[I-125] IBERIOTOXIN-D19Y Y36F, THE FIRST SELECTIVE, HIGH SPECIFIC ACTIVITY RADIOLIGAND FOR HIGH-CONDUCTANCE CALCIUM-ACTIVATED POTASSIUM CHANNELS/

Iberiotoxin (IbTX), a selective peptidyl ligand for high-conductance C a2+-activated K+ (maxi-K) channels cannot be radioiodinated in biologi cally active form due to the importance of Y36 in interacting with the channel pore. Therefore, an IbTX double mutant (IbTX-D19Y/Y36F) was e ngineered, expressed in Escherichia coli, purified to homogeneity, and radiolabeled to high specific activity with I-125. IbTX-D19Y/Y36F and [I-127]IbTX-D19Y/Y36F block maxi-K channels expressed in Xenopus laev is oocytes with equal potency as wild-type IbTX (K-d similar to 1 nM). Under low ionic strength conditions, [I-125]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 ana lysis), and with a binding site density of 0.45 pmol/mg of protein. Co mpetition studies with wild-type IbTX, IbTX-D19Y/Y36F or charybdotoxin (ChTX) result in complete inhibition of binding whereas toxins select ive for voltage-gated K+ channels (margatoxin (MgTX) or alpha-dendroto xin (alpha-DaTX)) do not have any effect on IbTX binding. Indole diter pene alkaloids, which are selective inhibitors of maxi-K channels, and potassium ions both modulate [I-125]IbTX-D19Y/Y36F binding in a compl ex manner. This pattern is also reflected during covalent incorporatio n 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 r at brain membranes, IbTX-D19Y/Y36F does not displace binding of [I-125 ]MgTX or [I-125]-alpha-DaTX to sites associated with voltage-gated Kchannels, nor do these latter toxins inhibit [I-125]IbTX-D19Y/Y36F bin ding. Taken together, these results demonstrate that [I-125]IbTX-D19Y/ Y36F is the first selective radioligand for maxi-K channels with high specific activity.