SYNTHETIC CHARYBDOTOXIN IBERIOTOXIN CHIMERIC PEPTIDES DEFINE TOXIN BINDING-SITES ON CALCIUM-ACTIVATED AND VOLTAGE-DEPENDENT POTASSIUM CHANNELS

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 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 [I-125]ChTX binding in sarcolemmal vesicles from smooth muscle (maxi-K binding) and [I-125]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 incoporated 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 [I-125]ChTX binding to sarcolemmal membranes from smooth muscle, and they both blocked the maxi-K channel in planar lipid bilayers. In contrast, [I-125]ChTX binding in brain and Kv1.3 currents expressed in oocytes were inhibited only by the chimera Ib-ChTX. Deletion of the first six amino acids from the N-terminus (ChTX7-37) significantly weakened the interaction of ChTX with the maxi-K channel but had little effect on toxin interaction with Kv1.3. These findings suggest that the C-terminal domain of the ChTX homologues defines the toxin-channel interaction which distinguishes between the maxi-K channel and Kv1.3. In contrast, the N-terminal domain of ChTX imparts of high-affinity interaction with the maxi-K channel but not with Kv1.3.