[125I]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 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.
Koschak
A
A
Institut für Biochemische Pharmakologie, Innsbruck, Austria.
Koch
R O
RO
Liu
J
J
Kaczorowski
G J
GJ
Reinhart
P H
PH
Garcia
M L
ML
Knaus
H G
HG
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D005260:Female; D007457:Iodine Radioisotopes / Q000378:metabolism; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D010649:Phenylalanine / Q000378:metabolism; D015221:Potassium Channels / Q000378:metabolism*; D011485:Protein Binding; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012508:Sarcolemma / Q000187:drug effects / Q000201:enzymology; D014132:Trachea; D014443:Tyrosine / Q000378:metabolism; D014982:Xenopus laevis
D007457:Iodine Radioisotopes; D010455:Peptides; D015221:Potassium Channels; D011994:Recombinant Proteins; D001224:Aspartic Acid; D014443:Tyrosine; D010649:Phenylalanine; C064719:iberiotoxin; D002118:Calcium