Population Patch Clamp Improves Data Consistency and Success Rates in the Measurement of Ionic Currents

Finkel, Alan S.; Wittel, Andrew; Yang, Naibo; Handran, Shawn; Hughes, Jan N.; Costantin, James L.

doi:10.1177/1087057106288050

Cited by 120 publications

(66 citation statements)

References 27 publications

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“…Although PPC can greatly improve the success rate and intrinsic averaging among many cells during automated electrophysiology experiments, 9 it had not yet been shown whether full dose responses for either agonists or antagonists that are represented by a sum of many uniform currents from multiple cells show good consistency with the results of conventional approaches that use singlecell recordings.…”

Section: Introductionmentioning

confidence: 99%

Validation of a High-Throughput, Automated Electrophysiology Platform for the Screening of Nicotinic Agonists and Antagonists

Graef¹,

Benson²,

Sidach³

et al. 2013

SLAS Discovery

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High-throughput compound screening using electrophysiology-based assays represents an important tool for biomedical research and drug discovery programs. The recent development and availability of devices capable of performing highthroughput electrophysiology-based screening have brought the need to validate these tools by producing data that are consistent with results obtained with conventional electrophysiological methods. In this study, we compared the response properties of hα3β4 and hα4β2 nicotinic receptors to their endogenous ligand acetylcholine (ACh) using three separate electrophysiology platforms: Dynaflow (low-throughput, manual system), PatchXpress 7000A (medium-throughput automated platform), and IonWorks Barracuda (high-throughput automated platform). We found that despite the differences in methodological approaches between these technologies, the EC 50 values from the ACh dose-response curves were consistent between all three platforms. In addition, we have validated the IonWorks Barracuda for both competitive and uncompetitive inhibition assays by using the competitive nicotinic antagonist dihydro-beta-erythroidin (DHβE) and uncompetitive nicotinic antagonist mecamylamine. Furthermore, we have demonstrated the utility of a custom-written algorithm for generating dose-response curves from multiple extrapolated current metrics that allows for discriminating between competitive and uncompetitive inhibition while maintaining high-throughput capacity. This study provides validation of the consistency of results using low-, medium-, and high-throughput electrophysiology platforms and supports their use for screening nicotinic compounds.

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Section: Introductionmentioning

confidence: 99%

Validation of a High-Throughput, Automated Electrophysiology Platform for the Screening of Nicotinic Agonists and Antagonists

Graef¹,

Benson²,

Sidach³

et al. 2013

SLAS Discovery

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“…21 Here, we have examined the use of the highest throughput automated planar array electrophysiology IonWorks system to this end. 22,23 Although perhaps nonintuitive, given the lack of simultaneous compound addition and read functionality, empirically we find that measurements after the peak response yield a highly acceptable pharmacological profile. With the 384-well throughput of this format and the electrophysiological readout, we suggest that this approach may be valuable for a focused set and secondary screening of GABA A receptors and other slow ligand-gated ion channels.…”

Section: Introductionmentioning

confidence: 99%

Population Patch-Clamp Electrophysiology Analysis of Recombinant GABAA α1β3γ2 Channels Expressed in HEK-293 Cells

