Validation of a Medium-Throughput Electrophysiological Assay for KCNQ2/3 Channel Enhancers Using IonWorks HT

Abstract

Enhancers of KCNQ channels are known to be effective in chronic pain models. To discover novel enhancers of KCNQ channels, the authors developed a medium-throughput electrophysiological assay by using the IonWorks platform. Screening of 20 CHO-K1 clones stably expressing KCNQ2/3 was performed on the IonWorks HT until the best clone (judged from seal rate, current level, and stability) was obtained. The KCNQ2/3 current amplitude in the cells was found to increase from 60 ± 15 pA to 473 ± 80 pA (at —10 mV), and the expression rate was increased by 56% when the cells were incubated at 27 °C overnight. The clone used for compound screening had a seal rate of greater than 90% and an overall success rate of greater than 70%. The voltage step protocol (hold cells at —80 mV and depolarize to —10 mV for 1 s) was designed to provide moderate current but still allow for pharmacological current enhancement. EC₅₀s were generated from 8-point concentration-response curves with a control compound on each plate using compounds that were also tested with conventional patch clamp. The authors found that there was a very good correlation (R ² > 0.9) between the 2 assays, thus demonstrating the highly predictive nature of the IonWorks assay. (Journal of Biomolecular Screening 2007;1059-1067)

Keywords

ion channel electrophysiology opener blocker efflux

References

Loussouarn G. , Park KH , Bellocq C. , Baro I. , Charpentier F. , Escande D. : Phosphatidylinositol-4,5-bisphosphate, PIP2, controls KCNQ1/KCNE1 voltage-gated potassium channels: a functional homology between voltage-gated and inward rectifier K+ channels. EMBO J 2003;22:5412-5421.

Suh B-C. , Hille B. : Recovery from muscarinic modulation of M current channels requires phosphatidylinositol 4,5-bisphosphate synthesis . Neuron 2002; 35:507-520.

Zhang H. , Craciun LC , Mirshahi T. , Rohacs T. , Lopes CMB , Jin T. , et al: PIP(2) activates KCNQ channels, and its hydrolysis underlies receptor-mediated inhibition of M currents. Neuron 2003;37:963-975.

Alaburda A. , Perrier J-F. , Hounsgaard J. : An M-like outward current regulates the excitability of spinal motoneurones in the adult turtle. J Physiol 2002;540:875-881.

Gu N. , Vervaeke K. , Hu H. , Storm JF : Kv7/KCNQ/M and HCN/h, but not KCa2/SK channels, contribute to the somatic medium after-hyperpolarization and excitability control in CA1 hippocampal pyramidal cells. J Physiol 2005; 566:689-715.

Rivera-Arconada I. , Lopez-Garcia JA : Effects of M-current modulators on the excitability of immature rat spinal sensory and motor neurones. Eur J Neurosci 2005;22:3091-3098.

Rivera-Arconada I. , Martinez-Gomez J. , Lopez-Garcia JA : M-current modulators alter rat spinal nociceptive transmission: an electrophysiological study in vitro. Neuropharmacology 2004;46:598-606.

Romero M. , Reboreda A. , Sanchez E. , Lamas JA : Newly developed blockers of the M-current do not reduce spike frequency adaptation in cultured mouse sympathetic neurons. Eur J Neurosci 2004;19:2693-2702.

Main MJ , Cryan JE , Dupere JR , Cox B. , Clare JJ , Burbidge SA : Modulation of KCNQ2/3 potassium channels by the novel anticonvulsant retigabine . Mol Pharmacol 2000;58:253-262.

10.

Rundfeldt C. , Netzer R. : The novel anticonvulsant retigabine activates M-currents in Chinese hamster ovary-cells transfected with human KCNQ2/3 subunits . Neurosci Lett 2000;282:73-76.

11.

Wickenden AD , Yu W. , Zou A. , Jegla T. , Wagoner PK : Retigabine, a novel anti-convulsant, enhances activation of KCNQ2/Q3 potassium channels . Mol Pharmacol 2000;58:591-600.

12.

Dupuis DS , Schroder RL , Jespersen T. , Christensen JK , Christophersen P. , Jensen BS , et al: Activation of KCNQ5 channels stably expressed in HEK293 cells by BMS-204352. Eur J Pharmacol 2002;437:129-137.

13.

Schroder RL , Jespersen T. , Christophersen P. , Strobaek D. , Jensen BS , Olesen SP : KCNQ4 channel activation by BMS-204352 and retigabine [published correction appears in Neuropharmacology. 2003;44(4):553]. Neuropharmacology 2001;40:888-898.

14.

Schroder RL , Strobaek D. , Olesen S-P. , Christophersen P. : Voltage-independent KCNQ4 currents induced by (+/—)BMS-204352. Pflugers Arch 2003;446:607-616.

15.

Hetka R. , Rundfeldt C. , Heinemann U. , Schmitz D. : Retigabine strongly reduces repetitive firing in rat entorhinal cortex . Eur J Pharmacol 1999;386:165-171.

16.

Yue C. , Yaari Y. : KCNQ/M channels control spike afterdepolarization and burst generation in hippocampal neurons. J Neurosci 2004;24:4614-4624.

17.

Blackburn-Munro G. , Dalby-Brown W. , Mirza NR , Mikkelsen JD , Blackburn-Munro RE : Retigabine: chemical synthesis to clinical application. CNS Drug Rev 2005;11:1-20.

