Lászlo Béress’s research while affiliated with Hannover Medical School and other places

(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.

Animal venoms are considered as a promising source of new drugs. Sea anemones release polypeptides that affect electrical activity of neurons of their prey. Voltage dependent sodium (Nav) channels are the common targets of Av1, Av2, and Av3 toxins from Anemonia viridis and CgNa from Condylactis gigantea. The toxins bind to the extracellular side of a channel and slow its fast inactivation, but molecular details of the binding modes are not known. Electrophysiological measurements on Periplaneta americana neuronal preparation revealed differences in potency of these toxins to increase nerve activity. Av1 and CgNa exhibit the strongest effects, while Av2 the weakest effect. Extensive molecular docking using a modern SMINA computer method revealed only partial overlap among the sets of toxins’ and channel’s amino acid residues responsible for the selectivity and binding modes. Docking positions support earlier supposition that the higher neuronal activity observed in electrophysiology should be attributed to hampering the fast inactivation gate by interactions of an anemone toxin with the voltage driven S4 helix from domain IV of cockroach Nav channel (NavPaS). Our modelling provides new data linking activity of toxins with their mode of binding in site 3 of NavPaS channel.

The human ether-à-go-go channel (hEag1 or KV10.1) is a cancer-relevant voltage-gated potassium channel that is overexpressed in a majority of human tumors. Peptides that are able to selectively inhibit this channel can be lead compounds in the search for new anticancer drugs. Here, we report the activity-guided purification and electrophysiological characterization of a novel KV10.1 inhibitor from the sea anemone Anthopleura elegantissima. Purified sea anemone fractions were screened for inhibitory activity on KV10.1 by measuring whole-cell currents as expressed in Xenopus laevis oocytes using the two-microelectrode voltage clamp technique. Fractions that showed activity on Kv10.1 were further purified by RP-HPLC. The amino acid sequence of the peptide was determined by a combination of MALDI- LIFT-TOF/TOF MS/MS and CID-ESI-FT-ICR MS/MS and showed a high similarity with APETx1 and APETx3 and was therefore named APETx4. Subsequently, the peptide was electrophysiologically characterized on KV10.1. The selectivity of the toxin was investigated on an array of voltage-gated ion channels, including the cardiac human ether-à-go-go-related gene potassium channel (hERG or Kv11.1). The toxin inhibits KV10.1 with an IC50 value of 1.1 μM. In the presence of a similar toxin concentration, a shift of the activation curve towards more positive potentials was observed. Similar to the effect of the gating modifier toxin APETx1 on hERG, the inhibition of Kv10.1 by the isolated toxin is reduced at more positive voltages and the peptide seems to keep the channel in a closed state. Although the peptide also induces inhibitory effects on other KV and NaV channels, it exhibits no significant effect on hERG. Moreover, APETx4 induces a concentration-dependent cytotoxic and proapoptotic effect in various cancerous and noncancerous cell lines. This newly identified KV10.1 inhibitor can be used as a tool to further characterize the oncogenic channel KV10.1 or as a scaffold for the design and synthesis of more potent and safer anticancer drugs.

APETx3, a novel peptide isolated from the sea anemone Anthopleura elegantissima, is a naturally occurring mutant from APETx1, only differing by a Thr to Pro substitution at position 3. APETx1 is believed to be a selective modulator of human ether-á-go-go related gene (hERG) potassium channels with a K(d) of 34 nM. In this study, APETx1, 2, and 3 have been subjected to an electrophysiological screening on a wide range of 24 ion channels expressed in Xenopus laevis oocytes: 10 cloned voltage-gated sodium channels (Na(V) 1.2-Na(V)1.8, the insect channels DmNa(V)1, BgNa(V)1-1a, and the arachnid channel VdNa(V)1) and 14 cloned voltage-gated potassium channels (K(V)1.1-K(V)1.6, K(V)2.1, K(V)3.1, K(V)4.2, K(V)4.3, K(V)7.2, K(V)7.4, hERG, and the insect channel Shaker IR). Surprisingly, the Thr3Pro substitution results in a complete abolishment of APETx3 modulation on hERG channels and provides this toxin the ability to become a potent (EC(50) 276 nM) modulator of voltage-gated sodium channels (Na(V)s) because it slows down the inactivation of mammalian and insect Na(V) channels. Our study also shows that the homologous toxins APETx1 and APETx2 display promiscuous properties since they are also capable of recognizing Na(V) channels with IC(50) values of 31 nM and 114 nM, respectively, causing an inhibition of the sodium conductance without affecting the inactivation. Our results provide new insights in key residues that allow these sea anemone toxins to recognize distinct ion channels with similar potency but with different modulatory effects. Furthermore, we describe for the first time the target promiscuity of a family of sea anemone toxins thus far believed to be highly selective.

