The amino acid sequence of MgTx was retrieved from the online protein (Uniprot P40755) database. The MgTx gene cassette was designed by placing the 6xHis-tag at the N-terminal to facilitate purification, and factor Xa protease site was introduced in between them to obtain native N-terminal MgTx, as demonstrated in Figure 1. The codon-optimized DNA sequence of this MgTx cassette for P. pastoris was generated according to the codon usage database available at www.kazusa.or.jp/codon and synthesized from Integrated DNA Technologies, Belgium. The codon-optimized MgTx cassette was cloned into yeast expression vector pPICZα A (Invitrogen, United States) by using EcoRI and XbaI restriction sites. In-frame ligation and nucleotide sequence of MgTx was confirmed by DNA sequencing by using plasmid-specific primers and aligning the obtained DNA sequence with the theoretical sequence of MgTx.

The expression plasmid was linearized by digesting with SacI endonuclease enzyme and transformed into P. pastoris X-33 competent cells using Pichia EasyComp Transformation Kit (Invitrogen, United States), following the protocol specified by the manufacturer. Transformed X-33 cells were spread on YPD agar plate (2% peptone, 1% yeast extract, 2% agar, 2% dextrose, and pH 7.0) containing 100 µg/ml of Zeocin. After 3-day incubation at 28°C, 24 prominent colonies were regrown on YPD plates supplemented with progressively increasing Zeocin 500, 1000, and 2000 µg/ml for the selection of the clone showing hyper-resistance against Zeocin. To confirm the integration of expression construct into the genome of Pichia transformants, survived on 2000 µg/ml Zeocin, colony PCR was performed by using plasmid-specific primers.

A selected clone from the YPD plate containing 2000 µg/ml of Zeocin was grown overnight in 5 ml of the YPD medium and diluted the next day to an OD₆₀₀ = 0.2 in 5 ml of BMGY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 × 10 − 5 % biotin, and 2% glycerol) for biomass production at 30°C with constant shaking (230 rpm) until the OD₆₀₀ reached between 15 and 20 (after 24–36 h). Cells were collected by centrifugation, resuspended in 5 ml of BMMY induction medium (same as BMGY with 0.5% methanol instead of glycerol), and grown for 5 days at 28°C with constant shaking (230 rpm). Absolute methanol (MeOH, 0.5%) was added every 24 h to maintain the induction, except when MeOH concentration dependence of induction was studied. To find the suitable amount of methanol induction, cells were induced with 0.5, 1, and 1.5% MeOH, and for pH optimization, cells were grown in media of different pH, i.e., 5, 6, and 7, without buffering. About 15 µl of the supernatant samples were taken at indicated time points and analyzed on 16% tricine–SDS-PAGE. The amount of His-tagged rMgTx (TrMgTx) in the gel image was determined by comparing the band intensities with the standards (TrMgTx with known concentration) using Image Lab tool (Bio-Rad). All the experiments were run in triplicates.

Large-scale flask-level production was executed following the optimized conditions as described earlier. The clone with the highest expression level of TrMgTx was inoculated in a 2-L flask containing 250 ml of the BMGY medium, and when the OD₆₀₀ reached between 15 and 20, the cells were shifted to 250 ml of the BMMY induction medium and induced with 0.5% MeOH for 72 h.

