ms) did not alter these parameters at concentrations sufficient to induce 60% block (data not shown).

To further characterize the blocking activity of ShK and D-allo-ShK on Kv1.3, we tested their ability to bind to the closed state of the channel. We allowed equilibration of the internal solution for 5 min before applying a 200-ms pulse from -80 to 40 mV to elicit a control Kv1.3 current (Fig. 3). We then perfused ShK (20 pM) or D-allo-ShK (50 nM) into the bath, whereas the channel was closed for 5 min before pulsing again at 30-s intervals. ShK blocked 60% of the Kv1.3 current at the first pulse after peptide incubation, and this blockade did not increase after applying a further 10 depolarizing pulses, indicating that ShK binds to the closed state of the Kv1.3 channel. In contrast, D-allo-ShK had no effect on the current at the first pulse after peptide incubation, and steady-state block was only reached after several depolarizing pulses, a phenomenon termed "use-dependent block," indicating that D-allo-ShK binds to an open or inactivated conformation of the channel.

The activities of both ShK and D-allo-ShK on the proliferation of rat Kv1.3^highKCa3.1^low T_EM and Kv1.3^lowKCa3.1^high naïve/T_CM lymphocytes were also tested. In keeping with its loss of affinity for Kv1.3 channels, D-allo-ShK was significantly less potent than ShK in inhibiting T_EM cells (p < 0.05 at 10^-1 nM and p < 0.01 at all other concentrations) (Fig. 4A), and both peptides were less effective in suppressing the proliferation of naïve/T_CM lymphocytes (Fig. 4B).

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Characterization of the effects of ShK and D-allo-ShK on Kv1.3 currents. ShK blocks the closed state of the channel, but D-allo-ShK does not. A Kv1.3 control current was elicited by a 200-ms depolarizing pulse to 40 mV. ShK (20 pM; middle panel; n = 3) or D-allo-ShK (50 nM; bottom panel; n = 6) was applied to the bath solution, whereas the membrane was held at -80 mV. After 5 min, consecutive 200-ms pulses were applied every 30 s. The dotted line shows the peak current amplitude before the addition of the blocker.

View larger version (28K): [in this window] [in a new window]   FIGURE 4. D-Allo-ShK preferentially suppresses the proliferation of TEM cells. Dose-dependent inhibition by ShK () and D-allo-ShK () of [3H]Tdr incorporation by rat TEM (A) and naïve and TCM (B) lymphocytes. IC50 on TEM cells 80 pM (ShK) and 10 nM (D-allo-ShK); IC50 on naïve and TCM cells 90 nM (ShK) and 10 µM (D-allo-ShK).

D-Allo-ShK Inhibits DTH Response in Rats—As an assessment of the immunosuppressive activity of D-allo-ShK in vivo, we tested its ability to inhibit a DTH reaction to ovalbumin mediated predominantly by skin-homing T_EM cells (58). All vehicle-treated control rats developed ear swelling 24 h after ovalbumin challenge in the ear, but the DTH reaction was significantly milder in animals treated with 1 mg/kg D-allo-ShK at the time of challenge in the ear (Fig. 5C). Thus, D-allo-ShK inhibited the T_EM-mediated DTH response.

Docking of D-Allo-ShK with Kv1.3—To examine how D-allo-ShK could block Kv1.3, we constructed models of the complexes of both D-allo-ShK and ShK with the pore vestibule region and inner helices of the channel. The models of the highest ranked predictions from the docking of ShK and the chiral analogue, D-allo-ShK, with the channel are presented in Fig. 6. In both models, Lys²² is located in the ion selectivity filter, blocking passage of K⁺ ions through the channel; its ammonium group forms hydrogen bonds with the backbone carbonyl oxygen atoms of the tetrad of Gly³⁹⁹ residues. Phe²⁷ packs alongside Lys²², in the space created by two Gly⁴⁰¹ residues of the ion selectivity filter from neighboring channel monomers (monomers A and B for ShK, and monomers C and D for D-allo-ShK, in Fig. 6), and the side chains of Asp⁴⁰² and His⁴⁰⁴. The side chain of Met²¹ occupies the equivalent pocket in the space diametrically opposite the pocket filled by Phe²⁷.

