Label-free cell phenotypic profiling decodes the composition and signaling of an endogenous ATP-sensitive potassium channel

Haiyan Sun; Ying Wei; Huayun Deng; Qiaojie Xiong; Joydeep Lahiri; Ye Fang

doi:10.1038/srep04934

journal article Open Access May 12, 2014

Label-free cell phenotypic profiling decodes the composition and signaling of an endogenous ATP-sensitive potassium channel

Haiyan Sun

Ying Wei

Huayun Deng Qiaojie Xiong Joydeep Lahiri Ye Fang

Scientific Reports Vol. 4 No. 1 · Springer Science and Business Media LLC

View at Publisher Save 10.1038/srep04934

Abstract

AbstractCurrent technologies for studying ion channels are fundamentally limited because of their inability to functionally link ion channel activity to cellular pathways. Herein, we report the use of label-free cell phenotypic profiling to decode the composition and signaling of an endogenous ATP-sensitive potassium ion channel (KATP) in HepG2C3A, a hepatocellular carcinoma cell line. Label-free cell phenotypic agonist profiling showed that pinacidil triggered characteristically similar dynamic mass redistribution (DMR) signals in A431, A549, HT29 and HepG2C3A, but not in HepG2 cells. Reverse transcriptase PCR, RNAi knockdown and KATPblocker profiling showed that the pinacidil DMR is due to the activation of SUR2/Kir6.2 KATPchannels in HepG2C3A cells. Kinase inhibition and RNAi knockdown showed that the pinacidil activated KATPchannels trigger signaling through Rho kinase and Janus kinase-3 and cause actin remodeling. The results are the first demonstration of a label-free methodology to characterize the composition and signaling of an endogenous ATP-sensitive potassium ion channel.

Topics

No keywords indexed for this article. Browse by subject →

References

73

[1]

Drug Discovery: A Historical Perspective

Jürgen Drews

Science 2000 10.1126/science.287.5460.1960

[2]

Imming, R., Sinning, C. & Meyer, A. Drugs, their targets and the nature and number of drug targets. Nat. Rev. Drug. Discov. 5, 821–834 (2006). 10.1038/nrd2132

[3]

Xu, J. et al. Ion-channel assay technologies: quo vadis? Drug Discov. Today 6, 1278–1287 (2001). 10.1016/s1359-6446(01)02095-5

[4]

Wood, C., Williams, C. & Waldron, G. J. Patch clamping by numbers. Drug Discov. Today 9, 434–441 (2004). 10.1016/s1359-6446(04)03064-8

[5]

Dunlop, J., Bowlby, M., Peri, R., Vasilyev, D. & Arias, R. High-throughput electrophysiology: an emerging paradigm for ion-channel screening and physiology. Nat. Rev. Drug Discov. 7, 358–368 (2008). 10.1038/nrd2552

[6]

Seino, S. ATP-sensitive potassium channels: A model of heteromultimeric potassium channel/receptor assemblies. Annu. Rev. Physiol. 61, 337–362 (1999). 10.1146/annurev.physiol.61.1.337

[7]

Grover, G. J. & Garlid, K. D. ATP-sensitive potassium channels: A review of their cardioprotective pharmacology. J. Mol. Cell Cardiol. 32, 677–695 (2000). 10.1006/jmcc.2000.1111

[8]

Rendell, M. The role of sulphonylureas in the management of type 2 diabetes mellitus. Drugs 64, 1339–1358 (2004). 10.2165/00003495-200464120-00006

[9]

Tucker, S. J. et al. Molecular determinants of KATP channel inhibition by ATP. EMBO J. 17, 3290–3296 (1998). 10.1093/emboj/17.12.3290

[10]

Enkvetchakul, D. & Nichols, C. G. Gating mechanism of KATP channels: function fits form. J. Gen. Physiol. 122, 471–480 (2003). 10.1085/jgp.200308878

[11]

Flagg, T. P., Enkvetchakul, D., Koster, J. C. & Nichols, C. G. Muscle KATP channels: Recent insights to energy sensing and myoprotection. Physiol. Rev. 90, 799–829 (2010). 10.1152/physrev.00027.2009

[12]

Mikhailov, M. V. et al. 3-D structural and functional characterization of the purified KATP channel complex Kir6.2-SUR1. EMBO J. 24, 4166–4175 (2005). 10.1038/sj.emboj.7600877

[13]

Aguilar-Bryan, L. & Bryan, J. Molecular biology of adenosine triphosphate-sensitive potassium channels. Endocrine Rev. 20, 101–135 (1999).

