AsKC11, a Kunitz Peptide from Anemonia sulcata, Is a Novel Activator of G Protein-Coupled Inward-Rectifier Potassium Channels

(1) Background: G protein-coupled inward-rectifier potassium (GIRK) channels, especially neuronal GIRK1/2 channels, have been the focus of intense research interest for developing drugs against brain diseases. In this context, venom peptides that selectively activate GIRK channels can be seen as a new source for drug development. Here, we report on the identification and electrophysiological characterization of a novel activator of GIRK1/2 channels, AsKC11, found in the venom of the sea anemone Anemonia sulcata. (2) Methods: AsKC11 was purified from the sea anemone venom by reverse-phase chromatography and the sequence was identified by mass spectrometry. Using the two-electrode voltage-clamp technique, the activity of AsKC11 on GIRK1/2 channels was studied and its selectivity for other potassium channels was investigated. (3) Results: AsKC11, a Kunitz peptide found in the venom of A. sulcata, is the first peptide shown to directly activate neuronal GIRK1/2 channels independent from Gi/o protein activity, without affecting the inward-rectifier potassium channel (IRK1) and with only a minor effect on KV1.6 channels. Thus, AsKC11 is a novel activator of GIRK channels resulting in larger K+ currents because of an increased chord conductance. (4) Conclusions: These discoveries provide new insights into a novel class of GIRK activators.


Introduction
G protein-coupled inward-rectifier potassium (GIRK/Kir3) channels are members of a large family of inward-rectifying potassium (Kir1-7) channels, which have been studied intensively for nearly three decades [1][2][3]. GIRK channels exist as tetrameric complexes comprising either identical (homomeric) or similar (heteromeric) subunits that encode a K + selectivity-filter-containing transmembrane pore (TMD for transmembrane domain) serving to conduct potassium ions and a cytoplasmic domain (CTD) [3,4]. The TMD and CTD are covalently linked by a tether, called the TMD-CTD linker [4]. GIRK channels are important regulators of the cellular excitability in the brain and cardiac cells, maintaining the resting membrane potential and regulating the shape and duration of the action potential in Sea anemones (phylum Cnidaria, class Anthozoa) produce various classes of peptide toxins that target a diverse array of ion channels/receptors, including Kunitz-type peptide toxins [22]. Some of these Kunitz peptide toxins, such as ShPI-1, HCRG21, and AsKC1, are modulators acting on different voltage-gated potassium (K V ) and transient receptor potential cation channel subfamily V member 1 (TRPV1) [23][24][25]. In the sequence of these peptide toxins, the Kunitz motif is a cysteine-rich peptide chain of~60 amino acid residues with an alpha and beta fold, stabilized by three conserved disulfide bridges [26]. Kunitztype peptides are not only found in sea anemones, but also snakes, spiders, scorpions, and cone snails [27]. They show diverse biological activities, such as inhibition of proteases and/or blocking or modulating ion channels [26]. For instance, some Kunitz-type peptides from venomous animals modulate type 2 vasopressin receptors and integrins [28,29] or TRPV1 [30], K V channels [23,25,[31][32][33], voltage-gated sodium (Na V ) channels [34], voltagegated calcium (Ca V ) channels [35], and acid-sensing ion channels (ASIC) [36]. None of the peptide toxins were shown to be an activator of GIRK channels.
In search of novel ligands that might have the potential for developing activators of GIRK1/2 channels to treat brain diseases, we screened venoms of sea anemones to find a novel activator for GIRK1/2 channels. Here, for the first time, we report the purification, identification, and electrophysiological characterization of the venom peptide of sea anemone Anemonia sulcata, called AsKC11. This Kunitz-type peptide is the first peptidic activator of GIRK1/2 channels. Interestingly, AsKC11 shows no effects on the inward-rectifier potassium channel 2.1 (Kir2.1 or IRK1) and exhibits low affinity for K V 1.6 channels. The discovery of AsKC11 represents a significant advancement in our ability to uncover a new class of activators of GIRK channels as well as providing a novel basis to find useful tools for fully probing the role and therapeutic potential of GIRK channels.

