title: Synthetic charybdotoxin-iberiotoxin chimeric peptides define toxin binding sites on calcium-activated and voltage-dependent potassium channels.
abstract: Charybdotoxin (ChTX) and iberiotoxin (IbTX) are highly charged peptidyl toxins which exhibit 68% sequence identity and share a similar three-dimensional structure. Despite these structural similarities, IbTX and ChTX differ in their selectivity for two types of potassium channels; large conductance calcium-activated potassium (maxi-K) channels and slowly inactivating voltage-gated (Kv1.3) potassium channels. ChTX blocks with high affinity both maxi-K and Kv1.3 channels, while IbTX blocks the maxi-K but not the voltage-gated channel. To identify regions of the toxins which impart this this selectivity, we have constructed by solid-phase synthesis two chimeric toxins, ChTX1-19IbTX20-37 (Ch-IbTX) and IbTX1-19ChTX20-37 (Ib-ChTX), as well as a truncated peptide, ChTX7-37. These peptides were assayed for their ability to inhibit [125I]ChTX binding in sarcolemmal vesicles from smooth muscle (maxi-K binding) and [125I]ChTX binding to plasma membranes from brain (Kv1.3 binding). The ability of the peptides to block the maxi-K channel was determined from recordings of single maxi-K channels incorporated into planar lipid bilayers. Block of Kv1.3 was determined from recordings of whole cell currents in Xenopus oocytes injected with mRNA encoding the cloned Kv1.3 channel. Both chimeric toxins inhibited [125I]ChTX binding to sarcolemmal membranes from smooth muscle, and they both blocked the maxi-K channel in planar lipid bilayers. In contrast, [125I]ChTX binding in brain and Kv1.3 currents expressed in oocytes were inhibited only by the chimera Ib-ChTX.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Membrane Biochemistry and Biophysics, Merck Research Laboratories, Rahway, New Jersey 07065.
authors.forename: K M
authors.initials: KM
authors.lastname: Giangiacomo
authors.forename: E E
authors.initials: EE
authors.lastname: Sugg
authors.forename: M
authors.initials: M
authors.lastname: Garcia-Calvo
authors.forename: R J
authors.initials: RJ
authors.lastname: Leonard
authors.forename: O B
authors.initials: OB
authors.lastname: McManus
authors.forename: G J
authors.initials: GJ
authors.lastname: Kaczorowski
authors.forename: M L
authors.initials: ML
authors.lastname: Garcia
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D018999:Charybdotoxin; D004594:Electrophysiology; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000378:metabolism; D012604:Scorpion Venoms / Q000138:chemical synthesis / Q000378:metabolism*
chemical_list: D010455:Peptides; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms; D018999:Charybdotoxin; C064719:iberiotoxin; D002118:Calcium