Two-step purification was used to efficiently isolate secreted TrMgTx from the culture. The cultured supernatant was collected by high-speed centrifugation, 2× diluted with buffer (50 mM potassium phosphate, pH 7.4), and 60 mM imidazole was added. The filtered supernatant was loaded on pre-equilibrated His-trap column packed with Ni Sepharose^TM High Performance affinity media (GE Healthcare, United Kingdom) with a binding buffer (50 mM potassium phosphate, 300 mM NaCl, and pH 7.4) at a flow rate of 3 ml/min using the liquid chromatography (LC) system (Shimadzu, Germany). After washing the column with three column volumes (CVs) of wash buffer (binding buffer + 60 mM imidazole), proteins were eluted by running three CVs of the elution buffer (binding buffer + 500 mM imidazole) and an additional three CVs of 1 M imidazole in isocratic mode. Fractions collected from the affinity column were directly applied on reversed-phase C₁₈ semi-prep column (10 mm) × 250 mm, 5 µM bead size, 300 Å pore size, Vydac® 218 TP, HiChrom, United Kingdom) using Prominence HPLC System (Shimadzu, Germany) at a flow rate of 1 ml/min. Then, a linear gradient of 10–30% of Solvent B (0.1% TFA in 95% acetonitrile) in Solvent A (0.1% TFA in deionized distilled water) was run for 30 min. Absorbance was monitored at 230 nm with a PDA detector. The peak fractions were collected manually and tested on 16% tricine–SDS-PAGE. The purity level was judged by reloading the fraction on the reversed-phase C₁₈ analytical column and was calculated by the equation: (area under the peak of interest)/(cumulated area under all the peaks) ×  x 100.

16% Tricine–SDS-PAGE was performed as described hitherto (Schägger, 2006). The protein sample was mixed with tricine sample buffer (Bio-Rad) at 1:1, incubated at 95°C for 5 min, and subsequently centrifuged at 10,000 rpm for 30 s before loading. Electrophoresis was carried out at constant 120 V for 90 min. For protein visualization, the gel was stained with Coomassie Brilliant Blue G-250 for 45 min and then destained by using 40% methanol and 10% acetic acid mixture for 2–3 h.

For Western blotting, the resolved proteins were electrotransferred in wet conditions onto a charged Immobilon-P PVDF membrane (Merck, Germany). Non–specific binding of antibodies in the subsequent steps was prevented by membrane blocking with 5% (w/v) skim milk in TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20), overnight at 4°C. The washed membrane was probed with mouse anti-histidine monoclonal antibodies conjugated with horseradish peroxidase (Bio-Rad, CA, United States) in TBST (1:2,500) and incubated for 1 h at room temperature. The bands were visualized using Pierce™ enhanced chemiluminescent (ECL) substrate (Thermo Scientific, MA, United States).

Hexahistidine residues fused at the N-terminal of TrMgTx were cleaved with factor Xa protease (Thermo Scientific, United States Cat# 32,521). About 300 µg of TrMgTx was mixed with factor Xa at an enzyme-to-substrate ratio of 1:100 in TBS buffer (50 mM Tris, 100 mM NaCl, 6 mM CaCl₂, pH 8.0) and incubated overnight at 25°C. The next day, the samples treated with or without enzyme were analyzed on 16% tricine/6 M urea–SDS-PAGE. To purify untagged rMgTx (UrMgTx), the cleaved His-tag and undigested peptide fragments were captured by using pre-charged Ni⁺ beads, centrifuged at a high speed for 1 min to remove the beads, and the supernatant was loaded on a reversed-phase C₁₈ analytical column (4.6 mm × 250 mm, 5 µM bead size, Vydac® 218TP) using the HPLC system (Shimadzu, Germany) and eluted with a linear gradient of 10–30% of Solvent B (0.1% TFA in 95% acetonitrile) in Solvent A (0.1% TFA in deionized distilled water) that was run for 25 min.

Mass spectrometric determinations were performed with an ESI-QTOF-MS instrument (maXis II UHR ESI-QTOF MS, Bruker, Bremen, Germany). The mass spectrometer was operated in a positive ionization mode; 0.5 bar nebulizer pressure, 200°C dry gas temperature, 4 L/min dry gas flow rate, 4000 V capillary voltage, 500 V end plate offset, 1 Hz spectra rate, and 500–2,500 m/z mass range were applied. ESI tuning mix (Agilent) calibrant injected after each run enabled internal m/z calibration. Mass spectra were processed and evaluated by Compass Data Analysis version 4.4 (Bruker).