Enhancement of K + Currents through GIRK1/2 Channels by the Venom Fraction from Anemonia Sulcata
To identify the novel activator of GIRK1/2 channels, we first tested the effects of venom fractions previously purified from the venom of Anemonia sulcata [25] on whole-cell currents in oocytes expressing GIRK1/2 channels. After screening, one of the venom fractions, named A12, was found to enhance the K + currents carried by the GIRK1/2 channels in the oocytes. The oocytes coinjected with GIRK1/2 cRNA were voltage-clamped at −90 mV. To increase the GIRK current at the negative potential, a high-potassium extracellular buffer (HK) was used, which allowed for more obvious currents and accurate measurement. Thus, after exchanging ND96 to HK, basal K + currents (I K,basal ) were observed and were carried by GIRK1/2 channels (Figure 1a). In the presence of HK, K + current enhancement was immediately evoked on the application of 0.5 mg/mL fraction A12 (I K,A ) and was reversible following washing out of this fraction (Figure 1a). Since no appreciable current response to this fraction could be recorded in noninjected oocytes (Figure 1b), the increase in K + currents in oocytes expressing GIRK1/2 channels can be attributed to the activation of GIRK1/2 channels.

Purification and Identification of AsKC11 from the Active Venom Fraction
The fraction A12 showed the ability to enhance the K + currents via GIRK1/2 channels and was further purified by reverse-phase high-performance liquid chromatography (RP-HPLC) (Figure 2a). This subpurification step allowed the separation of the major peptide B2, with an average mass of 6,851.6 Da. Figure 1. AsKC11 enhanced K + currents in oocytes coexpressing GIRK1/2 but not in noninjected 142 oocytes. (a) A representative current trace shows that the enhancement of K + currents (IK,A) through 143 GIRK1/2 channels was induced by the active venom fraction A12 in Xenopus laevis oocytes (n = 6). 144 (b) A representative current trace shows that no appreciable currents were induced by the active 145 venom fraction A12 in noninjected Xenopus laevis oocytes (n = 3). 146

