2004 MDC 8th International Drug Discovery Conf.

The 8th International Drug Discovery Conference was a huge success, thanks to all of our presenters and participants! The presentation topics are listed below. For those presentations having a downloadable (PDF) version, we have indicated the file size of the PDF.

Some presenters are not able provide a PDF of their presentations. Please contact presenters directly for more presentation information.

alternative reporter systems for FLIPR®

• Evaluation of a red-shifted Ca⁺⁺ dye (Rhod-2) in the FLIPR
  Duncan R. Groebe, Ph.D., Abbott Laboratories

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  Rhod-2 is a rhodamine-derived Ca⁺⁺ chelating fluorescent dye that was described in the same paper with fluo-3 (Minta et al., (1989) J. Biol. Chem. 264, 8171). At the time of its development, the fluorescent increase of rhod-2 upon Ca⁺⁺ binding was 10-fold less than that of fluo-3 with the result that attention focused on the use of fluo-3 for investigations of Ca⁺⁺ mobilization in cells. However, the protocol for purifying rhod-2 has been improved such that the fluorescent change in rhod-2 upon Ca⁺⁺ binding is now equivalent to that of fluo-3 (Hoagland, R. (1996) Handbook of Fluorescent Probes and Research Chemicals, 6th Edition, Chapter 20.3). This suggests that it is possible to employ rhod-2 as a red-shifted fluorescent dye in the FLIPR to characterize cell-surface receptor function.

  The activity of endogenous muscarinic acetylcholine receptors in HEK 293 cells were investigated using rhod-2 and fluo-4 on the FLIPR. To accommodate the excitation and emission requirements of rhod-2, the FLIPR was outfitted with an enhanced output coupler (Coherent, Santa Clara, CA) to enhance the 528.7 nm wavelength line of the argon laser and a 600 nm bandpass filter in the second position on the filter slide. The receptors were screened though a set of 9,600 compounds, which included a number of fluorescent compounds that interfered with fluo-4, and the LOPAC library (Sigma-Aldrich, St. Louis, MO). The performance of rhod-2 in the pilot screen was comparable to that of fluo-4 in discovering hits in the compound collections screened with the advantage that the fluorescent interference detected with fluo-4 was eliminated. A summary of the development and the results of the screen using rhod-2 will be presented.

• Photina™: an improved Ca²⁺-sensitive photoprotein
  Michela Stucchi, Ph.D., Axxam

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  Calcium-sensitive photoproteins are important tools for analysing all aspects of calcium-mediated signal transduction processes in mammalian cells. One of their characteristics is the immediate photon release (flash luminescence) upon calcium binding to the coelenterazine-photoprotein complex, which makes this system extremely useful for studying receptor-ligand interactions or ion channels if the mobilisation of calcium is involved.

  In order to create a very sensitive photoprotein, the sequence and structural similarities and differences of 7 characterized photoproteins were carefully analysed. Based on that analysis a photoprotein was created with a codon usage optimized for mammalian cells, with a reduced number of cysteine residues, and most importantly with a more general calcium binding site.

  This new photoprotein, called Photina™, allows the detection of light signals from as few as 100 cells and is well suited for all applications requiring a plate format of 1536. Data will also be shown demonstrating the versatility of Photina-containing cell lines using the luminescence detector on the FLIPR3 instrument.

• A simple, dual-laser FLIPR configuration for screening using the ratiometric voltage sensor probes:"FRETing" over increasing the sensitivity and throughput in ion channel drug discovery HTS
  Robert C. "Dutch" Boltz, Merck Research Laboratories

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• Novel role of a chemoattractant receptor family protein as a high affinity β-chemokine receptor
  Aram Elagox, Ph.D., Astra Zeneca