[14]

Inoue, I., Nagase, H., Kishi, K. & Higuti, T. ATP-sensitive K+ channel in the mitochandria inner membrane. Nature 352, 244–247 (1991). 10.1038/352244a0

[15]

Quesada, I., Rovira, J. M., Martin, F., Roche, E., Nadal, A. & Soria, B. Nuclear KATP channels trigger nuclear Ca2+ transients that modulate nuclear function. Proc. Natl. Acad. Sci. USA 99, 9544–9549 (2002). 10.1073/pnas.142039299

[16]

Foster, D. B. et al. Mitochondrial ROMK channel is a molecular component of mitoKATP . Circ. Res. 111, 446–454 (2012). 10.1161/circresaha.112.266445

[17]

Rines, A. K., Bayeva, M. & Ardehali, H. A new pROM king for the mito-KATP dance: ROMK takes the lead. Circ. Res. 111, 392–393 (2012). 10.1161/circresaha.112.275461

[18]

Wojtovich, A. P., Urciuoli, W. R., Chatterjee, S., Fisher, A. B., Nehrke, K. & Brookes, P. S. Kir6.2 is not the mitochondrial KATP channel but is required for cardioprotection by ischemic preconditioning. Am. J. Physiol. Heart Circ. Physiol. 304, H1439–1445 (2013). 10.1152/ajpheart.00972.2012

[19]

Rodrigo, G. C. & Standen, N. B. ATP-sensitive potassium channels. Curr. Pharm. Des. 11, 1915–1940 (2005). 10.2174/1381612054021015

[20]

Fang, Y., Ferrie, A. M., Fontaine, N. H., Mauro, J. & Balakrishnan, J. Resonant waveguide grating biosensor for living cell sensing. Biophys. J. 91, 1925–1940 (2006). 10.1529/biophysj.105.077818

[21]

Fang, Y., Ferrie, A. M., Fontaine, N. H. & Yuen, P. K. Characteristics of dynamic mass redistribution of EGF receptor signaling in living cells measured with label free optical biosensors. Anal. Chem. 77, 5720–5725 (2005). 10.1021/ac050887n

[22]

Verrier, F. et al. GPCRs regulate the assembly of a multienzyme complex for purine biosynthesis. Nat. Chem. Biol. 7, 909–915 (2011). 10.1038/nchembio.690

[23]

Kenakin, T. Cellular assays as portals to seven-transmembrane receptor-based drug discovery. Nat. Rev. Drug Discov. 8, 617–626 (2009). 10.1038/nrd2838

[24]

Fang, Y. The development of label-free cellular assays for drug discovery. Expert Opin. Drug Discov. 6, 1285–1298 (2011). 10.1517/17460441.2012.642360

[25]

Fang, Y. Troubleshooting and deconvoluting label-free cell phenotypic assays in drug discovery. J. Pharmacol. Tox. Methods 67, 69–81 (2013). 10.1016/j.vascn.2013.01.004

[26]

Ferrie, A. M., Sun, H., Zaytseva, N. & Fang, Y. Divergent label-free cell phenotypic pharmacology of ligands at the overexpressed β2-adrenergic receptors. Sci. Rep. 4, 3828 (2014). 10.1038/srep03828

[27]

Ferrie, A. M., Wang, C., Deng, H. & Fang, Y. A label-free optical biosensor with microfluidics identifies an intracellular signaling wave mediated through the β2-adrenergic receptor. Integr. Biol. 5, 1253–1261 (2013). 10.1039/c3ib40112j

[28]

Moreau, C., Jacquet, H., Prost, A. L., D'hahan, N. & Vivaudou, M. The molecular basis of the specificity of action of KATP channel openers. EMBO J. 19, 6644–6651 (2000). 10.1093/emboj/19.24.6644

[29]

Mannhold, R. KATP channel openers: structure-activity relationships and therapeutic potential. Med. Res. Rev. 24, 213–266 (2004). 10.1002/med.10060

[30]

Fang, Y., Li, G. & Ferrie, A. M. Non-invasive optical biosensor for assaying endogenous G protein-coupled receptors in adherent cells. J. Pharmacol. Tox. Methods 55, 314–322 (2007). 10.1016/j.vascn.2006.11.001

[31]

Treiman, M., Caspersen, C. & Christensen, S. B. A tool coming of age: thapsigargin as an inhibitor of sarco-endoplasmic reticulum Ca2+-ATPases. Trends Pharmacol. Sci. 19, 131–135 (1998). 10.1016/s0165-6147(98)01184-5

[32]

Moncoq, K., Trieber, C. A. & Young, H. S. The molecular basis for cyclopiazonic acid inhibition of the sarcoplasmic reticulum calcium pump. J. Biol. Chem. 282, 9748–9757 (2007). 10.1074/jbc.m611653200

[33]

Luckasen, J. R. Mitogenic properties of a calcium tonophore, A23187. Proc. Nat. Acad. Sci. USA 71, 5088–5090 (1974). 10.1073/pnas.71.12.5088

[34]