147
The fraction A12 showed the ability to enhance the K + currents via GIRK1/2 channels 148 and was further purified by reverse-phase high-performance liquid chromatography (RP-149 HPLC) (Figure 2a). This subpurification step allowed the separation of the major peptide 150 B2, with an average mass of 6,851.6 Da. 151 Searching LC-MS/MS spectra from reduced and alkylated and trypsin-digested B2 152 against a custom database consisting of 138,187 unique amino acid sequences, we identi-153 fied two putative transcripts (NCBI accession codes FK727748 and FK749190) from 154 Anemonia viridis encoding near-identical prepropeptides. Accurate mass measurement 155 showed that the monoisotopic molecular mass of the native peptide was 6,847.22 Da (Fig-156 ure 2b), while its sequence of 59 residues was determined by in-source decay during 157 MALDI-TOF using a 1,5-diaminonaphthalene matrix (Figure 2c). To further validate the 158 amino acid sequence of B2 we also manually examined the LC-MS/MS data, which yielded 159 complete and high-confidence coverage of the sequence of the mature B2 determined by 160 MALDI-ISD-MS ( Figure 3). 161 Figure 1. AsKC11 enhanced K + currents in oocytes coexpressing GIRK1/2 but not in noninjected oocytes. (a) A representative current trace shows that the enhancement of K + currents (I K,A ) through GIRK1/2 channels was induced by the active venom fraction A12 in Xenopus laevis oocytes (n = 6). (b) A representative current trace shows that no appreciable currents were induced by the active venom fraction A12 in noninjected Xenopus laevis oocytes (n = 3).
Searching LC-MS/MS spectra from reduced and alkylated and trypsin-digested B2 against a custom database consisting of 138,187 unique amino acid sequences, we identified two putative transcripts (NCBI accession codes FK727748 and FK749190) from Anemonia viridis encoding near-identical prepropeptides. Accurate mass measurement showed that the monoisotopic molecular mass of the native peptide was 6,847.22 Da (Figure 2b), while its sequence of 59 residues was determined by in-source decay during MALDI-TOF using a 1,5-diaminonaphthalene matrix (Figure 2c). To further validate the amino acid sequence of B2 we also manually examined the LC-MS/MS data, which yielded complete and highconfidence coverage of the sequence of the mature B2 determined by MALDI-ISD-MS ( Figure 3).
We next performed a standard protein Basic Local Alignment Search (BLAST, NCBI) using the blastp algorithm to identify hits. Among them, 33 hits were found in the Unipro-tKB Protein knowledgebase belonging to the Swiss-Prot section of UniProtKB. The Kunitz peptide U-actitoxin-Avd3n, also named AsKC11, found in A. viridis, which is a synonym of A. sulcata, was identified as sharing the same primary sequence of the peptide B2. Therefore, the peptide B2 was named AsKC11, belonging to the venom Kunitz-peptide family. The 20 hits showing more than 70% identity with AsKC11 are all from the Anemonia genus and most of them are from A. viridis. A multiple sequence alignment of AsKC11 with well-characterized venom Kunitz-type peptides and BPTI is shown in Figure 4.
To carry out more functional tests with more material, recombinant AsKC11 (rAsKC11) was produced. The yield of the rAsKC11 was about 330 µg from 1 L of the cellular culture. The recombinant peptide had the same retention time of 41 min by RP-HPLC, and when natural and recombinant peptides were coadministered, one HPLC peak was obtained. The molecular mass of rAsKC11 was 6,851.7 Da as the toxin B2. Additionally, it was found to increase the K + currents through the GIRK1/2 channels in the presence of HK, like the native peptide. These data indicate that the Kunitz peptide AsKC11 in the fraction A12 from the venom of A. sulcata is the active peptide responsible for enhancing the K + currents through GIRK1/2 channels.

