Specific and slow inhibition of the kir2.1 K+ channel by gambogic acid.

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 (EC(50) < or = 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 (EC(50) = approximately 10 mum) 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.

Voltage-gated k+ channel block by catechol derivatives: defining nonselective and selective pharmacophores.

High-throughput screening led to the identification of a 3-norbornyl derivative of catechol called 48F10 (3-bicyclo[2.2.1]hept-2-yl-benzene-1,2-diol) as a Kv2.1 K(+) channel inhibitor. By virtue of the involvement of Kv2.1 channels in programmed cell death, 48F10 prevents apoptosis in cortical neurons and enterocytes. This uncharged compound acts with an apparent affinity of 1 muM at the tetraethylammonium (TEA) site at the external mouth of the Kv2.1 channel but is ineffective on Kv1.5. Here we investigated the basis of this selectivity with structure-activity studies. We find that catechol (1,2-benzenediol), unlike 48F10, inhibits Kv2.1 currents with a Hill coefficient of 2 and slows channel activation. Furthermore, this inhibition, which requires millimolar concentrations, is unaffected by external TEA or by mutation of the external tyrosine implicated in channel block by TEA and 48F10. In addition, catechol does not distinguish between Kv2.1 and Kv1.5. Thus, catechol acts at conserved sites that are distinct from 48F10. We also tested 11 catechol derivatives based on hydrocarbon adducts including norbornyl substructures, a 48F10 isomer, and a 48F10 diastereomer. These compounds are more potent than catechol, but none replicated the marked selectivity of 48F10 for Kv2.1 over Kv1.5. We conclude that the targeting of 48F10 to the TEA site at the external mouth of the Kv2.1 pore and away from other sites involved in nonselective Kv channel block by catechol requires the norbornyl group in a unique position and orientation on the catechol ring.

Identification of gamma-aminobutyric acid receptor-interacting factor 1 (TRAK2) as a trafficking factor for the K+ channel Kir2.1.

To identify proteins that regulate potassium channel activity and expression, we performed functional screening of mammalian cDNA libraries in yeast that express the mammalian K(+) channel Kir2.1. Growth of Kir2.1-expressing yeast in media with low K(+) concentration is a function of K(+) uptake via Kir2.1 channels. Therefore, the host strain was transformed with a human cDNA library, and cDNA clones that rescued growth at low K(+) concentration were selected. One of these clones was identical to the protein of unknown function isolated previously as gamma-aminobutyric acid receptor-interacting factor 1 (GRIF-1) (Beck, M., Brickley, K., Wilkinson, H., Sharma, S., Smith, M., Chazot, P., Pollard, S., and Stephenson, F. (2002) J. Biol. Chem. 277, 30079-30090). GRIF-1 specifically enhanced Kir2.1-dependent growth in yeast and Kir2.1-mediated (86)Rb(+) efflux in HEK293 cells. Quantitative microscopy and flow cytometry analysis of immunolabeled surface Kir2.1 channel showed that GRIF-1 significantly increased the number of Kir2.1 channels in the plasma membrane of COS and HEK293 cells. Physical interaction of Kir2.1 channel and GRIF-1 was demonstrated by co-immunoprecipitation from HEK293 lysates and yeast two-hybrid assay. In vivo association of Kir2.1 and GRIF-1 was demonstrated by co-immunoprecipitation from brain lysate. Yeast two-hybrid assays showed that an N-terminal region of GRIF-1 interacts with a C-terminal region of Kir2.1. These results indicate that GRIF-1 binds to Kir2.1 and facilitates trafficking of this channel to the cell surface.

Attenuation of apoptosis in enterocytes by blockade of potassium channels.

Apoptosis plays an important role in maintaining the balance between proliferation and cell loss in the intestinal epithelium. Apoptosis rates may increase in intestinal pathologies such as inflammatory bowel disease and necrotizing enterocolitis, suggesting pharmacological prevention of apoptosis as a therapy for these conditions. Here, we explore the feasibility of this approach using the rat epithelial cell line IEC-6 as a model. On the basis of the known role of K+ efflux in apoptosis in various cell types, we hypothesized that K+ efflux is essential for apoptosis in enterocytes and that pharmacological blockade of this efflux would inhibit apoptosis. By probing intracellular [K+] with the K+-sensitive fluorescent dye and measuring the efflux of 86Rb+, we found that apoptosis-inducing treatment with the proteasome inhibitor MG-132 leads to a twofold increase in K+ efflux from IEC-6 cells. Blockade of K+ efflux with tetraethylammonium, 4-aminopyridine, stromatoxin, chromanol 293B, and the recently described K+ channel inhibitor 48F10 prevents DNA fragmentation, caspase activation, release of cytochrome c from mitochondria, and loss of mitochondrial membrane potential. Thus K+ efflux occurs early in the apoptotic program and is required for the execution of later events. Apoptotic K+ efflux critically depends on activation of p38 MAPK. These results demonstrate for the first time the requirement of K+ channel-mediated K+ efflux for progression of apoptosis in enterocytes and suggest the use of K+ channel blockers to prevent apoptotic cell loss occurring in intestinal pathologies.

Novel neuroprotective K+ channel inhibitor identified by high-throughput screening in yeast.

Discovery of K+ channel modulators is limited by low-throughput capacity of existing K+ channel assays. To enable high-throughput screening for novel pharmacological modulators of K+ channels, we developed an assay based on growth of yeast that functionally expresses mammalian Kir2.1 channels. Screening of 10,000 small molecules from a combinatorial chemical library yielded 42 potential Kir2.1 inhibitors. One compound, 3-bicyclo[2.2.1]hept-2-yl-benzene-1,2-diol, was confirmed to inhibit K+ channels in patch-clamp measurements in mammalian cells with EC50 values of 60 and 1 microM for Kir2.1 and Kv2.1 channels, respectively. Inhibition of Kv2.1 channels decreased in the presence of the external pore blocker tetraethylammonium (TEA) and depended on a residue required for extracellular TEA action, suggesting that the identified compound targets the external mouth of the channel. Furthermore, at the nontoxic concentration of 3 microM, the identified compound completely abolished in vitro neuronal apoptosis mediated by Kv2.1 channels. Therefore, yeast-based screening has identified a novel uncharged neuroprotective mammalian K+ channel inhibitor.