Abstract
A peptidyl inhibitor of the high conductance Ca(2+)-activated K+ channels (KCa) has been purified to homogeneity from the venom of the scorpion Androctonus mauretanicus mauretanicus. The peptide has been named kaliotoxin (KTX). It is a single 4-kDa polypeptide chain. Its complete amino acid sequence has been determined. KTX displays sequence homology with other scorpion-derived inhibitors of Ca(2+)-activated or voltage-gated K+ channels: 44% homology with charybdotoxin (CTX), 52% with noxiustoxin (NTX), and 44% with iberiotoxin (IbTX). Electrophysiological experiments performed in identified nerve cells from the mollusc Helix pomatia showed that KTX specifically suppressed the whole cell Ca(2+)-activated K+ current. KTX had no detectable effects on voltage-gated K+ current (delayed rectifier and fast transient A current) or on L-type Ca2+ currents. KTX interacts in a one-to-one way with KCa channels with a Kd of 20 nM. Single channel experiments were performed on high conductance KCa channels excised from the above Helix neurons and from rabbit coeliac ganglia sympathetic neurons. KTX acted exclusively at the outer face of the channel. KTX applied on excised outside-out KCa channels induced a transient period of fast-flicker block followed by a persistent channel blockade. The KTX-induced block was not voltage-dependent which suggests differences in the blockade of KCa channels by KTX and by CTX. Comparison of KTX and CTX sequences leads to the identification of a short amino acid sequence (26-33) which may be implicated in the toxin-channel interaction. KTX therefore appears to be a useful tool for elucidating the molecular pharmacology of the high conductance Ca(2+)-activated K+ channel.
Highlights
Halim Zerrouk and Abdellah Benslimane From theZnstitut Pasteur du Maroc, Laboratoire de Purification des ProtCkianseasb,lanca, Morocco
A peptidyl inhibitor of the high conductance Ca2+- Potassium-selective channels are extraordinarilydiverse as activated K+ channels (KCa) has been purified to ho- regards to their gating mechanism, pharmacology, ionic conmogeneity from the venom of the scorpion Androc- duction properties, and regulation
cyanogen bromide (CNBr)-cleavedpeptides were separated by HPLC on column prepacked with 5 pm of Lichrosphere 100 RP-18 with isocratic desalt in 100% solvent A (0.1% trifluoroacetic acid) in 70 min followed by a linear gradient from 0 to 40% solvent B insolvent A in 100 min; flow rate 1ml/ min; absorbance reading at 215 nm
Summary
Polyacrylamide gel electrophoresis of basic proteins was performed a t pH 4.1 on 20%homogeneous Phast-Gel using aPhast-System (Pharmacia, Sweden) as defined in the Pharmacia application file No 300. Proteins were stained with Coomassie Blue under nondenaturating conditions using the Pharmacia development technique No 200 for native polyacrylamide gel electrophoresis. The toxin was reduced with dithioerythritol and S-alkylated with iodoacetic acid as described previously [20]. The reduced carboxymethylated toxin (RCM-toxin) was desalted by performing dialysis against 50 mM ammonium bicarbonate, pH 8.0
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