Potency of AsKC11 at Enhancing GIRK1/2 Channel-Carried Inward K + Currents
The potency of AsKC11 to enhance inward K + currents through GIRK1/2 channels was studied by applying increasing concentrations of AsKC11 (2.4, 12, 24, and 48 µM) to oocytes expressing GIRK1/2 channels. These oocytes were subjected to a 1-s voltage ramp protocol from −150 to +60 mV from a holding potential of −20 mV. The K + current enhancement elicited by AsKC11 was concentration dependent, as shown in Figure 5a.  172 We next performed a standard protein Basic Local Alignment Search (BLAST, NCBI) 173 using the blastp algorithm to identity hits. Among them, 33 hits were found in the Uni-174 protKB Protein knowledgebase belonging to the Swiss-Prot section of UniProtKB. The Ku-175 nitz peptide U-actitoxin-Avd3n, also named AsKC11, found in A. viridis, which is a syno-176 nym of A. sulcata, was identified as sharing the same primary sequence of the peptide B2. 177 Therefore, the peptide B2 was named AsKC11, belonging to the venom Kunitz-peptide 178 family. The 20 hits showing more than 70% identity with AsKC11 are all from the Anemo-179 nia genus and most of them are from A. viridis. A multiple sequence alignment of AsKC11 180 with well-characterized venom Kunitz-type peptides and BPTI is shown in Figure 4. polylepis, Uniprot accession number: P00979. Cysteine residues are indicated in red, key residues for KV channel inhibition are 194 boxed in yellow, key residues for trypsin inhibition are boxed in green. 195 To carry out more functional tests with more material, recombinant AsKC11 196 (rAsKC11) was produced. The yield of the rAsKC11 was about 330 µ g from 1 L of the 197 cellular culture. The recombinant peptide had the same retention time of 41 min by  HPLC, and when natural and recombinant peptides were coadministered, one HPLC 199 peak was obtained. The molecular mass of rAsKC11 was 6,851.7 Da as the toxin B2. Ad-200 ditionally, it was found to increase the K + currents through the GIRK1/2 channels in the 201 presence of HK, like the native peptide. These data indicate that the Kunitz peptide 202 AsKC11 in the fraction A12 from the venom of A. sulcata is the active peptide responsible 203 for enhancing the K + currents through GIRK1/2 channels. The potency of AsKC11 to enhance inward K + currents through GIRK1/2 channels 206 was studied by applying increasing concentrations of AsKC11 (2.4, 12, 24, and 48 µ M) to 207 oocytes expressing GIRK1/2 channels. These oocytes were subjected to a 1-s voltage ramp 208 protocol from −150 to +60 mV from a holding potential of −20 mV. The K + current enhance-209 ment elicited by AsKC11 was concentration dependent, as shown in Figure 5a. Furthermore, according to the currents measured with the voltage ramp protocol, we could observe that the reversal potentials measured for basal and AsKC11-induced currents through GIRK1/2 channels were comparable and close to 0 mV (Figure 5b). Thus, AsKC11 did not affect the reversal potential of the measured currents, but simply increased its amplitude. This current enhancement was presumably a result of increased GIRK1/2 channel chord conductance. The impact on the inward rectification of GIRK1/2 channels was hardly observed in the presence of 48 µM AsKC11 (Figure 5c).
In addition to heterotetrameric GIRK1/2 channels, the GIRK2 channel can also form homomultimers in the human body. To further address the relationship between AsKC11 and GIRK1/2 channels, we investigated the effect of 48 µM AsKC11 on inward K + currents through the homotetrameric GIRK2 channel. In oocytes expressing the GIRK2 channel, 48 µM AsKC11 enhanced K + currents carried by the GIRK2 channel to approximately half the extent of amplitudes (43.7 ± 14.7% enhancement in inward K + currents, n = 6) compared to heterotetrameric GIRK1/2 channels (74.1 ± 12.5% enhancement in inward K + currents, n = 4) (data not shown).