The human venous blood from anonymized healthy donors was obtained from a blood bank. The peripheral blood mononuclear cells were isolated through Histopaque1077 (Sigma-Aldrich Hungary, Budapest, Hungary) density gradient centrifugation. Cells obtained were resuspended in RPMI 1640 medium containing 10% fetal calf serum (Sigma-Aldrich), 100 µg/ml penicillin, 100 µg/ml streptomycin, and 2 mM _l glutamine, seeded in a 24-well culture plate at a density of 5   x   10 5 cells per ml, and grown in a 5% CO₂ incubator at 37°C for 2–5 days. Phytohemagglutinin A (Sigma-Aldrich) was added in 5, 7, and 10 µg/ml concentrations to the medium to boost the potassium ion channel expression.

CHO cells were transiently transfected with the vector pCMV6-GFP (OriGene Technologies, Germany) encoding the human Kv1.2 ion channel using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following the manufacturer's protocol, and grown under the standard conditions as used previously (Bagdány et al., 2005). GFP-positive transfectants were identified with Nikon TE 2000U fluorescence microscope (Nikon, Tokyo, Japan), and currents were recorded after 24 h post transfection.

Electrophysiological measurements were performed by using the patch-clamp technique in whole-cell or outside-out patch configuration and voltage clamp mode using the Multiclamp 700B amplifier and Axon Digidata1440 digitizer (Molecular Devices, Sunnyvale, CA). Micropipettes were pulled from GC150F-15 borosilicate capillaries (Harvard Apparatus Kent, United Kingdom), resulting in 3–5 MΩ resistance in the bath solution. The extracellular solution contained 145 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2.5 mM CaCl₂, 5.5 mM glucose, and 10 mM HEPES, with a pH of 7.35 and an osmolarity between 302 and 308 mOsM/L. When the toxins were dissolved at different molar concentrations in bath solution, it was supplemented with 0.1 mg/ml of BSA. The pipette filling (intracellular) solution consisted of 140 mM KF, 2 mM MgCl₂, 1 mM CaCl₂, 10 mM HEPES, and 11 mM EGTA, with a pH of 7.22 and an osmolarity of 295 mOsM/L.

To record the hKv1.3 and hKv1.2 currents, 15- to 200-ms-long depolarization impulses were applied at +50 mV from a holding potential of −120 mV every 15 s. The pClamp 10.1 software package was used to acquire and analyze the measured data. Current traces were low-pass filtered by the analog four-pole Bessel filters of the amplifiers, and the sampling frequency was set at 20 kHz, at least twice that of the filter cutoff frequency. The effect of the toxin at a given molar concentration was calculated as the remaining current fraction (RCF = I/I₀, where I₀ is the peak current in the absence of the toxin and I is the peak current at equilibrium block at a given toxin concentration). The data points on dose–response curves represent the mean of 3–4 individual measurements. The data points were fitted with a three-parameter [inhibition] vs response model, RCF = Bottom + [Top–Bottom)/(1+([toxin]/K _d )], where Top and Bottom values were constrained to 1 and 0, respectively, and [toxin] is the concentration of the toxin. The best fit curve gave K _d of the given toxin.

To examine the effect of additional residues of TrMgTx on binding to the ion channel, the association and dissociation rate constants of both the versions of the toxin were determined for the Kv1.3 channel of the activated human T lymphocytes and the hKv1.2 channel transiently expressed in the CHO cell. Whole-cell currents were recorded after applying the toxin (200 pM for Kv1.3 and 100 pM for Kv1.2) to the extracellular solution until the equilibrium block was achieved (wash-in), and then removed it by perfusing the toxin-free control solution (washout). Peak currents at a time point t [I(t)] were normalized to the peak current in the absence of the toxin [I_norm(t) = I(t)/I₀], and these were plotted as a function of time. The association time constant (τ _on ) was determined by fitting the data points during the wash-in procedure in the one-phase decay equation: I n o r m ( t ) = ( ( 1 − R C F ) × e − t τ o n ) + R C F , with the RCF = I/I₀ at the equilibrium block as defined in the Electrophysiology section. The dissociation time constant (τ _off ) was determined by fitting the following equation to the data points during the washout procedure: I n o r m ( t ) = R C F + ( 1 − e − t τ o f f ) .

