title: Characterization of Kir1.1 channels with the use of a radiolabeled derivative of tertiapin.
abstract: Inward rectifier potassium channels (Kir) play critical roles in cell physiology. Despite representing the simplest tetrameric potassium channel structures, the pharmacology of this channel family remains largely undeveloped. In this respect, tertiapin (TPN), a 21 amino acid peptide isolated from bee venom, has been reported to inhibit Kir1.1 and Kir3.1/3.4 channels with high affinity by binding to the M1-M2 linker region of these channels. The features of the peptide-channel interaction have been explored electrophysiologically, and these studies have identified ways by which to alter the composition of the peptide without affecting its biological activity. In the present study, the TPN derivative, TPN-Y1/K12/Q13, has been synthesized and radiolabeled to high specific activity with (125)I. TPN-Y1/K12/Q13 and mono-iodo-TPN-Y1/K12/Q13 ([(127)I]TPN-Y1/K12/Q13) inhibit with high affinity rat but not human Kir1.1 channels stably expressed in HEK293 cells. [(125)I]TPN-Y1/K12/Q13 binds in a saturable, time-dependent, and reversible manner to HEK293 cells expressing rat Kir1.1, as well as to membranes derived from these cells, and the pharmacology of the binding reaction is consistent with peptide binding to Kir1.1 channels. Studies using chimeric channels indicate that the differences in TPN sensitivity between rat and human Kir1.1 channels are due to the presence of two nonconserved residues within the M1-M2 linker region. When these results are taken together, they demonstrate that [(125)I]TPN-Y1/K12/Q13 represents the first high specific activity radioligand for studying rat Kir1.1 channels and suggest its utility for identifying other Kir channel modulators.
authors.affiliation: Department of Ion Channels, Merck Research Laboratories, Post Office Box 2000, Rahway, New Jersey 07065, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Felix
authors.forename: Jessica
authors.initials: J
authors.lastname: Liu
authors.forename: William A
authors.initials: WA
authors.lastname: Schmalhofer
authors.forename: Timothy
authors.initials: T
authors.lastname: Bailey
authors.forename: Maria A
authors.initials: MA
authors.lastname: Bednarek
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kinkel
authors.forename: Adam B
authors.initials: AB
authors.lastname: Weinglass
authors.forename: Martin
authors.initials: M
authors.lastname: Kohler
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Kaczorowski
authors.forename: Birgit T
authors.initials: BT
authors.lastname: Priest
authors.forename: Maria L
authors.initials: ML
authors.lastname: Garcia
mesh_terms: D000818:Animals; D001483:Base Sequence; D001514:Bee Venoms / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D002468:Cell Physiological Phenomena; D002478:Cells, Cultured; D004594:Electrophysiology / Q000379:methods; D006801:Humans; D007457:Iodine Radioisotopes / Q000737:chemistry*; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D013997:Time Factors
chemical_list: D001514:Bee Venoms; D007457:Iodine Radioisotopes; C086989:KCNJ1 protein, human; C510692:Kcnj1 protein, rat; D024661:Potassium Channels, Inwardly Rectifying; C029738:tertiapin