AsKC11-Evoked Activation of GIRK1/2 Channels Independent from Gi/o
To assess whether AsKC11-induced GIRK channel activation requires Gi/o GPCRs, we investigated AsKC11-elicited K + currents carried by GIRK1/2 channels in the absence and presence of pertussis toxin (PTX) which directly inactivates Gi/o by ADP ribosylation. First, 2.5 ng of PTX was injected into the oocytes expressing GIRK1/2 channels one hour before the measurement. These oocytes were then voltage-clamped at −90 mV for measuring. As shown in Figure 6, in oocytes expressing GIRK1/2 channels, PTX treatment did not affect the response amplitudes evoked by 48 µM AsKC11. Thus, unlike the activation of GIRK channels mediated by GPCRs through Gi/o, we found that the effects of AsKC11 are not affected by the PTX treatment. These data support the concept that AsKC11 acts directly at the GIRK1/2 channels and does not require the presence of an activated Gi/o GPCR. Therefore, AsKC11 could be considered as a novel activator of GIRK1/2 channels. and GIRK1/2 channels, we investigated the effect of 48 μM AsKC11 on inward K currents 229 through the homotetrameric GIRK2 channel. In oocytes expressing the GIRK2 channel, 48 230 μM AsKC11 enhanced K + currents carried by the GIRK2 channel to approximately half 231 the extent of amplitudes (43.7 ± 14.7% enhancement in inward K + currents, n = 6) compared 232 to heterotetrameric GIRK1/2 channels (74.1 ± 12.5% enhancement in inward K + currents, n 233 = 4) (data not shown). 234

