Synthesis of a Biotin Derivative of Iberiotoxin:  Binding Interactions with Streptavidin and the BK Ca²⁺-Activated K⁺ Channel Expressed in a Human Cell Line

Abstract

Iberiotoxin (IbTx) is a scorpion venom peptide that inhibits BK Ca 2+ -activated K + channels with high affinity and specificity. Automated solid phase synthesis was used to prepare a biotin-labeled derivative (IbTx-LC-biotin) of IbTx by substitution of Asp19 of the native 37-residue peptide with N-ε-(d-biotin-6-amidocaproate)-L-lysine. Both IbTx-LC-biotin and its complex with streptavidin (StrAv) block single BK channels from rat skeletal muscle with nanomolar affinity, indicating that the biotin-labeled residue, either alone or in complex with StrAv, does not obstruct the toxin binding interaction with the BK channel. IbTx-LC-biotin exhibits high affinity (K D = 26 nM) and a slow dissociation rate (k off = 5.4 × 10 -4 s -1 ) in a macroscopic blocking assay of whole-cell current of the cloned human BK channel. Titration of IbTx-LC-biotin with StrAv monitored by high performance size exclusion chromatography is consistent with a stoichiometry of two binding sites for IbTx-LCbiotin per StrAv tetramer, indicating that steric interference hinders simultaneous binding of two toxin molecules on each of the two biotin-binding faces of StrAv. In combination with fluorescent conjugates of StrAv or anti-biotin antibody, IbTx-LC-biotin was used to image the surface distribution of BK channels on a transfected cell line. Fluorescence microscopy revealed a patchlike surface distribution of BK channel protein. The results support the feasibility of using IbTx-LCbiotin and similar biotin-tagged K + channel toxins for diverse applications in cellular neurobiology.

Riluzole-induced block of voltage-gated Na+ current and activation of BKCa channels in cultured differentiated human skeletal muscle cells

“…Consistent with previous studies (Nimigean and Magleby, 1999;Tricarico et al, 2005;Bingham et al, 2006), this study clearly demonstrated the presence of BK Ca channels in human skeletal muscle cells. Paxilline or iberitoxin was found to block I K(Ca) in these cells.…”

“…Paxilline, an indole alkaloid isolated from Penicillium paxilli, has been known as an inhibitor of BK Ca channels . In single-channel recordings, the single-channel conductance of BK Ca channels seen in HSkMCs, was 176 ± 7 pS (n = 9), a value that is similar to that of prototypical BK Ca channels present in skeletal muscle cells (Moczydlowski and Latorre, 1983;Tricarico et al, 2005;Bingham et al, 2006). However, this value is apparently greater than that of small-conductance Ca 2+ -activated K + channels found in differentiated H9c2 myoblast (Wang et al, 1999).…”

Synthesis of an iberiotoxin derivative by chemical ligation: A method for improved yields of cysteine-rich scorpion toxin peptides

“…MH + 4265.4 Da, Calc. MH + 4265.8 Da) demonstrated a K d of 1.6 nM via lipid bilayer (Table 1), and demonstrated characteristic discrete long-lived blocked states upon assay by single BK Ca channels as observed in other αKTx1 toxins (see [11,2]; data not shown). Synthetic yields of IbTx-D19C-Acm were ~2% per 100 mg peptide oxidized.…”

“…Combining the structure–activity profiles of ChTx [33,34], IbTx [5,8,19], IbTx-chimeras [12], and our previous IbTx derivative studies [2], a suitable thiol-ester ligation construct was designed, containing an orthogonally protected ‘spinster thiol’. Native chemical ligation requires a C-terminal peptide segment (Peptide B) to contain an N-terminal Cys (see Fig.…”

“…An N-terminal Flag-BK version of the human K Ca 1.1 channel (HSlo) was stably expressed in a human cell line (HEK293) as previously described [2]. Channel activity was monitored by the whole-cell patch clamp electrophysiological technique [18] using an Axon 200B amplifier and Clampex software (Axon Instruments) for voltage pulse programming and data acquisition.…”