The association rate constant (k _on ) and dissociation rate constant (k _off ) were calculated from the measured time constants, assuming a simple bimolecular interaction between the channel and toxin, and by using the following equations (Peter et al., 2001), with τ_on and τ_off defined above, and [toxin] as the toxin concentration: k o n = 1 − ( τ o n × k o f f ) τ o n × [ t o x i n ] , k o f f = 1 τ o f f .

The mononuclear cells were isolated from anonymized healthy donors as described earlier. Prior to CD4⁺ T_EM cells separation, dead cells were removed using the Dead Cell Removal Microbead Kit (Miltenyi Biotec B.V & CO. KG, Bergisch Gladbach, Germany). Untouched CD4⁺ T_EM lymphocytes were purified through magnetic cell sorting (negative selection) with the CD4⁺ Effector Memory T Cell Isolation Kit (Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany) according to the manufacturer's protocol.

For TCR-specific stimulation, anti-human CD3 monoclonal antibodies (clone OKT3, BioLegend, San Diego, CA) were bound to the surface of 24-well cell culture plates at a density of 5 µg/well in phosphate-buffered saline (PBS) overnight at 4°C. Before seeding the cells, the wells were washed twice with PBS to remove the unbound antibodies. The CD4⁺ T_EM cells were divided into four groups: 1) unstimulated, 2) stimulated, 3) stimulated + TrMgTx (8.5 nM), and 4) stimulated + UrMgTx (5 nM). The cells were seeded at a density of 0.5 × 10 6 cells/ml per well, and when indicated, the cells were preincubated with the toxins for 5 min prior to the stimulation. The plate was incubated at 37°C in 5% CO₂ for 24 h. Each experiment was performed in technical duplicates on three different donors.

For quantifying the extent of T_EM cell stimulation, cells were washed with PBS buffer containing 1% of fetal bovine serum (FBS) and stained with fluorescein isothiocyanate (FITC)–labeled anti-human CD154 (CD40L) antibody (clones 24–31, BioLegend, San Diego, CA) and PerCP/Cyanine5.5–labeled anti-human CD25 (IL2R) antibody (clone BC96, BioLegend) at 4°C for 20 min. Cells were washed with the PBS + 1% FBS buffer and resuspended in 150 µl in PBS + 1% FBS. Samples were measured with NovoCyte 3000 RYB flow cytometer (ACEA Bioscience Inc.), FITC and PerCP/Cyanine5.5 were excited by using blue laser (488 nm), and 530/30 nm and 695/40 nm emission filters were used, respectively. Flow cytometry data were analyzed using FCS Express 6.0 (De Novo Software, Glendale, CA). Briefly, cells were gated on the basis of their FSC and SSC parameters, and then, the histograms corresponding to CD40L and CD25 were plotted as peak-normalized overlays. Mean fluorescent intensities (MFIs) were computed from the histograms and normalized to that of their stimulated (S) but not treated control. The negative (unlabeled) and unstimulated control (US) were always used for comparison.

Data analyses and graph generation were performed using the GraphPad Prism software (version 9.1, La Jolla CA, United States). Statistical comparisons were made by using one-way ANOVA with Tukey's test and unpaired t test. For all the experiments, data are reported with standard error of mean (SEM).

The expression cassette, consisting of six histidines followed by the factor Xa cleavage site and coding DNA sequence of MgTx (Figure 1B), was synthesized by using favorable codons for P. pastoris and cloned into pPICZαA expression vector under the control of alcohol oxidase I (AOX1) promoter. In-frame ligation to the α-factor secretion signal and nucleotide sequence of the TrMgTx was verified by DNA Sanger sequencing. A schematic illustration of the TrMgTx–pPICZαA recombinant plasmid was created by using the in silico SnapGene^® cloning tool (Figure 1A). The recombinant plasmid was linearized with the SacI enzyme and transformed into Pichia X-33 competent cells. Following 3 days of incubation at 30°C, more than 40 prominent colonies were observed on the YPD agar plate, containing 100 µg/ml of Zeocin. Hyper-resistant clones against Zeocin were selected by growing the initially chosen colonies on the YPD agar plates augmented with gradually increasing Zeocin to 0.5, 1, and 2 mg/ml (Supplementary Figure S1). The transformants that survived against the highest concentration of Zeocin (2 mg/ml) were selected, and colony PCR was performed which confirmed the integration of TrMgTx–pPICZαA plasmid by a single crossover at the 5′ AOX1 locus of the P. pastoris genome (Supplementary Figure S2).