AsKC11-Evoked Activation of GIRK1/2 Channels Independent from Gi/o 235
To assess whether AsKC11-induced GIRK channel activation requires Gi/o GPCRs, 236 we investigated AsKC11-elicited K + currents carried by GIRK1/2 channels in the absence 237 and presence of pertussis toxin (PTX) which directly inactivates Gi/o by ADP ribosylation. 238 First, 2.5 ng of PTX was injected into the oocytes expressing GIRK1/2 channels one hour 239 before the measurement. These oocytes were then voltage-clamped at −90 mV for meas-240 uring. As shown in Figure 6, in oocytes expressing GIRK1/2 channels, PTX treatment did 241 not affect the response amplitudes evoked by 48 μM AsKC11. Thus, unlike the activation 242 of GIRK channels mediated by GPCRs through Gi/o, we found that the effects of AsKC11 243 are not affected by the PTX treatment. These data support the concept that AsKC11 acts 244 directly at the GIRK1/2 channels and does not require the presence of an activated Gi/o 245 GPCR. Therefore, AsKC11 could be considered as a novel activator of GIRK1/2 channels. 246 247 Figure 6. Summary of PTX effect on the response induced by AsKC11 (48 µ M) in oocytes expressing 248 GIRK1/2 channels. The amplitude of the IK,A was normalized to the amplitude of the IK,basal. No sig-249 nificant difference in the current response between the experimental groups GIRK1/2: AsKC11 (n = 250 Figure 6. Summary of PTX effect on the response induced by AsKC11 (48 µM) in oocytes expressing GIRK1/2 channels. The amplitude of the I K,A was normalized to the amplitude of the I K,basal . No significant difference in the current response between the experimental groups GIRK1/2: AsKC11 (n = 5) and GIRK1/2: AsKC11 + PTX (n = 3) (parametric unpaired t-test with Welch's correction, p = 0.167). The gray column representing the control group Non-injection: AsKC11 is invisible since no appreciable current was induced by AsKC11 in noninjected oocytes (n = 3).

Affinity of AsKC11 to GIRK1/2 Channels
As shown in the abovementioned results, AsKC11 binding was reversible. We next explored whether the AsKC11-elicited activation of GIRK1/2 channels followed a kinetic behavior of a simple bimolecular reaction. The current activation upon AsKC11 application and recovery upon AsKC11 removal followed a single exponential time course compatible with a bimolecular reaction scheme (See Section 4). As required by the biomolecular scheme, for the 48 µM AsKC11-evoked activation of GIRK1/2 channels, the apparent first-order association rate constant (k on ) was 5.71 × 10 −3 s −1 , and the first-order dissociation rate constant (k off = β) was 6.33 × 10 −3 s −1 . The second-order association rate constant (α) was 7.82 × 10 −5 µM −1 s −1 . When using these values to calculate the affinity of AsKC11 to GIRK1/2 channels, a k d value of 80.9 µM is found. Assuming the relationship between the channel occupancy and response is linear, k d equals EC 50 = 80.9 µM.

Effect of AsKC11 on Different Potassium Ion Channels
To explore the selectivity of AsKC11 for GIRK1/2, we tested it on oocytes expressing the closely related potassium channel IRK1 as well as five voltage-gated potassium channels, K V 1.1-1.4 and K V 1.6. At a concentration of 48 µM, AsKC11 was inactive on IRK1 (n = 3) (Figure 7). For KV1 channels, most marine toxins exert obvious effects with ~1 μM [22]. In this 275 experiment, no significant effect was observed for KV1.1-1.4 channels, although a small 276 inhibition was recorded for KV1.6 (13.5 ± 2.9%) (n ≥ 3) by applying 1 μM AsKC11 (