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  Human formyl peptide-receptor-like 1 (FPRL-1) is a promiscuous G protein-coupled receptor (GPCR), and belongs to a chemoattractant receptor family protein. This receptor has been reported to interact with various host-derived peptides and lipids involved in inflammatory responses. We described here, a novel role for FPRL-1 as a high affinity β-chemokine receptor for an N-terminally truncated form of the CKβ8 (CCL23/MPIF-1) splice variant CKβ8-1 (aa22-137). RT-PCR analysis of human tissues and cells mRNA revealed a predominant expression of FPRL-1 in inflammatory cells, particularly in neutrophils. Intracellular calcium mobilisation assay, used as screening tool, in recombinant Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293s) cells co-expressing FPRL-1 and G_α16, demonstrated FPRL-1 is a functional high affinity receptor for CKβ8-1 (aa46-137, sCKβ8-1), with pEC₅₀ values of 9.13 and 8.85 respectively. In recombinant CHO-K1 cells, sCKβ8-1 induced FPRL-1 activation was regulated by G_αi/G_αo proteins, as revealed by pertussis toxin sensitivity and inhibition of forskolin-induced cyclic AMP accumulation. Binding experiments were performed with a radioiodinated synthetic peptide, [125-I]-WKYMVm, known potent FPRL-1 agonist. CHO-K1 cell membranes expressing FPRL-1 bound [125-I]-WKYMVm with a Kd of 9.34. From various FPRL-1 agonists tested, sCKβ8-1 was the most effective non-synthetic ligand to displace the radiolabeled agonist, with a pIC₅₀ of 7.97. The functional significance of sCKβ8-1 interaction with FPRL-1 was further demonstrated by the activation of polymorpho-nuclear leukocytes (PMNs) calcium mobilisation and chemotaxis. These interactions were shown to be via FPRL-1 by specific blockade of PMNs activation in the presence of an FPRL-1 antibody.

novel applications with FLIPR

• Case study: automated FLIPR screening to identify agonists for a GPCR target
  Ralf Heilker, Boehringer Ingelheim Pharma GmbH & Co.

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  FLIPR screening has broadened the technology platform of the pharmaceutical industry for specific functional formats. We believe that the implementation of this technology is of strategic importance in order to extend the scope of information provided to the exploratory projects to support lead compound generation and lead compound selection.

  In this case study, we have compared various Molecular Devices FLIPR Calcium Assay kits using different assay conditions to establish a GPCR agonist screen. The optimized assay was successfully transferred to a FLIPR3 system, integrated into an automated HTS environment. A throughput of >140 384 well-format MTP per assay day was achieved, corresponding to about 54.000 datapoints/day. The presentation will include data both on the assay development and on the screening campaign. Further, the counterscreening strategy to identify non-specifically active compounds will be described.

• FLIPR multiplex HTS: screen one GPCR get one half off
  Maura Charlton, Pfizer

  The GPCR gene family has a proven track record of providing valuable drug targets, and is the focus of lead identification efforts at a large number of biotechnology and pharmaceutical companies. Given the large number of attractive GPCRs and the steady increase in the size of compound files for screening, we have been exploring ways to make the process of gene family lead discovery faster and more cost efficient. This presentation will discuss our experiences with cell-based assay development for GPCRs, with a focus on the measurement of intracellular Ca2+ flux. It will also include an example of a successful multiplex HTS that has been run using the FLIPR, and discuss possible extensions of this approach. We have found that screening GPCRs in this way can lead to significant savings in time and resources, and allows a preliminary determination of selectivity at the level of the primary screen.

• Act One: a novel real-time cAMP assay using the FLIPR
  Chris Langmead, Nisha Patel, Elena Ratti, Jennie Heath, Martyn Wood, GlaxoSmithKline

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  Modulation of intracellular cAMP levels is a signal transduction mechanism of a number of G protein coupled receptors targeted in psychiatric disorders. GPCRs coupled to Gs stimulate of adenylyl cyclase to increase cAMP levels, whereas Gi/Go activation leads to a reduction cAMP levels. Current technologies to measure cAMP are end-point assays, often requiring cell lysis, antibody detection and lengthy incubations. The Act One cAMP uses a cyclic nucleotide gated (CNG) ion channel as a biosensor and the Molecular Devices membrane potential dye as a reporting system. Co-expression of the Gi coupled dopamine D2 receptor with the CNG channel enables the pharmacology of anti-psychotic drugs to be studied using this novel system. Forskolin stimulates cAMP production, which is inhibited in a concentration-dependent fashion by the dopamine receptor agonists quinpirole and bromocriptine. Quinpirole induced inhibition of cAMP levels was reversed in a concentration-dependent manner by a range of D2 receptor antagonists, including haloperidol, olanzapine, clozapine, risperidone and aripiprazole, with pKb values in line with those in the literature. Aripiprazole did not display any partial agonist activity in this system. Further studies examined Gs coupled GPCRs in HEK293 cells expressing the CNG channel. NECA and noradrenaline both gave concentration-dependent increases in cAMP as a result of stimulation of the endogenous adenosine A2b receptor and b1 adrenceptor, respectively. Stimulation of the cell line transiently transfected with the 5-HT6 receptor with 5-CT and SKF100786 also lead to a concentration dependent increases in cAMP levels. This demonstrates the versatility of the Act One system in drug discovery.