18.

Blackburn-Munro G. , Jensen BS : The anticonvulsant retigabine attenuates nociceptive behaviours in rat models of persistent and neuropathic pain. Eur J Pharmacol 2003;460:109-116.

19.

Dost R. , Rostock A. , Rundfeldt C. : The anti-hyperalgesic activity of retigabine is mediated by KCNQ potassium channel activation. Naunyn-Schmiedebergs Arch Pharmacol 2004;369:382-390.

20.

Passmore GM , Selyanko AA , Mistry M. , Al-Qatari M. , Marsh SJ , Matthews EA , et al: KCNQ/M currents in sensory neurons: significance for pain therapy. J Neurosci 2003;23:7227-7236.

21.

Bridgland-Taylor MH , Hargreaves AC , Easter A. , Orme A. , Henthorn DC , Ding M. , et al: Optimisation and validation of a medium-throughput electrophysiology-based hERG assay using IonWorks™ HT. J Pharmacol Toxicol Methods 2006;54:189-199.

22.

Guthrie H. , Livingston FS , Gubler U. , Garippa R. : A place for high-throughput electrophysiology in cardiac safety: screening hERG cell lines and novel compounds with the IonWorks HT™ system. J Biomol Screen 2005;10:832-840.

23.

Sorota S. , Zhang X-S. , Margulis M. , Tucker K. , Priestley T. : Characterization of a hERG screen using the IonWorks HT: comparison to a hERG rubidium efflux screen. Assay Drug Dev Technol 2005;3:47-57.

24.

John VH , Dale TJ , Hollands EC , Chen MX , Partington L. , Downie DL , et al: Novel 384-well population patch clamp electrophysiology assays for Ca2+-activated K+ channels. J Biomol Screen 2007;12:50-60.

25.

Schroeder K. , Neagle B. , Trezise DJ , Worley J. : Ionworks HT: a new high-throughput electrophysiology measurement platform . J Biomol Screen 2003 ;8:50-64.

26.

Tatulian L. , Brown DA : Effect of the KCNQ potassium channel opener retigabine on single KCNQ2/3 channels expressed in CHO cells. J Physiol 2003;549:57-63.

27.

Wang H-S. , Pan Z. , Shi W. , Brown BS , Wymore RS , Cohen IS , et al: KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel. Science 1998;282:1890-1893.

28.

Wang K. , McIlvain B. , Tseng E. , Kowal D. , Jow F. , Shen R. , et al: Validation of an atomic absorption rubidium ion efflux assay for KCNQ/M-channels using the ion Channel Reader 8000. Assay Drug Dev Technol 2004;2:525-534.

29.

Molecular adsorption to polypropylene compound plate may reduce potency measurements. Retrieved from http://www.moleculardevices.com/pages/instruments/ionworks_techsupport.html.

30.

Armijo JA , Shushtarian M. , Valdizan EM , Cuadrado A. , de las Cuevas I. , Adin J. : Ion channels and epilepsy. Curr Pharm Design 2005;11:1975-2003.

31.

Dost R. , Rundfeldt C. : The anticonvulsant retigabine potently suppresses epileptiform discharges in the low Ca++ and low Mg++ model in the hippocampal slice preparation. Epilepsy Res 2000; 38:53-66.

32.

Gribkoff VK : The therapeutic potential of neuronal KCNQ channel modulators. Expert Opin Ther Targets 2003;7:737-748.

33.

Korsgaard Mpg , Hartz BP , Brown WD , Ahring PK , Strobaek D. , Mirza NR : Anxiolytic effects of Maxipost (BMS-204352) and retigabine via activation of neuronal Kv7 channels . J Pharmacol Exp Ther 2005;314:282-292.

34.

Lerche H. , Weber YG , Jurkat-Rott K. , Lehmann-Horn F. : Ion channel defects in idiopathic epilepsies. Curr Pharm Des 2005;11:2737-2752.

35.

Armand V. , Rundfeldt C. , Heinemann U. : Effects of retigabine (D-23129) on different patterns of epileptiform activity induced by 4-aminopyridine in rat entorhinal cortex hippocampal slices . Naunyn-Schmiedebergs Arch Pharmacol 1999 ;359:33-39.

36.

Armand V. , Rundfeldt C. , Heinemann U. : Effects of retigabine (D-23129) on different patterns of epileptiform activity induced by low magnesium in rat entorhinal cortex hippocampal slices . Epilepsia 2000;41:28-33.

37.

Harrison PK , Sheridan RD , Green AC , Tattersall JEH : Effects of anticonvulsants on soman-induced epileptiform activity in the guinea-pig in vitro hippocampus. Eur J Pharmacol 2005 ;518:123-132.

38.

Piccinin S. , Randall AD , Brown JT : KCNQ/Kv7 channel regulation of hippocampal gamma-frequency firing in the absence of synaptic transmission . J Neurophysiol 2006;95:3105-3112.

39.

Rekling JC : Neuroprotective effects of anticonvulsants in rat hippocampal slice cultures exposed to oxygen/glucose deprivation. Neurosci Lett 2003;335:167-170.

40.

Straub H. , Kohling R. , Hohling J. , Rundfeldt C. , Tuxhorn I. , Ebner A. , et al: Effects of retigabine on rhythmic synchronous activity of human neocortical slices. Epilepsy Res 2001;44:155-165.