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
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
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.