Specific and Slow Inhibition of the Kir2.1 K+ Channel by Gambogic Acid

Abstract

Although Kir2.1 channels are important in the heart and other excitable cells, there are virtually no specific drugs for this K+ channel. In search of Kir2.1 modulators, we screened a library of 720 naturally occurring compounds using a yeast strain in which mammalian Kir2.1 enables growth at low [K+]. One of the identified compounds, gambogic acid (GA), potently (EC50 ≤ 100 nm) inhibited Kir2.1 channels in mammalian cells when applied chronically for 3 h. This potent and slow inhibition was not seen with Kv2.1, HERG or Kir1.1 channels. However, acutely applied GA acted as a weak (EC50 = ∼10 μm) non-selective K+ channel blocker. Intracellular delivery of GA via a patch pipette did not potentiate the acute effect of GA on Kir2.1, showing that slow uptake is not responsible for the delayed, potent effect. Immunoblots showed that total Kir2.1 protein expression was not altered by GA. Similarly, immunostaining of intact cells expressing Kir2.1 with an extracellular epitope tag demonstrated that GA does not affect Kir2.1 surface expression. However, the 3-h treatment with GA caused redistribution of Kir2.1 and Kv2.1 from the Triton X-100-insoluble to the Triton X-100-soluble membrane fraction. Thus, GA changes the K+ channel membrane microenvironment resulting in potent, specific, and slow acting inhibition of Kir2.1 channels. Although Kir2.1 channels are important in the heart and other excitable cells, there are virtually no specific drugs for this K+ channel. In search of Kir2.1 modulators, we screened a library of 720 naturally occurring compounds using a yeast strain in which mammalian Kir2.1 enables growth at low [K+]. One of the identified compounds, gambogic acid (GA), potently (EC50 ≤ 100 nm) inhibited Kir2.1 channels in mammalian cells when applied chronically for 3 h. This potent and slow inhibition was not seen with Kv2.1, HERG or Kir1.1 channels. However, acutely applied GA acted as a weak (EC50 = ∼10 μm) non-selective K+ channel blocker. Intracellular delivery of GA via a patch pipette did not potentiate the acute effect of GA on Kir2.1, showing that slow uptake is not responsible for the delayed, potent effect. Immunoblots showed that total Kir2.1 protein expression was not altered by GA. Similarly, immunostaining of intact cells expressing Kir2.1 with an extracellular epitope tag demonstrated that GA does not affect Kir2.1 surface expression. However, the 3-h treatment with GA caused redistribution of Kir2.1 and Kv2.1 from the Triton X-100-insoluble to the Triton X-100-soluble membrane fraction. Thus, GA changes the K+ channel membrane microenvironment resulting in potent, specific, and slow acting inhibition of Kir2.1 channels. K+ channels of the inwardly rectifying family (Kir) 4The abbreviations used are:KirK+ channels of the inwardly rectifying familyGAgambogic acidHTShigh throughput screeningTX100Triton X-100HAhemagglutinin. play key roles in the electric activity of many cell types. Kir2.1 channels are particularly important in the heart where they set the resting potential and contribute to the terminal phase of action potential repolarization. Mutations in the Kir2.1 gene cause Andersen syndrome, a triad of periodic paralysis, arrhythmia, and dysmorphic features (1.Plaster N.M. Tawil R. Tristani-Firouzi M. Canún S. Bendahhou S. Tsunoda A. Donaldson M.R. Iannaccone S.T. Brunt E. Barohn R. Clark J. Deymeer F. George Jr., A.L. Fish F.A. Hahn A. Nitu A. Ozdemir C. Serdaroglu P. Subramony S.H. Wolfe G. Fu Y.H. Ptácek L.J. Cell. 2001; 105: 511-519Abstract Full Text Full Text PDF PubMed Scopus (838) Google Scholar), as well as short QT syndrome (2.Priori S.G. Pandit S.V. Rivolta I. Berenfeld O. Ronchetti E. Dhamoon A. Napolitano C. Anumonwo J. di Barletta M.R. Gudapakkam S. Bosi G. Stramba-Badiale M. Jalife J. Circ. Res. 2005; 96: 800-807Crossref PubMed Scopus (542) Google Scholar). Kir channel activity is regulated by endogenous magnesium and polyamines (3.Matsuda H. Saigusa A. Irisawa H. Nature. 1987; 325: 156-15