Supernatant samples were collected from the cultures every 24 h post methanol induction for 5 days and were analyzed by tricine–SDS-PAGE. A peptide band of ∼6.5 kDa MW was detectable by using R-250 Coomassie Brilliant blue (CBB) after 24 h of methanol induction. This band gradually attained maximal density after 72 h of induction. However, the amount of secreted peptide toxin declined on the fourth and fifth day following induction (Figures 2A,B). The molecular mass of TrMgTx estimated from the gel was slightly higher than the predicted mass (5.9 kDa), most likely because of its higher PI value of 9.10. The highest concentration of peptide in the supernatant was 78 ± 7 mg/L after 72 h of induction. This yield was, in all pairwise multiple comparison (Tukey's test), significantly higher than the yield at any other time point (p < 0.05).

For optimization of methanol induction, biomass production of a TrMgTx clone was induced with 0.5, 1, and 1.5% MeOH, using a medium of pH 6. The gel scanning assay revealed that the amount of TrMgTx was the largest in the supernatant after inducing with 0.5% methanol for 72 h (71 ± 13 mg/L) (Figure 2C). This yield is significantly higher than that of the other two samples induced with 1 and 1.5% MeOH (all pairwise multiple comparison (Tukey's test), p < 0.05). Similarly, to find a suitable pH of the medium for improved expression of the peptide, biomass was induced with 0.5% MeOH in a media having pH 5, 6, or 7, or “not adjusted” for 72 h (Figure 2D). The supernatant of the culture at pH 6 showed 76 ± 8 mg/L expression of the peptide toxin which is considerably higher than for other samples from cultures with a pH 5 or 7 or “not adjusted” (all pairwise multiple comparison (Tukey's test), p < 0.05).

A Pichia X-33 clone that produced large amounts of TrMgTx in small cultures was subjected to large-scale fermentation in a medium of pH 6, and the expression was induced with 0.5% methanol for 3 days. The culture medium was centrifuged, filtered through a 0.45-µm membrane to remove cell debris, and then loaded on the Ni⁺ Sepharose column using the LC system as the first step of the two-step purification protocol applied. After removing the unbound proteins, His-tagged peptides were eluted with 0.5 and 1 M imidazole in isocratic mode. A very large peak appeared in the chromatogram with 0.5 M imidazole as compared with the peak obtained with 1 M imidazole, confirming that 0.5 M imidazole removed most of the bonded TrMgTx from the resin (Figure 3A). When the collected fractions were analyzed on tricine–SDS-PAGE, a clear and dense band of TrMgTx around ∼6.5 kDa was observed in the elution fraction (with 0.5 M imidazole) and a low-intensity band of the same size appeared in the fraction eluted with 1 M imidazole. On the contrary, no such band was observed in the fractions collected during the loading of the supernatant (flow though) and washing of the column (Figure 3B), demonstrating that resin had efficiently captured all His-tagged peptides from the cultured supernatant.