The interactions between residues at the interface between ShK and the channel were assessed in relation to earlier experimental mutant cycle analysis (16). Mutant cycle analysis had indicated that ShK was strongly coupled with His⁴⁰⁴ of the channel. In their model of ShK complexed to Kv1.3, Lanigan et al. (16) noted that the distance of closest approach of Arg¹¹ of ShK with His⁴⁰⁴ of Kv1.3 was 11 Å. In the current model this separation is 4.3 Å (Arg¹¹ N with N). The channel residues Asp³⁸⁶ and Ser³⁷⁹ were also implicated in coupling with Arg¹¹ of ShK in the complex. In the present model the distance between the N of Arg¹¹ and the O of is 2.6 Å. Notably, the and y that contact Arg¹¹ are on a different channel monomer from the also close to Arg¹¹.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Circulating half-life of D-allo-ShK and effect on DTH. A, a single dose of 1 mg/kg of D-allo-ShK was injected subcutaneously into five rats. Blood was drawn at the indicated times and tested by patch clamp to determine the amount of D-allo-ShK. B, data fit to a single exponential decay. Half-life was 40 min. C, DTH reaction was elicited against ovalbumin and rats (n = 6/group) were treated with saline or 1 mg/kg D-allo-ShK dissolved in saline. Ear swelling was measured 24 h later. Statistical analysis was carried out using the Mann-Whitney U test.

Although the side chains of Tyr⁴⁰⁰ of the channel are not in close contact with the N of Lys²², these groups help maintain the shape of the selectivity filter of the channel, in particular ensuring close contact with N of Lys²² and the backbone carbonyl of Gly³⁹⁹. The cooperativity observed in the mutant cycle analysis between Tyr⁴⁰⁰ and Lys²² reflects the structural role played by the side chains of Tyr⁴⁰⁰. Similarly, the backbone atoms of Asp⁴⁰² form part of the selectivity filter; the side chains of these residues contribute to the structure of the filter. A summary of several internuclear distances for the two diasteromeric models of ShK complexed with the channel is presented in supplemental Table S3.

The overlay of the two complexes in Fig. 6A illustrates the approximate mirror symmetry (along the horizontal axis, between monomers A and B, and monomers C and D) of the binding of the two diastereomers to the channel. Residues of the toxins that contact the channel and lie along this plane share common binding interactions in the two diastereomers. Additionally, residues of the toxins that contact the channel along the channel monomer interface perpendicular to this plane also share common binding modes in the two diastereomers.

The structure of the complex with ShK presented here is similar to an earlier model (16), and the proximity of channel and ShK residues is consistent with the mutant cycle analysis data used to derive that model. In the complex of ShK with the channel, the side chains of several residues form hydrogen bonds with the channel, including the hydroxyl of Tyr²³, which hydrogen bonds with the backbone carbonyl oxygen atom of of the selectivity filter, and the side chain imidazole of . Tyr²³ is completely buried in the interface between channel and ShK, In contrast, Tyr²³ of D-allo-ShK, although in close proximity to , remains exposed to solvent in the complex. Similarly, the hydroxyl groups of Thr⁶ and Ser²⁶ participate in hydrogen bonding interactions with the channel in the complexes of both diastereomers of ShK, yet the residues on the channel they interact with are different in the two complexes. Arg²⁴ lies in close proximity to His⁴⁰⁴ in both cases. The loss of solvent-accessible surface area upon complex formation of ShK is 1981 Ų. This compares favorably with the earlier model of Lanigan et al. (16) of 1600 Ų. The complex of D-allo-ShK with the channel buries 1987 Ų surface in the interface. Thus, the models suggest that D-allo-ShK and ShK bury roughly the same surface area upon channel binding.

View larger version (63K): [in this window] [in a new window]   FIGURE 6. Docking of D-allo-ShK and ShK with a model of the Kv1.3 channel. The top panel shows an overlay of ShK (magenta) and D-allo-ShK (cyan), represented as tubes, as they were docked onto the channel (surface representation in silver). The middle and bottom panels show the side chains of residues implicated by mutant cycle analysis (16) to be involved in interactions between the channel and ShK. The side chains of the channel residues are color-coded as follows: red, Asp; green, Ser; blue, His. Channel monomers A-D, are distinguished by subscripts. The side chains of the ShK peptides are colored yellow. The view in the top and central panels is down the 4-fold symmetry (ion conduction) axis of the channel, whereas the bottom panels present views from the side of the channel.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Polypeptides that are mirror images of their naturally occurring (all-L) homologues are expected to show no activity against their natural targets when those targets are other proteins. However, D-allo-ShK blocked the Kv1.3 channel with nM affinity, making it a weaker blocker than the native toxin but nonetheless still a potent inhibitor. It also maintained the selectivity profile of the native peptide but interestingly did not block the closed state of the channel, unlike ShK. D-Allo-ShK was also able to block T cell proliferation. It is highly unusual for an all-D analogue of a folded polypeptide or protein that acts at a specific binding site on a target protein to retain activity. Kent and co-workers (54) found that an all-D analogue of human immunodeficiency virus, type 1 protease was capable of cleaving an all-D peptide substrate but was inactive against the natural all-L peptide substrate. Similarly, Nielsen et al. (56) found that an all-D version of the plant trypsin inhibitor EETI-II was inactive against trypsin, even though its conformation as judged by NMR was identical to that of the native all-L polypeptide. Recently, an intriguing exception to the lack of activity of all-D polypeptides was found in the case of the tarantula toxin GsMTx4, a selective inhibitor of stretch-activated cation channels (59). Both GsMTx4 and its enantiomer modified channel gating of stretch-activated cation channels, a result interpreted as indicating that this polypeptide acted at the channel-lipid interface rather than at a specific binding site exclusively associated with the channel protein.

