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

• Published in: Biochemistry (Easton) 1997-02, Vol.36 (7), p.1943-1952
• Main Authors: Koschak, Alexandra, Koch, Robert O, Liu, Jessica, Kaczorowski, Gregory J, Reinhart, Peter H, Garcia, Maria L, Knaus, Hans-Günther
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Aspartic Acid - metabolism; Calcium - pharmacology; Cattle; Female; Iodine Radioisotopes - metabolism; Molecular Sequence Data; Muscle, Smooth - drug effects; Muscle, Smooth - enzymology; Oxidation-Reduction; Peptides - chemistry; Peptides - isolation & purification; Peptides - pharmacology; Phenylalanine - metabolism; Potassium Channels - metabolism; Protein Binding; Radioligand Assay; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Sarcolemma - drug effects; Sarcolemma - enzymology; Trachea; Tyrosine - metabolism; Xenopus laevis
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi962074m

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.