Discussion
Currently, the activation of neuronal GIRK channels, specifically GIRK1/2, is considered a promising pharmaceutical strategy against many neurological and psychiatric conditions, such as epilepsy, hyperalgesia, and neurodegenerative diseases [1,3,5,6]. In this article, we report the identification of AsKC11, a Kunitz toxin/peptide found in Anemonia sulcata, that activates GIRK channels. Kunitz-scaffold toxins have also been found in the venoms of snakes, spiders, solitary wasps, scorpions, polychaete worms, cone snails, and gorgonian corals [37]. They show diverse biological activities such as inhibition of proteases and/or blocking or modulating ion channels [26]. AsKC11 is the first peptide-type activator of neuronal GIRK1/2 channels with a deduced EC 50 of 80.9 µM. The other known peptide ligand of GIRK channels is a selective and potent GIRK inhibitor tertiapin (TPN), a peptide toxin found in the venom of honeybee Apis mellifera [38]. TPN inhibits the GIRK1/4 channels with nanomolar affinities [38]. Thereafter, the stable derivative of the bee venom toxin TPN was generated and was named TPNQ [39]. So far, TPNQ has been one of the most useful molecular probes for studying GIRK channels.
Sea anemones are rich in various bioactive neuropeptide toxins [40]. These peptide toxins have been applied to neuroscience research tools or directly developed as marine drugs [40]. Typically, neuropeptide toxins are among the main peptide/protein components of sea anemone venoms and act on Na V channels, K V channels, ASIC, and other ion channels [40,41]. AsKC11 is unique because it is a sea anemone Kunitz toxin active on neuronal GIRK1/2 channels. This peptide is a major component and its role as a part of the sea anemone venom has not been determined yet.
In terms of the structure, AsKC11 belongs to K V type 2 anemone toxins which all contain a Kunitz-type motif. New K V type 2 anemone toxins are supposed to be protease inhibitors and K V channel blockers, inferred from the sequence similarity according to the UniprotKB Protein knowledgebase and NCBI BLAST [22,42]. Thus, AsKC11 was assumed to inhibit K V 1 channels at the beginning of the study. However, data of the experiments show that AsKC11 at 1 µM was inactive on K V 1.1-1.4 channels, while the K V 1.6 channel was mildly blocked by 1 µM AsKC11. Compared to the most marine toxins significantly blocking K V 1 channels in a nanomolar range [22], AsKC11 shows much less potency on K V 1 channels. This could be partly explained by the lack of sufficient key residues which are essential for the high affinity of Kunitz peptides to K V 1 channels. According to the sequence homology shown in the alignment (Figure 4), only two positively charged amino acid residues (arginine and lysine) important for K V channel activity are conserved in AsKC11 [23,43,44]. Moreover, AsKC11 most likely exerts protease inhibition activity since most of the major and minor protease binding sites are conserved, as shown in Figure 4 [23,43,45].
Previously, the only reported exogenous GIRK activators were small non-peptidic molecules, ethanol and the natural product-naringin-activating GIRK channels with very low potency, as well as some compounds of undisclosed structure [3,38,46]. These activators are weak and nonspecific since they exert effects in the millimolar range and target several types of ion channels at the same range of concentrations [3,[46][47][48]. Thus, early investigation of the pathological and physiological relevance of GIRK channels was hampered partly by the lack of potent and selective probes [39]. Technologies that enable the screening of large libraries consisting of thousands of compounds boosted the discovery of many small molecules that specifically activate GIRK channels in the absence of an active Gi/o-coupled GPCR [3]. ML297, as the prototype of selective and potent GIRK activators, is now one of the most used pharmacological tools to probe GIRK functions. The activation of GIRK1/2 evoked by ML297 required the presence of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate instead of Gi/o, therefore exhibiting a GPCR-independent mechanism of channel activation [2]. Like ML297, AsKC11 directly activates GIRK1/2 channels, independent from Gi/o. Additionally, AsKC11 significantly activated the homotetrameric GIRK2 channel. In contrast, ML297 has been reported to not affect the GIRK2 channel [2,16]. Scientists have been making efforts to ameliorate the pharmacokinetic profile of the small molecules specifically activating GIRK channels and improving their selectivity towards neuronal GIRK1/2. Though AsKC11 activated GIRK1/2 with low potency in the current study, it sheds light on a new horizon for discovering a novel class of GIRK activators.
Approximately 5 µg AsKC11 was diluted in 8 µL 10% ACN 100 mM ammonium bicarbonate, pH 8, reduced with 5 mM dithiothreitol at 70 • C for 5 min, and alkylated with 10 mM iodoacetamide at 30 • C for 30 min. The reduced and alkylated AsKC11 was digested with trypsin (20 ng/µL in 10 µL) at 37 • C overnight and the resulting tryptic peptides were desalted using a C18 ZipTip (Thermo Fisher, USA), dried by vacuum centrifugation, and redissolved in 0.5% formic acid. Then, 1 µg of reduced, alkylated, and digested AsKC11 was analyzed by LC-MS/MS on a Q Exactive Fourier transform mass spectrometer (Thermo Fisher, USA) coupled to an Ultimate 3000 nano-UHPLC system (Dionex, USA). Peptides were separated on an Acclaim PepMap 100 column (C18, 3 µm particles, 100 Å pore size, 75 µm inner diameter) (Dionex, USA) using a flow rate of 300 nL/min and a gradient of 3-80% solvent B (90% ACN, 0.1% FA) in 0.1% FA over 27 min. Survey MS spectra were acquired across 400-2,000 m/z at a resolution of 70,000 at 200 m/z. Higher-energy collisional dissociation (HCD) MS2 scans were performed at 200-2,000 m/z at a resolution of 17,000 using an isolation width of 2.0 m/z, a threshold of 1e5 ion counts, and a maximum accumulation time of 60 ms.
To measure the mass of AsKC11, 200 ng peptide was analyzed by LC-ESI-MS as above and by MALDI-TOF-MS using an UltraFlex II (Bruker) operated in positive linear mode with α-cyano-4-hydroxycinnamic acid (CHCA) (7 mg/mL in 50% ACN, 0.2% TFA) as the matrix. To obtain amino acid sequence information from the intact, native AsKC11 by MALDI-ISD-MS, we spotted 1 µL peptide (1 µg/µL in 1% formic acid 50 % ACN) with 1 µL saturated 1,5-diaminonaphthalene (1,5-DAN) solution in 100% methanol. Spectra were acquired from 800 to 7,000 m/z using the same Bruker UltraFlex II operated in linear positive mode using approximately 10% higher laser power than with CHCA. Fragment ions were used for both manual de novo sequencing and manually compared to theoretical fragment ion mass lists obtained using the Protein Prospector MS-Product tool (https: //prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msproduct, accessed on 10 January 2022).

