A Potassium‐Channel Toxin From the Sea Anemone Bunodosoma Granulifera, An Inhibitor for Kv1 Channels — Revision of the Amino Acid Sequence, Disulfide‐Bridge Assignment, Chemical Synthesis, and Biological Activity

Abstract

The potassium channel toxin secreted by the sea anemone Bunodosoma granulifera (BgK) is a 37‐amino‐acid peptide containing three disulfide bridges. Because a synthetic peptide corresponding to the reported sequence of BgK was found not to fold properly, the sequence was determined again. The new sequence differed from the previous one in the C‐terminal tetrapeptide, which contains two cysteines involved in disulfide bridging. The revised sequence is: V C R D W F K E T A C R H A K S L G N C R T S Q K Y R A N C A K T C E L C. The toxin BgK was synthesized according according to the new sequence and folded successfully. Disulfide bridges were assigned by peptide mapping on both natural and synthetic forms to be between Cys2‐Cys37, Cys11‐Cys30 and Cys20‐Cys34. The toxin contains a C‐terminal free carboxylate as shown by comparing the native toxin with two synthetic peptides containing the C‐terminus in either the carboxylate or carboxamido form. Synthetic BgK inhibits binding of 125I‐α‐dendrotoxin to rat brain synaptosomal membranes, similarly to natural BgK (nanomolar range). No activity was observed on maxi‐K+ channels incorporated into planar lipid bilayers. The ability of BgK to block voltage‐dependent K+ channels was determined from recordings of whole cell currents in Xenopus oocytes injected with cRNA encoding three cloned Kv1 channels (Kv1.1, Kv1.2, Kv1.3) and one Kv3 (Kv3.1) channel. The Shaker‐related Kv1 channels are equally affected by BgK, while the Shaw‐related channel Kv3.1 is insensitive up to 0.125 pM toxin. Indeed, half blockage of the current through the three Kv1 channels tested occurred in the same concentration range (Kd= 6 nM for Kv1.1, 15 nM for Kv1.2, 10 nM for Kv1.3). The specificity of BgK for the Shaker‐related K+ channels indicates that BgK is able to discriminate a large group of neuronal Kv1 channels in situ. The sequence, the disulfide bridge pattern, the secondary structure and the biological activity of BgK demostrated that the sea anemone toxins, i.e. BgK, ShK and Kaliseptine, constitute novel molecular probes useful for investigating K+ channel properties.

Potassium Channel Modulation by a Toxin Domain in Matrix Metalloprotease 23

“…Peptide toxins have helped to define the molecular mechanisms underlying K ϩ channel function and to determine the relationship between K ϩ currents in native tissues and specific genes. The cnidarian toxins ShK and BgK are potent inhibitors of voltage-gated and calcium-activated K ϩ channels (15,16,(51)(52)(53)(54)(55). SMART has identified domains in a vast number of proteins that resemble ShK and BgK (see the SMART Web site).…”

“…Amide exchange rates were monitored by dissolving freezedried material in 2 H 2 O at pH 5.2 and then recording a series of one-dimensional spectra, followed by 70-ms TOCSY, 50-ms NOESY, and an exclusive correlation (E-COSY) spectra, all at 5°C. In addition, 1 H-13 C HSQC spectra for the assignment of 13 C chemical shifts and a 1 H-15 N HSQC spectrum for the assignment of 15 N chemical shifts (28,30,31) were collected at 20°C on the Bruker DRX-600 and a Bruker Avance 500 spectrometer equipped with a TXI-cryoprobe, respectively. Diffusion measurements were performed at 5 and 20°C using a pulsed field gradient longitudinal eddy-current delay pulse sequence (31,32) as implemented (33).…”

“…Indeed, venomous creatures possess toxins with homology to several proteins, including acetylcholinesterases (5), phospholipases (6, 7), nerve growth factor (8), endothelins (9), Lynx-1 (10, 11), Kunitz-type serine protease inhibitors (12), and the ion channel regulatory (ICR) 5 domains of cysteinerich secretory proteins (CRISPs) (3,13,14). Mammalian proteins containing toxin-like domains (TxDs) that block K ϩ channels have not been characterized previously.BgK, a 37-residue peptide toxin from the sea anemone Bunodosoma granulifera (15,16), and ShK, a 35-residue peptide toxin from the sea anemone Stichodactyla helianthus (17,18) are potent inhibitors of K ϩ channels. The Simple Modular Architecture Research Tool (SMART) (available on the World Wide Web) predicts the existence of a large superfamily of proteins that contain domains (referred to as ShKT domains in the SMART data base) resembling these two toxins (Fig.…”

“…BgK, a 37-residue peptide toxin from the sea anemone Bunodosoma granulifera (15,16), and ShK, a 35-residue peptide toxin from the sea anemone Stichodactyla helianthus (17,18) are potent inhibitors of K ϩ channels. The Simple Modular Architecture Research Tool (SMART) (available on the World Wide Web) predicts the existence of a large superfamily of proteins that contain domains (referred to as ShKT domains in the SMART data base) resembling these two toxins (Fig.…”

Allurin: Exploring the Activity of a Frog Sperm Chemoattractant in Mammals

“…Last is the ion channel regulatory (ICR) domain at the C-terminal (magenta), named for its ability to block or modulate a variety of voltage-dependent potassium and calcium channels as well as ryanodine receptors (Gibbs et al 2006 ). The ICR domain is similar in tertiary structure to ion channelblocking peptides from sea anenomes (Pennington et al 1999 ;Alessandri-Haber et al 1999 ;Cotton et al 1997 ).…”