Sea anemone venom is known to contain toxins that are active on voltage-sensitive Na+ channels, as well as on delayed rectifier K+ channels belonging to the Kv1 family. This report describes the properties of a new set of peptides from Anemonia sulcata that act as blockers of a specific member of the Kv3 potassium channel family. These toxins, blood depressing substance (BDS)-I and BDS-II, are 43 amino acids long and differ at only two positions. They share no sequence homologies with other K+ channel toxins from sea anemones, such as AsKS, AsKC, ShK, or BgK. In COS-transfected cells, the Kv3.4 current was inhibited in a reversible manner by BDS-I, with an IC50 value of 47 nM. This inhibition is specific because BDS-I failed to block other K+ channels in the Kv1, Kv2, Kv3, and Kv4 subfamilies. Inward rectifier K+ channels are also insensitive to BDS-I. BDS-I and BDS-II share the same binding site on brain synaptic membranes, with K0.5 values of 12 and 19 nM, respectively. We observed that BDS-I and BDS-II have some sequence homologies with other sea anemone Na+ channels toxins, such as AsI, AsII, and AxI. However, they had a weak effect on tetrodotoxin-sensitive Na+ channels in neuroblastoma cells and no effect on Na+ channels in cardiac and skeletal muscle cells. BDS-I and BDS-II are the first specific blockers identified so far for the rapidly inactivating Kv3.4 channel.

Diochot et al (1998) Sea anemone peptides with a specific blocking activity against the fast inactivating potassium channel K_v3.4. J.Biol.Chem. PMID: 9506974.

The aim of the present study was to investigate whether K(V)3.4 channel subunits are involved in neuronal death induced by neurotoxic beta-amyloid peptides (Abeta). In particular, to test this hypothesis, three main questions were addressed: 1) whether the Abeta peptide can up-regulate both the transcription/translation and activity of K(V)3.4 channel subunit and its accessory subunit, MinK-related peptide 2 (MIRP2); 2) whether the increase in K(V)3.4 expression and activity can be mediated by the nuclear factor-kappaB (NF-kappaB) family of transcriptional factors; and 3) whether the specific inhibition of K(V)3.4 channel subunit reverts the Abeta peptide-induced neurodegeneration in hippocampal neurons and nerve growth factor (NGF)-differentiated PC-12 cells. We found that Abeta(1-42) treatment induced an increase in K(V)3.4 and MIRP2 transcripts and proteins, detected by reverse transcription-polymerase chain reaction and Western blot analysis, respectively, in NGF-differentiated PC-12 cells and hippocampal neurons. Patch-clamp experiments performed in whole-cell configuration revealed that the Abeta peptide caused an increase in I(A) current amplitude carried by K(V)3.4 channel subunits, as revealed by their specific blockade with blood depressing substance-I (BDS-I) in both hippocampal neurons and NGF-differentiated PC-12 cells. The inhibition of NF-kappaB nuclear translocation with the cell membrane-permeable peptide SN-50 prevented the increase in K(V)3.4 protein and transcript expression. In addition, the SN-50 peptide was able to block Abeta(1-42)-induced increase in K(V)3.4 K(+) currents and to prevent cell death caused by Abeta(1-42) exposure. Finally, BDS-I produced a similar neuroprotective effect by inhibiting the increase in K(V)3.4 expression. As a whole, our data indicate that K(V)3.4 channels could be a novel target for Alzheimer’s disease pharmacological therapy.

Pannaccione et al (2007) Up-regulation and increased activity of KV3.4 channels and their accessory subunit MinK-related peptide 2 induced by amyloid peptide are involved in apoptotic neuronal death. Mol.Pharmacol. PMID: 17495071.

Martina M., et al. (2007) Voltage-dependent potassium currents during fast spikes of rat cerebellar Purkinje neurons: inhibition by BDS-I toxin. J. Neurophysiol. PMID: 17065256

Kv3 potassium channels, with their ultra-rapid gating and high activation threshold, are essential for high-frequency firing in many CNS neurons. Significantly, the Kv3.4 subunit has been implicated in the major CNS disorders Parkinson’s and Alzheimer’s diseases, and it is claimed that selectively targeting this subunit will have therapeutic utility. Previous work suggested that BDS toxins (“blood depressing substance,” from the sea anemone Anemonia sulcata) were specific blockers for rapidly inactivating Kv3.4 channels, and consequently these toxins are increasingly used as diagnostic agents for Kv3.4 subunits in central neurons. However, precisely how selective are these toxins for this important CNS protein? We show that BDS is not selective for Kv3.4 but markedly inhibits current through Kv3.1 and Kv3.2 channels. Inhibition comes about not by “pore block” but by striking modification of Kv3 gating kinetics and voltage dependence. Activation and inactivation kinetics are slowed by BDS-I and BDS-II, and V(1/2) for activation is shifted to more positive voltages. Alanine substitution mutagenesis around the S3b and S4 segments of Kv3.2 reveals that BDS acts via voltage-sensing domains, and, consistent with this, ON gating currents from nonconducting Kv3.2 are markedly inhibited. The altered kinetics and gating properties, combined with lack of subunit selectivity with Kv3 subunits, seriously affects the usefulness of BDS toxins in CNS studies. Furthermore, our results do not easily fit with the voltage sensor “paddle” structure proposed recently for Kv channels. Our data will be informative for experiments designed to dissect out the roles of Kv3 subunits in CNS function and dysfunction.

Shuk Yin M. Yeung, Dawn Thompson, Zhuren Wang, David Fedida, Brian Robertson. Modulation of Kv3 subfamily potassium currents by the sea anemone toxin BDS: significance for CNS and biophysical studies. The Journal of Neuroscience 25, 8735-8745 (2005).

Blood-depressing substance I (BDS-I), a 43 amino-acid peptide from sea anemone venom, is used as a specific inhibitor of Kv3-family potassium channels. We found that BDS-I acts with even higher potency to modulate specific types of voltage-dependent sodium channels. In rat dorsal root ganglion (DRG) neurons, 3 μM BDS-I strongly enhanced tetrodotoxin (TTX)-sensitive sodium current but weakly inhibited TTX-resistant sodium current. In rat superior cervical ganglion (SCG) neurons, which express only TTX-sensitive sodium current, BDS-I enhanced current elicited by small depolarizations and slowed decay of currents at all voltages (EC(50) ∼ 300 nM). BDS-I acted with exceptionally high potency and efficacy on cloned human Nav1.7 channels, slowing inactivation by 6-fold, with an EC(50) of approximately 3 nM. BDS-I also slowed inactivation of sodium currents in N1E-115 neuroblastoma cells (mainly from Nav1.3 channels), with an EC(50) ∼ 600 nM. In hippocampal CA3 pyramidal neurons (mouse) and cerebellar Purkinje neurons (mouse and rat), BDS-I had only small effects on current decay (slowing inactivation by 20-50%), suggesting relatively weak sensitivity of Nav1.1 and Nav1.6 channels. The biggest effect of BDS-I in central neurons was to enhance resurgent current in Purkinje neurons, an effect reflected in enhancement of sodium current during the repolarization phase of Purkinje neuron action potentials. Overall, these results show that BDS-I acts to modulate sodium channel gating in a manner similar to previously known neurotoxin receptor site 3 anemone toxins but with different isoform sensitivity. Most notably, BDS-I acts with very high potency on human Nav1.7 channels.

Pin Liu, Sooyeon Jo, Bruce P. Bean. Modulation of neuronal sodium channels by the sea anemone peptide BDS-I. Journal of Neurophysiology 107, 3155-3167 (2012).