Recombinant Synthesis of rAsKC11
The construction pET32b(+)/AsKC11 was synthesized by JSC Eurogen (Russia). The recombinant plasmid with target genes was used for the transformation of BL21(DE3) Escherichia coli cells by electroporation. The transformed cells were cultured in LB medium (600 mL) containing carbenicillin (100 µg/mL) at 37 • C to the optical density of A600 0.6-0.8. Isopropyl-β,D-thiogalactopyranoside (IPTG) was added to the final concentration of 0.3 mM for the expression induction. The cells were grown for 17 h at 18 • C. The bacterial cells were precipitated from the solution by centrifugation at 6,000 rpm for 7 min.
The fusion protein that contained thioredoxin and peptide rAsKC11 was isolated by metal affinity chromatography on the Ni-NTA-agarose in the native condition from the soluble fraction of the cellular lysate after its ultrasound treatment or treatment using a French press. The fusion protein was cleaved by treatment with cyanogen bromide. The target peptide, rAsKC11, was purified by RP-HPLC on a Jupiter C4 column (10 × 250 mm, Phenomenex, Torrance, CA, USA), equilibrated by 0.1% TFA, pH 2.2, in a gradient of ACN concentration (0-70%) for 70 min at 1.5 mL/min.

Oocyte Preparation
All procedures for the use and handling of adult female Xenopus laevis frogs (CRB Xénopes, Rennes, France) were approved by the Animal Ethics Committee of the KU Leuven (Project No. P186/2019) following regulations of the European Union (EU) concerning the welfare of laboratory animals, as declared in Directive 2010/63/EU. Oocytes were isolated from ovarian tissue surgically removed during hypothermia and 0.1% (m/v) tricaine (Sigma-Aldrich Chemical, St. Louis, MO, USA)-induced anesthesia. After recovery from anesthesia, frogs were returned to their tanks in the Aquatic Facility of KU Leuven and were monitored daily.
The oocytes were enzymatically defolliculated by collagenase type IA (3 mg/mL) (Sigma-Aldrich Chemical, St. Louis, MO, USA) digestion at 16°C on a rocker platform in a Ca 2+ -free ND96 solution. Isolated stage V-VI oocytes were then maintained in ND96 solution containing Theofylline and Gentamicin at 16 • C. The ND96 solution was composed of 96 mM NaCl, 2 mM MgCl 2 , 2 mM KCl, 5 mM HEPES, and 1.8 mM CaCl 2 , with a final pH of 7.5.

Electrophysiological Measurements
For the expression of potassium channels (hK V 1.1, hK V 1.2, hK V 1.3, rK V 1.4, rK V 1.6, and mGIRK1/2, mGIRK2 alone, or IRK1) in Xenopus oocytes, the linearized plasmids were transcribed using the T3, T7, or SP6 mMESSAGEmMACHINE transcription kit (Ambion, Austin, TX, USA). Following the cRNA injection into oocytes and 1-4 days of incubation at 16 • C, electrophysiological experiments were conducted using the two-electrode voltageclamp (TEVC) GeneClamp 500B (Axon Instruments, San Jose, CA, USA). Electrodes were fabricated from borosilicate glass tubes (1.14 mm outside diameter, 0.7 mm inside diameter) by a programmable microelectrode puller, PUL-1 (World Precision Instruments, Sarasota, FL, USA). Electrodes were filled with 3 M KCl and had tip resistances from 0.8 to 1.5 MΩ. Membrane currents from voltage-clamped oocytes were digitized using a Digidata 1550 low-noise data-acquisition system (Axon Instruments, San Jose, CA, USA) and a Dell PC running pCLAMP 10.4 software (Axon Instruments, San Jose, CA, USA).
For measuring the GIRK channels and IRK1, oocytes were placed in a 0.2 mL recording chamber continuously perfused with ND96 solution. After electrode impalement and clamping the membrane potential to −90 mV, the perfusion solution was changed to the HK solution composed of 96 mM KCl, 2 mM NaCl, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 5 mM HEPES with a final pH of 7.5. The resulting increase in inward K + current represents a basal K + current that is following primarily G protein-independent GIRK channel activity. The direct application of AsKC11 to the bath was followed by the ceased perfusion of HK for 40-60 s and then AsKC11 was washed out by HK. This produced the reversible GIRK-dependent K + current. Flow, through the perfusion system, was gravity-driven. K V 1.x currents were evoked by 500 ms depolarizations to 0 mV followed by a 500 ms pulse to −50 mV, from a holding potential of −90 mV. All recordings were performed at room temperature (21 to 23 • C).
The spectrophotometric determination of AsKC11 concentration in the solution of the fraction A12 was conducted using the NanoDrop (ND-1000 UV/Vis, De Meern, the Netherlands).

Data Analysis
All electrophysiological data are presented as means ± standard deviation (SD) of n ≥ 3 independent experiments unless otherwise indicated. All data were acquired using pClamp Clampex 10.4 (Axon Instruments, San Jose, CA, USA) and analyzed using pClamp Clampfit 10.4 (Axon Instruments, San Jose, CA, USA) and GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA, USA). The statistical comparison between two experimental groups was performed by parametric unpaired t-test with Welch's correction where p < 0.05 was considered significant.
The biomolecular reaction scheme which is compatible with a single exponential time course is referred to in a previous article [49], shown below: The biomolecular reaction scheme which is compatible with a single exponential time 442 course is referred to in a previous article [49], shown below: 443 444 = × [ 11] (1) C = channel, C:AsKC11 = channel with bound AsKC11. k on and k off are the apparent first-order association and first-order disassociation rate constants, respectively. α and β are the second-and first-order rate constants of association and dissociation, respectively. τ on and τ off are the time constants for the approach to equilibrium upon wash-in and wash-out, respectively.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.