[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 (K_d ∼ 1 nM). Under low ionic strength conditions, [¹²⁵I]IbTX-D19Y/Y36F binds with high affinity to smooth muscle sarcolemmal maxi-K channels (K_d 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 α-dendrotoxin (α-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 β-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]-α-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.