• Examining phosphoinositide 3-kinase (PI3K) in excitation-contraction coupling through fluorescent plate reader analysis
  Susan A. McDowell, Ph.D., Ball State University

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  Excitation-contraction (EC) coupling in cardiomyocytes involves a tightly regulated system of membranes, channels, receptors and organelles. To examine whether the phosphoinositide 3-kinase (PI3K) pathway exerts control over this tightly regulated system, EC-coupling was examined in neonatal rat ventricular myocytes using the pharmacologic PI3K inhibitor, LY294002. In the presence of LY294002, spontaneous, steady-state contraction frequency diminished in a dose-dependent manner. As this decrease in contraction could be due to a decrease in intracellular Ca²⁺, fluorescent plate reader (FLIPR) analysis was used to assess intracellular Ca²⁺ content. Intracellular Ca²⁺ decreased in a dose dependent manner with LY294002, suggesting that the decrease in myocyte contraction was due to diminished intracellular Ca²⁺. To determine which of the proteins involved in EC-coupling are under the regulation of PI3K, the effects of compounds acting at the L-type Ca²⁺ channel, SERCA-2a, the ryanodine receptor, and the Na-Ca exchanger (NCX), were compared to LY294002. Intracellular Ca²⁺ increased in response to the L-type Ca²⁺ channel agonist BAY K 8644. LY294002 attenuated the response to BAY K 8644, suggesting PI3K regulation of this channel. Ca²⁺ reuptake from the cytoplasm into the sarcoplasmic reticulum via SERCA-2a was inhibited by thapsigargin as indicated by increased basal Ca²⁺ levels. LY294002 had no detectable effect on basal Ca²⁺ levels, suggesting SERCA-2a activity is sustained in the presence of LY294002. The minimal effect of LY294002 with inhibition of the ryanodine receptor or the NCX suggests that the primary effect of LY294002 on EC-coupling occurs through inhibition of PI3K mediated L-type Ca²⁺ channel activity.

improving HTS efficiency using Analyst® and IMAP®

• Genome-wide RNAi screens against unrelated viruses identify distinct host factors that regulate pathogenesis
  Sara Cherry, Matthew Tudor, Susan Armknecht, Sean Whelan and Norbert Perrimon, Harvard Medical School

  Because viruses are obligate intracellular parasites with limited coding capacity, they co-opt host proteins and processes to complete their life cycle. Whereas biochemical approaches have traditionally been used to identify host factors involved in the infectious process, a genetic approach would be expected to provide new, complementary information about such factors. To this end, we have performed genome-wide RNAi screens to identify host factors that affect the growth of two different viruses, Drosophila C Virus and Vesicular Stomatitis Virus. We used two different screening approaches: DCV replication was assayed using immunostaining followed by automated microscopy, whereas recombinant VSV infection was quantitated using a luminometer. In each screen we identified approximately 1% of the genome that, when knocked down, protects cells from infection. These genes fall into a number of categories that have revealed processes co-opted by each virus for replication, and found very little overlap between the host genes required by each virus. By using this comparative approach, we aim to identify general factors involved in the anti-viral response, as well as those factors specific for the replication cycle of individual viruses.