Deng, H., Hu, H., Ling, S., Ferrie, A. M. & Fang, Y. Discovery of natural phenols as G protein-coupled receptor-35 (GPR35) agonists. ACS Med. Chem. Lett. 3, 165–169 (2012). 10.1021/ml2003058

[35]

Taniguchi, Y., Tonai-Kachi, H. & Shinjo, K. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid is a GPR35 agonist. Pharmacology 82, 245–249(2008). 10.1159/000157625

[36]

Jenkins, L. et al. Identification of novel species-selective agonists of the G-protein-coupled receptor GPR35 that promote recruitment of β-arrestin-2 and activate Gα13 . Biochem. J. 432, 451–459 (2010). 10.1042/bj20101287

[37]

Deng, H. & Fang, Y. The three catecholics benserazide, catechol and pyrogallol are GPR35 agonists. Pharmaceuticals 6, 500–509 (2013). 10.3390/ph6040500

[38]

Deng, H. & Fang, Y. Discovery of nitrophenols as GPR35 agonists. MedChemComm 3, 1270–1274(2012). 10.1039/c2md20210g

[39]

Deng, H., Hu, H. & Fang, Y. Multiple tyrosine metabolites are GPR35 agonists. Sci. Rep. 2, 373 (2012). 10.1038/srep00373

[40]

Grant, T. L., Ohnmacht, C. J. & Howe, B. B. Anilide tertiary carbinols: a novel series of K+ channel openers. Trends Pharmacol. Sci. 15, 402–404 (1994). 10.1016/0165-6147(94)90082-5

[41]

Roth, B. L. et al. Binding of typical and atypical antipsychotic agents to 5-hydroxytryptamine-6 and 5-hydroxytryptamine-7 receptors. J. Pharmacol. Exp. Ther. 268, 1403–1410 (1994). 10.1016/s0022-3565(25)38596-4

[42]

Hoffmann, H. H., Palese, P. & Shaw, M. L. Modulation of influenza virus replication by alteration of sodium ion transport and protein kinase C activity. Antiviral Res. 80, 124–134 (2008). 10.1016/j.antiviral.2008.05.008

[43]

Kenakin, T. Functional selectivity and biased receptor signaling. J. Pharmacol. Exp. Ther. 336, 296–302 (2011). 10.1124/jpet.110.173948

[44]

Malhi, H. et al. KATP channels regulate mitogenically induced proliferation in primary rat hepatocytes and human liver cell lines. J. Biol. Chem. 275, 26050–26057 (2000). 10.1074/jbc.m001576200

[45]

Liu, G. J. et al. ATP-sensitive potassium channels induced in liver cells after transfection with insulin cDNA and the GLUT2 transporter regulate glucose-stimulated insulin secretion. FASEB J. 17, 1682–1684 (2003). 10.1096/fj.02-0051fje

[46]

Surah-Narwal, S. et al. Block of human aorta Kir6.1 by the vascular KATP channel inhibitor U37883A. Br. J. Pharmacol. 128, 667–672 (1999). 10.1038/sj.bjp.0702862

[47]

Serrano-Martín, X., Payares, G. & Mendoza-León, A. Glibenclamide, a blocker of K+ATP channels, shows antileishmanial activity in experimental murine cutaneous leishmaniasis. Antimicrob. Agents Chemother. 50, 4214–4216 (2006). 10.1128/aac.00617-06

[48]

Paucek, P., Mironova, G., Mahdi, F., Beavis, A. D., Woldegiorgis, G. & Garlid, K. D. Reconstitution and partial purification of the glibenclamide-sensitive, ATP-dependent K+ channel from rat liver and beef heart mitochondria. J. Biol. Chem. 267, 26062–26069 (1992). 10.1016/s0021-9258(18)35717-x

[49]

Sampson, L. J., Hayabuchi, Y., Standen, N. B. & Dart, C. Caveolae localize protein kinase A signaling to arterial ATP-sensitive potassium channels. Circ. Res. 95, 1012–1018 (2004). 10.1161/01.res.0000148634.47095.ab

[50]

Lin, Y. F., Jan, Y. N. & Jan, L. Y. Regulation of ATP-sensitive potassium channel function by protein kinase A-mediated phosphorylation in transfected HEK293 cells. EMBO J. 19, 942–955 (2000). 10.1093/emboj/19.5.942

Showing 50 of 73 references

Metrics

18

Citations

73

References

Details

Published: May 12, 2014
Vol/Issue: 4(1)
License: View

Authors

Cite This Article

Haiyan Sun, Ying Wei, Huayun Deng, et al. (2014). Label-free cell phenotypic profiling decodes the composition and signaling of an endogenous ATP-sensitive potassium channel. Scientific Reports, 4(1). https://doi.org/10.1038/srep04934

Label-free cell phenotypic profiling decodes the composition and signaling of an endogenous ATP-sensitive potassium channel

You May Also Like