[¹²⁵I]Iberiotoxin-D19Y/Y36F, the First Selective, High Specific Activity Radioligand for High-Conductance Calcium-Activated Potassium Channels

Abstract

Iberiotoxin (IbTX), a selective peptidyl ligand for high-conductance Ca2(+)-activated K+ (maxi-K) channels cannot be radioiodinated in biologically active form due to the importance of Y36 in interacting with the channel pore. Therefore, an IbTX double mutant (IbTX-D19Y/Y36F) was engineered, expressed in Escherichia coli, purified to homogeneity, and radiolabeled to high specific activity with 125I. IbTX-D19Y/Y36F and [127I]IbTX-D19Y/Y36F block maxi-K channels expressed in Xenopus laevis oocytes with equal potency as wild-type IbTX (Kd approximately 1 nM). Under low ionic strength conditions, [125I]IbTX-D19Y/Y36F binds with high affinity to smooth muscle sarcolemmal maxi-K channels (Kd of 5 pM as determined by either equilibrium binding or kinetic binding analysis), and with a binding site density of 0.45 pmol/mg of protein. Competition studies with wild-type IbTX, IbTX-D19Y/Y36F or charybdotoxin (ChTX) result in complete inhibition of binding whereas toxins selective for voltage-gated K+ channels (margatoxin (MgTX) or alpha-dendrotoxin (alpha-DaTX) do not have any effect on IbTX binding. Indole diterpene alkaloids, which are selective inhibitors of maxi-K channels, and potassium ions both modulate [125I]IbTX-D19Y/Y36F binding in a complex manner. This pattern is also reflected during covalent incorporation of the radiolabeled toxin into the 31 kDa beta-subunit of the maxi-K channel in the presence of a bifunctional cross-linking reagent. In rat brain membranes, IbTX-D19Y/Y36F does not displace binding of [125I]MgTX or [125I]-alpha-DaTX to sites associated with voltage-gated K+ channels, nor do these latter toxins inhibit [125I]IbTX-D19Y/Y36F binding. Taken together, these results demonstrate that [125I]IbTX-D19Y/Y36F is the first selective radioligand for maxi-K channels with high specific activity.

Regional Differences in Distribution and Functional Expression of Small-Conductance Ca2+-Activated K+Channels in Rat Brain

“…The difficulties obtaining suitable SK channel antibodies with high selectivity were mostly attributable to the very low expression density of SK channel protein in mammalian brain (based on radioligand binding data using the SK channel ligand [ 125 I]apamin). This toxin labels only 20 -40 fmol of binding sites/mg of protein, a density 10 times lower than for BKchannels (as measured by [ 125 I]iberiotoxin binding; Koschak et al, 1997) or only 1% of the density of toxin-sensitive voltagegated K ϩ channels (e.g., measured by [ 125 I]hongotoxin binding; Koschak et al, 1998) in the same membrane preparation.…”

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

“…Previous studies have shown that measured equilibrium constants for block and binding of charybdotoxin and IbTx are strongly dependent upon the ionic strength of the extracellular assay medium (Anderson et al, 1988;Garcia-Calvo et al, 1991;Vasquez et al, 1989;Koschak et al, 1997) and the association of particular isoforms of four known accessory beta subunits with the BK channel alpha subunit in native tissues (Hanner et al, 1997;Wallner et al, 1999;Giangiacomo et al, 2000;Meera et al, 2000). For example when the BK channel is coexpressed with the β1 subunit, the dissociation rate of ChTx is slowed by ∼10-fold (Hanner et al, 1997;Gianciacomo et al, 2000).…”

Coassembly of Big Conductance Ca2+-activated K+ Channels and L-type Voltage-gated Ca2+ Channels in Rat Brain

“…Materials-An iberiotoxin double mutant was engineered, purified, and radioiodinated with 125 I ( 125 I-IbTX-D19Y/Y36F) as described previously (29). Isradipine ((ϩ)-3 H-PN200 -110) was obtained from PerkinElmer Life Sciences.…”

“…1A, rat brain membranes were labeled with the modified BK-specific scorpion toxin IbTX ( 125 I-IbTX-D19Y/Y36F). In earlier studies this double mutated radiolabeled toxin has been demonstrated to interact specifically with BK channels in a manner no different from native IbTX (29,33). Labeled membranes were subsequently precipitated with antibodies raised against different kinds of voltage-gated Ca 2ϩ channels.…”