Characterization of Kir1.1 Channels with the Use of a Radiolabeled Derivative of Tertiapin John P. Felix, ‡,§ Jessica Liu, ‡,§ William A. Schmalhofer, ‡,§ Timothy Bailey, ‡,§ Maria A. Bednarek, | Stephanie Kinkel, ‡ Adam B. Weinglass, ‡ Martin Kohler, ‡ Gregory J. Kaczorowski, ‡ Birgit T. Priest, ‡ and Maria L. Garcia* ,‡ Department of Ion Channels, and Medicinal Chemistry, Merck Research Laboratories, Post Office Box 2000, Rahway, New Jersey 07065 ReceiVed March 14, 2006; ReVised Manuscript ReceiVed May 25, 2006 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. Potassium channels regulate many critical cellular func- tions such as the electrical pattern of neurons, muscle contraction, hormone and neurotransmitter release, electrolyte movement, and cell proliferation (1). The large family of potassium channels contains, as a common structural feature, two transmembrane domains connected by a pore loop, the M1-M2 linker, where the potassium selectivity sequence resides. Four of these subunits assemble to form a functional tetrameric channel. Other structurally distinct domains can associate with the pore domain to yield channels that gate in response to different stimuli. The high-resolution X-ray structure of potassium channels has provided detailed information concerning the molecular mechanisms involved in ion permeation (2, 3). Inward rectifier potassium channels (Kir) 1 are the prototype of a minimum potassium channel structure in that they only consist of two transmembrane segments and a pore domain. Many residues in the N- and C-terminal regions of these proteins are important for the regulation of channel activity, as well as for their trafficking and interaction with other subunits. Inward rectification, defined as the capacity to carry more current in the inward than in the outward direction, is due to the voltage-dependent block of the channel by intracellular cations such as Mg 2+ and polyamines (4, 5). The molecular basis for inward rectification is well- understood from functional studies as well as from the high- resolution X-ray structure of the bacterial homologue of Kir1.1 (6, 7). The functional importance of inward rectifier potassium channels is highlighted by the fact that mutations in the genes encoding these proteins result in neuronal degeneration (Kir3.2), cardiac arrhythmias (Kir2.1), defective renal salt absorption (Kir1.1), and defective insulin secretion from pancreatic  cells (Kir6.2) (8, 9). Despite the prominent role that Kir channels play in cell physiology, the molecular pharmacology of these channels remains mostly undeveloped. The only high-affinity probe for some members of the Kir family is tertiapin (TPN), a 21 amino acid peptide isolated from bee venom (10). TPN blocks Kir1.1 and Kir3.1/3.4 channels with high affinity by binding to residues present in the external end of the ion-conduction pore of these channels through a reversible bimolecular reaction. Other * To whom correspondence should be addressed. Telephone: (732) 594-7564. Fax: (732) 594-3925. E-mail: maria_garcia@merck.com. ‡ Department of Ion Channels. § These authors contributed equally to this work. | Medicinal Chemistry. 1 Abbreviations: Kir, inward rectifier potassium channel; TFA, trifluoroacetic acid; TPN, tertiapin; [ 125 I]-TPN-Y1/K12/Q13, mono- iodotyrosine-TPN-Y1/K12/Q13; Kd, equilibrium dissociation constant; Ki, equilibrium inhibition constant; k1, association rate constant; k-1, dissociation rate constant; MCDP, mast cell degranulating peptide; ChTX, charybdotoxin; IbTX, iberiotoxin; MgTX, margatoxin; DTX, dendrotoxin; ShK, Stichodactyla helianthus peptide. 10129 Biochemistry 2006, 45, 10129-10139 10.1021/bi060509s CCC: $33.50 © 2006 American Chemical Society Published on Web 07/25/2006