Sea anemones are sources of biologically active proteins and peptides. However, up to date few peptidomic studies of these organisms are known; therefore most species and their peptide diversity remain unexplored. Contrasting to previous venom peptidomic works on sea anemones and other venomous animals, in the present study we combined pH gradient ion-exchange chromatography with gel filtration and reversed-phase chromatography, allowing the separation of the 1-10 kDa polypeptides from the secretion of the unexplored sea anemone Phymanthus crucifer (Cnidaria/Phymanthidae). This multidimensional chromatographic approach followed by MALDI-TOF-MS detection generated a peptide fingerprint comprising 504 different molecular mass values from acidic and basic peptides, being the largest number estimated for a sea anemone exudate. The peptide population within the 2.0-3.5 kDa mass range showed the highest frequency whereas the main biomarkers comprised acidic and basic peptides with molecular masses within 2.5-6.9 kDa, in contrast to the homogeneous group of 4-5 kDa biomarkers found in sea anemones such as B. granulifera and B. cangicum (Cnidaria/Actiniidae). Our study shows that sea anemone peptide fingerprinting can be greatly improved by including pH gradient ion-exchange chromatography into the multidimensional separation approach, complemented by MALDI-TOF-MS detection. This strategy allowed us to find the most abundant and unprecedented diversity of secreted components from a sea anemone exudate, indicating that the search for novel biologically active peptides from these organisms has much greater potential than previously predicted.

An improved chromatographic method was developed to isolate and purify polypeptides and proteins from the crude venom of the Taiwan cobra Naja naja atra. The procedure devised is simple, easy to reproduce, and enables large scale isolation of almost all polypeptides and proteins in this cobra venom. Six pure polypeptide fractions of the venom were isolated and characterized using gel filtration on Sephadex G50 (medium), ion exchange chromatography on SP-Sephadex C25, desalting on Sephadex G25 (fine) and preparative HPLC on a RPC 18 column. The neuromuscular activity of these fractions was tested on the chick biventer cervicis nerve-muscle preparation and their toxicity (LD(50)) was determined after i.v. administration in mice. Their antinociceptive activity was tested in the mouse abdominal test by i.v. application. Two of these polypeptide samples had major physiological effects: one acted as a cardiotoxin causing reversible myocardial contractures with no effect on muscle twitches elicited by nerve stimulation (NS); another was a neurotoxin that blocked muscle contractions in response to NS and exogenously added acetylcholine. The cardiotoxic fraction was identified as CTX I, a well-known cardiotoxin present in this venom, and the neurotoxin was identified as neurotoxin-α with an LD50 in mice of 0.075 mg/kg.

Sea anemone venom is a known source of interesting bioactive compounds, including peptide toxins which are invaluable tools for studying structure and function of voltage-gated potassium channels. APETx3 is a novel peptide isolated from the sea anemone Anthopleura elegantissima, containing 42 amino acids cross-linked by 3 disulfide bridges. Sequence alignment reveals that APETx3 is a natural occurring mutant from APETx1, only differing in 1 amino acid at position 3. APETx1 is believed to be a selective modulator of human ether-a-go-go related (hERG) potassium channels. In this study, APETx1, 2 and 3 have been subjected to an electrophysiological screening on a wide range of 21 ion channels expressed in Xenopus leavis oocytes: 10 cloned voltage-gated sodium channels (NaV1.2-NaV1.8, the insect channels DmNaV1, BgNaV1-1a and the arachnid channel VdNaV1) and 11 cloned voltage-gated potassium channels (KV2.1, KV3.1, KV4.2, KV4.3, KV7.1, KV7.2, KV7.3, KV7.4, KV7.5, hERG, the insect channel Shaker IR). Surprisingly, the Thr3Pro substitution results in a complete abolishment of APETx3 modulation on hERG channels. However, the same substitution provides this toxin the ability to become a potent modulator of voltage-gated sodium channels (NaVs). APETx3 slows down the inactivation of mammalian and insect channels similar to site 3 toxins such as α-scorpion toxins and sea anemone NaVs toxins. Our screening reveals that the homologous toxins APETx1 and APETx2 display promiscuous properties as they are also capable of recognizing NaV channels, causing an inhibition of the sodium conductance.All together, these data provide new insights in key residues which allow these toxins to recognize distinct ion channels with similar potency but with different modulatory effects. Furthermore, we describe for the first time the target promiscuity of a family of sea anemone toxins believed to be highly selective.