We had originally proposed that the homotetrameric Kv1.3 channel could accommodate an enantiomer or diastereomer of the native toxin, and its ability to block the channel with nM affinity affirms our underlying hypothesis. However, the lack of a center of inversion within the 4-fold symmetric channel precludes D-allo-ShK from binding in a manner that is the mirror (or inverse) of its native enantiomeric partner. It is not surprising then that D-allo-ShK is not equipotent with native ShK in its ability to block Kv1.3. The overlay of the polypeptide trace of the two peptides in Fig. 6 illustrates how the two peptides engage the channel differently and without any formal symmetry relationship between them. Nonetheless, the two enantiomeric peptides make many similar interactions with the channel in the two models (for example Lys²² fills the ion selectivity filter, and Arg²⁴ and Arg²⁹ of the peptides interact with His⁴⁰⁴ of the channel), although these interactions are not identical in the two models. Because symmetry plays no role in determining the binding of D-allo-ShK to Kv1.3, and D-allo-ShK showed the same selectivity for Kv1.3 over Kv1.1 and other K channels as ShK, it is likely to be active against heterotetrameric channels consisting of mixtures of various K⁺ channel subunits.

ShK has a circulating half-life of 30 min (25). Such a rapid clearance from the blood could be due to renal elimination and/or proteolysis. The finding that D-allo-ShK has a similar half-life (40 min) implies that the disappearance of ShK and D-allo-ShK is due to rapid renal clearance because endogenous proteases can only cleave L forms of polypeptide chains and are therefore unable to proteolyze D-allo-ShK. Moreover, the kidney allows peptides up to 5 kDa to pass through without filtration and both forms of ShK are smaller than this cut-off. Even though it has a similar circulating half-life to ShK and is less potent as a blocker of Kv1.3 channels, D-allo-ShK may be useful in some in vitro experimental situations where its resistance to proteolysis prolongs its lifetime. Moreover, it is expected not to be recognized by the immune system because it should be resistant to proteolytic processing by T cells for presentation on major histocompatibility complex complexes and therefore is not likely to be antigenic in vivo. Further modification of the all-D polypeptide to enhance Kv1.3 selectivity, as achieved for the native toxin (14, 17, 19) should yield a selective, protease-resistant, and nonimmunogenic reagent for studies of channel structure and function. More generally, our results demonstrate that mirror image polypeptides can retain potent activity and may have broader applications in both in vitro and in vivo studies than hitherto appreciated.

	FOOTNOTES

 
^* This work was supported in part by National Institutes of Health Grant NS-48252 (to K. G. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1-S3 and supplemental Fig. S1.

¹ These authors contributed equally to this work.

² Recipient of a fellowship by the National Health and Medical Research Council of Australia. To whom correspondence should be addressed. Tel.: 61-3-9345-2306; Fax: 61-3-9345-2686; E-mail: ray.norton{at}wehi.edu.au.

³ The abbreviations used are: Fmoc, N-(9-fluorenyl)methoxycarbonyl; NOESY, nuclear Overhauser enhancement spectrometry; RP, reversed phase; HPLC, high pressure liquid chromatography; MD, molecular dynamics; DTH, delayed-type hypersensitivity; T_CM, central memory T; T_EM, effector memory T.

	ACKNOWLEDGMENTS

 
We thank Yen Yee Tan for help with the NMR analysis, Evelyne Beraud for the generous gift of the PAS T cell line, and Jacqui Gulbis for helpful discussions.

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