Characterization of Kir1.1 Channels with the Use of a Radiolabeled Derivative of Tertiapin

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• 作者：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 ; Maria L. Garcia
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：August 22, 2006
• 年：2006
• 卷：45
• 期：33
• 页码：10129 - 10139
• 全文大小：160K
• 年卷期：v.45,no.33(August 22, 2006)
• ISSN：1520-4995

文摘

Inward rectifier potassium channels (Kir) play critical roles in cell physiology. Despiterepresenting the simplest tetrameric potassium channel structures, the pharmacology of this channel familyremains largely undeveloped. In this respect, tertiapin (TPN), a 21 amino acid peptide isolated from beevenom, has been reported to inhibit Kir1.1 and Kir3.1/3.4 channels with high affinity by binding to theM1-M2 linker region of these channels. The features of the peptide-channel interaction have been exploredelectrophysiologically, and these studies have identified ways by which to alter the composition of thepeptide 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 ¹²⁵I. TPN-Y1/K12/Q13 andmono-iodo-TPN-Y1/K12/Q13 ([¹²⁷I]TPN-Y1/K12/Q13) inhibit with high affinity rat but not human Kir1.1channels stably expressed in HEK293 cells. [¹²⁵I]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 fromthese cells, and the pharmacology of the binding reaction is consistent with peptide binding to Kir1.1channels. Studies using chimeric channels indicate that the differences in TPN sensitivity between rat andhuman Kir1.1 channels are due to the presence of two nonconserved residues within the M1-M2 linkerregion. When these results are taken together, they demonstrate that [¹²⁵I]TPN-Y1/K12/Q13 representsthe first high specific activity radioligand for studying rat Kir1.1 channels and suggest its utility foridentifying other Kir channel modulators.