To achieve high purity and homogeneity of the recombinantly produced TrMgTx, the partially purified fraction (i.e., eluted by 0.5 mM imidazole, see the Purification of Tagged Recombinant MgTx section for details) was applied on the C₁₈ RP-HPLC semi-preparative column (second step of the purification protocol) and eluted with a gradient of 10–30% of acetonitrile in distilled water for 30 min (Figure 3C). Tricine–SDS-PAGE analyses of the collected HPLC fractions (Figure 3D) showed that a single band of TrMgTx at ∼6.5 kDa MW appeared in the fraction corresponding to the peak eluted at the retention time (R_T) of ∼28 min (indicating to peak 2 in the chromatogram shown in Figure 3C). The average molecular mass of 5980.86 Da was determined for TrMgTx by ESI-QTOF-MS which is in full agreement with the predicted average mass (5980.96 Da) of the peptide (Figure 3E). The quality of the peptide after the two-step purification was assessed by using the anti-His antibody in the Western blot (Figure 4A) and HPLC (Figure 4B). The Western blot verified the purity and identity of TrMgTx by detecting a single band with anti-His antibodies (Figure 4A) of the appropriate size (cf. Figures 3B,D). The TrMgTx is more than 98% pure after the two-step purification as assessed by the C₁₈ RP-HPLC analytical column (Figure 4B). Table 1 summarizes the purification scheme; on average, a total of 9.1 mg of TrMgTx was produced with 43% net recovery from 250 ml P. pastoris culture under optimized conditions.

TrMgTx contains 14 additional N-terminal amino acid residues (EFHHHHHHLQIEGR). This mainly consists of 6x histidines used for affinity purification and the protease cleavage site for factor Xa. Factor Xa protease cleaves the tagged peptides without leaving any extra amino acids at the N-terminal (Waugh, 2011). To get rMgTx with native N-terminal, all the additional residues were removed by digesting the tagged peptide with factor Xa protease overnight. Tricine–SDS-PAGE analyses (Figure 5A) revealed that a band of ∼4.1 kDa (the MW of native MgTx) was present in the overnight digested sample, confirming the successful removal of additional residues. The UrMgTx was purified using the C₁₈ RP-HPLC column after separating the 6xHis fragments with Ni⁺ beads. The peak at R_T 21.6 min in the chromatogram (Figure 5B) indicates the elution of UrMgTx. The determined average mass (4178.95 Da) of UrMgTx is equivalent to the theoretical mass (4179.018 Da) of native MgTx, proving that there is no extra residue at either terminal of UrMgTx after cleaving the tag (Figure 5C).

Pharmacological activity of both versions of rMgTx was evaluated by studying their effect on the hKv1.2 and hKv1.3 ion channels. The hKv1.2 current was recorded in CHO cells, which heterologously expressed the hKv1.2 ion channel, and hKv1.3 current was recorded in the human peripheral blood lymphocytes. The stimulation of the hKv1.3 channel expression (activation of isolated mononuclear cells by PHA, see the Materials and Methods section) and recording conditions (no Ca²⁺ in the pipette to elicit Ca²⁺-activated K⁺ channels) guaranteed that the current recorded in these cells is K⁺ current through Kv1.3 (Varga et al., 2012; Bartok et al., 2014; Tajti et al., 2021). A custom-built perfusion system was used to apply toxins at a very small perfusion rate of 200 µl/min. The speed and complete solution exchange in the recording chamber at this perfusion rate were tested frequently using fully reversible blockers as a positive control. Around 50% reduction in the peak current upon perfusion of the positive control in concentrations equal to the respective K _d values, i.e., 10 mM tetraethylammonium for hKv1.3 and 14 nM charybdotoxin for hKv1.2, proved the complete exchange of solution (data not shown).

