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

Biochemistry. 1997 Feb 18;36(7):1943-52. 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 (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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 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


  • Iodine Radioisotopes
  • Peptides
  • Potassium Channels
  • Recombinant Proteins
  • Aspartic Acid
  • Tyrosine
  • Phenylalanine
  • iberiotoxin
  • Calcium