Hollands¹,

Dale²,

Baxter³

et al. 2009

SLAS Discovery

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γ-Amino butyric acid (GABA)—activated Cl— channels are critical mediators of inhibitory postsynaptic potentials in the CNS. To date, rational design efforts to identify potent and selective GABAA subtype ligands have been hampered by the absence of suitable high-throughput screening approaches. The authors describe 384-well population patch-clamp (PPC) planar array electrophysiology methods for the study of GABAA receptor pharmacology. In HEK293 cells stably expressing human α1β3γ2 GABAA channels, GABA evoked outward currents at 0 mV of 1.05 ± 0.08 nA, measured 8 s post GABA addition. The IGABA was linear and reversed close to the theoretical ECl (—56 mV). Concentration-response curve analysis yielded a mean pEC50 value of 5.4 and Hill slope of 1.5, and for a series of agonists, the rank order of potency was muscimol > GABA > isoguvacine. A range of known positive modulators, including diazepam and pentobarbital, produced concentration-dependent augmentation of the GABA EC 20 response (1 µM). The competitive antagonists bicuculline and gabazine produced concentration-dependent, parallel, rightward displacement of GABA curves with pA2 and slope values of 5.7 and 1.0 and 6.7 and 1.0, respectively. In contrast, picrotoxin (0.2-150 µM) depressed the maximal GABA response, implying a non-competitive antagonism. Overall, the pharmacology of human α1β3γ2 GABAA determined by PPC was highly similar to that obtained by conventional patch-clamp methods. In small-scale single-shot screens, Z′ values of >0.5 were obtained in agonist, modulator, and antagonist formats with hit rates of 0% to 3%. The authors conclude that despite the inability of the method to resolve the peak agonist responses, PPC can rapidly and usefully quantify pharmacology for the α1β3γ2 GABAA isoform. These data suggest that PPC may be a valuable approach for a focused set and secondary screening of GABAA receptors and other slow ligand-gated ion channels. ( Journal of Biomolecular Screening 2009:769-780)

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“…[19][20][21] The primary approach has involved the use of surrogate ions such as rubidium (Rb þ ) or thallium (Tl þ ), which may be detected by atomic absorption spectrometry 22,23 or fluorescent dyes. 8,10,11 With the advent of automated patch-clamps, 6,[24][25][26][27] it is now possible to consider whether the more direct measurements of channel electrophysiological activity obtained from the automated patch-clamp justifies its higher implementation cost in high-throughput screens. It is therefore important to obtain more specific and quantitative information to assess the cost and benefit comparatives between assays employing electrophysiological methods versus surrogate ion flux measurements.…”

mentioning

confidence: 99%

Profiling Diverse Compounds by Flux- and Electrophysiology-Based Primary Screens for Inhibition of Human Ether-à-go-go Related Gene Potassium Channels

Zou

Babcock

et al. 2010

ASSAY and Drug Development Technologies

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Compound effects on cloned human Ether-à-go-go related gene (hERG) potassium channels have been used to assess the potential cardiac safety liabilities of drug development candidate compounds. In addition to radioactive ligand displacement tests, two other common approaches are surrogate ion-based flux assays and electrophysiological recordings. The former has much higher throughput, whereas the latter measures directly the effects on ionic currents. Careful characterization in earlier reports has been performed to compare the relative effectiveness of these approaches for known hERG blockers, which often yielded good overall correlation. However, cases were reported showing significant and reproducible differences in potency and/or sensitivity by the two methods. This raises a question concerning the rationale and criteria on which an assay should be selected for evaluating unknown compounds. To provide a general basis for considering assays to profile large compound libraries for hERG activity, we have conducted parallel flux and electrophysiological analyses of 2,000 diverse compounds, representative of the 300,000 compound collection of NIH Molecular Library Small Molecular Repository (MLSMR). Our results indicate that at the conventional testing concentration 1.0 mM, the overlap between the two assays ranges from 32% to 50% depending on the hit selection criteria. There was a noticeable rate of false negatives by the thallium-based assay relative to electrophysiological recording, which may be greatly reduced under modified comparative conditions. As these statistical results identify a preferred method for cardiac safety profiling of unknown compounds, they suggest an efficient method combining flux and electrophysiological assays to rapidly profile hERG liabilities of large collection of naive compounds.

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Population Patch Clamp Improves Data Consistency and Success Rates in the Measurement of Ionic Currents

Cited by 120 publications

References 27 publications

Validation of a High-Throughput, Automated Electrophysiology Platform for the Screening of Nicotinic Agonists and Antagonists

Validation of a High-Throughput, Automated Electrophysiology Platform for the Screening of Nicotinic Agonists and Antagonists

Population Patch-Clamp Electrophysiology Analysis of Recombinant GABAA α1β3γ2 Channels Expressed in HEK-293 Cells

Profiling Diverse Compounds by Flux- and Electrophysiology-Based Primary Screens for Inhibition of Human Ether-à-go-go Related Gene Potassium Channels

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