TrMgTx and UrMgTx at the 200 pM concentration inhibited ∼70 and ∼80% of the whole-cell hKv1.3 current upon reaching the block equilibrium, respectively. Figures 6A,B show the current traces recorded in the presence and absence of the respective peptide toxins. Figures 6C,D demonstrate the development and recovery from the block of the hK1.3 current with TrMgTx and UrMgTx as indicated using the colored bars. In case of hKv1.3, the block equilibrium develops slower for TrMgTx than for UrMgTx upon application of 200 pM of toxin in bath solution, as reported by the time constants (τ _on ) obtained by fitting a single exponential function to the decay of the peak currents in the presence of the blockers (for TrMgTx τ _on = 168 ± 19 s and for UrMgTx τ _on = 116 ± 10 s were obtained, n = 4). Almost full recovery of the peak currents was attained upon perfusing the bath medium with toxin-free solution with similar time constants for washout (τ _off ) for the two peptides (for TrMgTx τ _off = 568 ± 84 s and for UrMgTx τ _off = 560 ± 28 s were obtained, n = 4). To reveal the impact of additional amino acids at the N-terminal of TrMgTx on the interaction with hKv1.3, blocking parameters k _on , k _off , and equilibrium dissociation constant K _d were calculated (Table 2) from the measured time constant values and plotted on the bar graph in Figure 6F. The k _on rate of UrMgTx was significantly higher than was for TrMgTx (in unpaired t test comparison, n = 4, p < 0.001); however, the k _off rate of the peptides was statistically the same (p > 0.05, n = 4). The hKv1.3 blocking potency of both tagged and UrMgTx was obtained by determining their k _d values by using dose–response relationships as well. The data points were fitted using a three-parameter [inhibition] vs response equation, and the best fit gave the k _d 50 and 86 pM for UrMgTx and TrMgTx, respectively, as is shown in Figure 6E. Based on the dose–response relationship, the tagged version of the toxin, TrMgTx, is slightly less potent for hKv1.3 than the tag-free version of the peptide, UrMgTx.

A similar set of experiments were repeated for the hKv1.2 ion channel expressed in CHO cells. TrMgTx and UrMgTx at 100 pM concentrations blocked about 60 and 90% of the whole-cell current of hK1.2, respectively, as shown by the current traces recorded in the presence and absence of the respective peptide toxins (Figures 7A,B). The change in the peak current of hK1.2 upon application and washout of the TrMgTx and UrMgTx (colored bars) is shown in Figures 7C,D. Like hKv1.3, the equilibrium block of hKv1.2 with TrMgTx developed on a slower time course than with the UrMgTx at identical (100 pM) concentrations (for TrMgTx τ _on = 202 ± 2.3 s and for UrMgTx τ _on = 191 ± 12 s). Perfusion of the bath with toxin-free solution results in slow but full recovery (τ _off = 532 ± 69 s) from the block in case of TrMgTx. On the contrary, in case of UrMgTx, slow and partial recovery (τ _off = 1308 ± 351 s) from the block was observed. Blocking parameters k _on , k _off , and K _d for hKv1.2 were calculated from measured time constant values as given in Table 2 and plotted on a bar graph (Figure 7F). The k _on obtained for UrMgTx is slightly higher than that of TrMgTx (unpaired t test, n = 3−4, p < 0.01); however, the k _off rate of UrMgTx is substantially lower than that of TrMgTx (unpaired t test, n = 3−4, p < 0.001). In the dose–response relationship, the best fit of data points results in K _d = 64 pM for TrMgTx and K _d = 14 pM for UrMgTx (Figure 7E). Tagged TrMgTx is nearly fivefold less potent for hKv1.2 than tag-free UrMgTx.

Upon T lymphocyte stimulation, the expression of activation markers in the cell membrane, such as IL2R and CD40 ligand, are upregulated. High-affinity blockers of Kv1.3 inhibit the activation and proliferation of human T_EM cells through decreasing the driving force for the Ca²⁺ influx (Chandy et al., 2004; Cahalan and Chandy, 2009). In this study, it has been investigated whether recombinantly produced MgTx (with tag or without tag) would affect the expression of IL2R and CD40L, as activation markers in CD4⁺ T_EM cells upon TCR ligation for 24 h. The presence of either toxin TrMgTx or UrMgTx (at ∼100× concentration of their respective K _d values) during the T_EM cell stimulation significantly reduced the upregulation of IL2R and CD40L expression. About 8.5 nM of TrMgTx showed ∼39% inhibition of IL2R expression and ∼36% inhibition of CD40L expression. Similarly, 5 nM of UrMgTx hampered expression of both activation markers by ∼45% (Figures 8A-D). It is important to note that CD40L expression of the stimulated cells after treatment with either TrMgTx or UrMgTx was comparable to that of unstimulated cells (Figure 8D). These results suggest that both TrMgTx and UrMgTx are biologically active peptides; however, TrMgTx has a slightly lesser efficacy than UrMgTx due to the presence of a His-tag at the N-terminus.