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

Citation
J. Cotton et al., 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, European journal of biochemistry, 244(1), 1997, pp. 192-202
Citations number
60
Categorie Soggetti
Biology
ISSN journal
00142956
Volume
244
Issue
1
Year of publication
1997
Pages
192 - 202
Database
ISI
SICI code
0014-2956(1997)244:1<192:APTFTS>2.0.ZU;2-B
Abstract
The potassium channel toxin secreted by the sea anemone Bunodosoma gra nulifera (BgK) is a 37-amino-acid peptide containing three disulfide b ridges. Because a synthetic peptide corresponding to the reported sequ ence of BgK was found not to fold properly the sequence was determined again. The new sequence differed from the previous one in the C-termi nal tetrapeptide, which contains two cysteines involved in disulfide b ridging. 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 a ccording to the new sequence and folded successfully. Disulfide bridge s were assigned by peptide mapping on both natural and synthetic farms to be between Cys(2)-Cys(37), Cys11-Cys30 and Cys20-Cys34. The toxin contains a C-terminal free carboxylate as shown by comparing the nativ e toxin with two synthetic peptides containing the C-terminus in eithe r the carboxylate or carboxamido form. Synthetic BgK inhibits binding of I-125-alpha-dendrotoxin to rat brain synaptosomal membranes, simila rly to natural BgK (nanomolar range). No activity was observed on maxi -K+ channels incorporated into planar lipid bilayers. The ability of B gK to block voltage-dependent K+ channels was determined from recordin gs of whole cell currents in Xenopus oocytes injected with cRNA encodi ng 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 mu M toxin. Indeed, half blockage of the current through the three Kv1 cha nnels tested occurred in the same concentration range (K-d = 6 nM for Kv-1.1, 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 discrimin ate a large group of neuronal Kv1 channels in situ. The sequence, the disulfide bridge pattern, the secondary structure and the biological a ctivity of BgK demonstrated that the sea anemone toxins, i.e. BgK, ShK and Kaliseptine, constitute novel molecular probes useful for investi gating K+ channel properties.