*4.7. Electrophysiology*

For the expression of Nav channels (mammalian rNav1.2, rNav1.4, hNav1.5, mNav1.6 and hNav1.8 channels; the insect channel *Bg*Nav1 from *Blattella germanica*; and the auxiliary subunits rβ1, hβ1, and TipE) and Kv channels (mammalian rKv1.1, rKv1.2, hKv1.3, rKv1.4, rKv1.5, rKv1.6, rKv2.1, hKv3.1, rKv4.2, hKv10.1, and h*ERG*; and *Drosophila Shaker*'s IR) in *Xenopus laevis* oocytes; the linearized plasmids were transcribed using the T7 or SP6 mMESSAGE-mMACHINE transcription kit (Ambion, Carlsbad, CA, USA). The harvesting of stage V–VI oocytes from anesthetized female *Xenopus laevis* frog was previously described [58]. Oocytes were injected with 50 nL of cRNA at a concentration of 1 ng/nL using a microinjector (Drummond Scientific, Broomall, PA, USA). The oocytes were incubated in a solution containing 96-mM NaCl, 2-mM KCl, 1.8-mM CaCl2, 2-mM MgCl2, and 5-mM HEPES (pH 7.4), supplemented with 50 mg/L gentamycin sulfate.

Two-electrode voltage-clamp recordings were performed at room temperature (18–22 ◦C) using a Geneclamp 500 amplifier (Molecular Devices, Downingtown, PA, USA) controlled by a pClamp data acquisition system (Axon Instruments, Union City, CA, USA). Whole cell currents from oocytes were recorded 1–4 days after injection. The bath solution's composition was 96-mM NaCl, 2-mM KCl, 1.8-mM CaCl2, 2-mM MgCl2, and 5-mM HEPES (pH 7.4). Toxins were applied directly to the bath. Resistances of both electrodes were kept between 0.8 and 1.5 MΩ. The elicited currents were sampled at 20 kHz (Nav) or 2 kHz (Kv), filtered at 2 kHz (Nav) or 0.5 kHz (Kv) using a four-pole low-pass Bessel filter. Leak subtraction was performed using a -P/4 protocol. Only data obtained from cells exhibiting currents with peak amplitudes below 2 μA were considered for analysis. For the electrophysiological analysis, a number of protocols were applied from a holding potential of −90 mV with a start-to-start interval of 0.2 Hz. Kv1.1–Kv1.6 and Shaker currents were evoked by 250-ms depolarizations to 0 mV followed by a 250 ms pulse to −50 mV from a holding potential of −90 mV. Current traces of hERG channels were elicited by applying a +40 mV prepulse for 2 s followed by −120 mV for 2 s. Kv2.1, Kv3.1, and Kv4.2 currents were elicited by 250 ms pulses to +20 mV from a holding potential of −90 mV. Kv10.1 currents were evoked by 2-s depolarizing pulses to 0 mV from a holding potential of −90 mV. Sodium current traces were evoked by 100-ms depolarization to the voltage corresponding to maximal sodium current in control conditions. All data were analyzed using pClamp Clampfit 10.0 (Molecular Devices, Downingtown, PA, USA) and Origin 7.5 software (Originlab, Northampton, MA, USA).

#### *4.8. Determination of Ki Against Porcine Pancreatic and Human Saliva* α*-Amylases*

Kinetic assays were carried out at 37 ◦C in 50 mM sodium phosphate and 100 mM sodium chloride (pH 7.0). 2-Chloro-4-nitrophenyl-α-maltotrioside (CNPG3) (Sigma Aldrich, St. Louis, MO, USA) was used as the substrate and the optical absorption of the 2-chloro-4-nitrophenol was measured at 405 nm. Reactions were run with final [CNPG3] = 1 mM ([*S*]/*K*<sup>M</sup> = 1.41, *KM* = 0.71 mM), nominal [*E*] = 20 nM for PPA, [CNPG3] = 3.3 mM ([*S*]/*KM* = 0.97, *KM* = 3.4 mM), and nominal [*E*] = 100 nM for HSA to provide sufficient analytical signal. Inhibitor dilution schemes were optimized considering recommendations in [34].

The enzyme was pre-incubated with the inhibitor for 10 minutes before the addition of substrate which launched the reaction. Reactions were monitored on an xMark microplate spectrophotometer (BioRad, USA) in the kinetic mode for 30 min. The initial linear steady state region provided initial rate values for each inhibitor concentration (υ), along with uninhibited rate values (υ*o*). Measurements were run in triplicate. Nonlinear least squares regression was carried out with GraphPad Prism 7.00 (San Diego, CA, USA). Fractional rates (υ/υ*o*) were plotted against inhibitor concentrations and the set of data points was fitted by the Morrison Ki regression algorithm [34,35]. Ki and [*E*] were simultaneously treated as adjustable parameters following the approach described in [59]. Derived enzyme active sites concentrations showed physically meaningful values close to nominal (34.6 and 132.5 nM for PPA and HSA respectively). Best-fit constant values were presented as mean ± SE (*n* = 3).

**Author Contributions:** Conceptualization, O.S., I.G., M.M., J.T., and E.L. Data curation, O.S., I.G., and E.L. Investigation, O.S., I.G., A.K., E.Z., N.K., L.S., and S.P. Methodology, O.S., S.P., and E.L. Supervision, J.T., E.K., and E.L. Validation, O.S., A.K., E.Z., N.K., L.S., S.P., and E.L. Visualization, O.S., A.K., E.Z., and N.K. Writing—original draft, O.S., I.G., A.K., E.Z., and E.L. Writing—review and editing, O.S., I.G., A.K., E.Z., M.M., S.P., J.T., E.K., and E.L. Authors guarantee the reliability of obtained data in any part of the work. All authors read and approved the final manuscript.

**Funding:** This research was partially (when obtaining the recombinant peptide and for determination of Ki) supported by RFBR grant number 18-38-00389. The MS and CD spectra were carried out on the equipment of the Collective Facilities Center «The Far Eastern Center for Structural Molecular Research (NMR/MS) PIBOC FEB RAS». S.P. was funded by postdoctoral grant PDM/19/164 (KU Leuven, Belgium). J.T. was funded by grants CELSA/17/047 (KU Leuven, Belgium), G0A4919N, and G0C2319N (FWO-Vlaanderen, Belgium).

**Acknowledgments:** We gratefully acknowledge Valery Mikhailov for conducting the antimicrobial activity research and Stanislav Anastyuk for obtaining of MS data. We would like to thank Alexandra Litavrina for the revision of the English text and Academician Valentin Stonik for editorial improvements of this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.