• IMAP®: a practical view of protein kinase assay development for HTS
  Kety Huberman, OSI Pharmaceuticals

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  A large area of focus in drug discovery is that of the regulation of activity of disease-related protein kinases. The most common platform for this effort is to perform HTS on a biochemical assay designed to measure the phosphorylating activity of the kinase of interest. The presentation will highlight the advantages/disadvantages and challenges of different assay technologies for the development of kinase specific targets for HTS and discuss considerations and strategies taken when developing assays for these targets. Two specific cases of kinase targets developed using IMAP technology will be described.

• IMAP: a generic assay for most kinases
  James Beasley, Ph.D., Pharmacopeia

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  Several IMAP assays have been developed and utilized at Pharmacopiea. Some were developed and screened against Pharmacopeia's ECLIPS combinatorial libraries encompassing more than 5 million compounds. Others were used as selectivity counter-screens. Most of these assays were miniaturized into 8 µL 1536-well format. Screen quality parameters for the IMAP assays such as assay reproducibility, delta mP, Z factor, and assay sensitivity as well as the advantages and disadvantages of the IMAP system, including the new progressive buffer system, will be discussed.

• HTS experience using IMAP technology
  David Pechter, Shering-Plough Research Institute
• Development of an enzyme-coupled reaction for high throughput screening on the Analyst GT
  Grant Carr, Albany Molecular

  An Analyst GT has been used to develop a relatively complex screen based on a two stage assay. In the first stage, the target of interest converts ATP and a second substrate into product. In the second stage, the products ability to modulate the activity of a second enzyme is used to measure the amount of product produced in stage 1. Decreases in activity infer the presence of an inhibitory sample. The strategy behind the design of the assay, implications that design has on the types of active found and the secondary assays required to identify samples of interest are discussed.

• Cytochrome P450 enzyme inhibition assays—putting the tools to work
  Robert Singleton, Ph.D., Aventis

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  Cytochrome P450 enzymes play a critical role in the metabolism of a broad range of xenobiotics. Hence, the understanding of Cytochrome P450 enzyme and compound interactions are critical and vitally important in the development of drug molecules. In order to facilitate these studies, a method utilizing Gentest Cytochrome P450 enzyme assays, a Beckman Biomek FX robotic system, and a Molecular Devices Analyst GT has been developed. This method provides quality data for SAR analysis, which in-turn is utilized to remediate compound liabilities.

• Utilizing the ScreenStation AQ for uHTS and post uHTS compound profiling studies on novel target enzymes.
  Brian Polizzi, GlaxoSmithKline Pharmaceuticals

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  Robust, low volume screening platforms are an integral part of any successful high throughput screening facility. The ScreenStation AQ delivers high precision, non-contact dispensing of liquids combined with the detection versatility of the Acquest uHTS plate reader. Four reagent banks, each consisting of eight individually controlled syringes, are capable of simultaneously dispensing volumes as low as 1 µL. Microtiter plates can then be quickly delivered to the Acquest where up to eight detection technologies are available. The entire system is controlled through a single software package, which can integrate liquid handling routines and detection protocols for a complete uHTS script. Applications for the ScreenStation AQ can include bulk reagent dispensing into 96, 384, and 1536 well plates, complete uHTS scheduling, and mode of action studies requiring accurate serial dilutions of assay substrates. Gravimetric and fluorimetric studies indicate the ScreenStation AQÔ reliably delivers requested volumes with less than 4% coefficient of variation across all MTP formats. CV and Z' assay statistics have translated well from QC dye evaluations to various assay platforms. One example is a fluorescence polarization assay, where approximately 3000 compounds evaluated within 2 hours, yielded an average CV and Z' of 3.30% and 0.65, respectively. These values remain consistent with assay statistics achieved via manual pipetting. The ScreenStation AQ can achieve a throughput capacity of up to 100,000 wells per day, which can easily accommodate our demanding research environment.

advances in high content analysis using Discovery-1

• Integration of high throughput and high content tools into a fully automated primary screening system
  John Lee, Ph.D., Infinity Pharmaceuticals

  Infinity Pharmaceuticals, Inc., is a drug discovery company that is developing and integrating unique approaches and capabilities in synthetic chemistry, chemical genetics, informatics, and biological screening. In concert, these technologies are being developed to transform the drug discovery process while addressing the pharmaceutical industry's shortage of biologically active and selective drug candidates directed against biologically well-validated targets.

  Infinity aims to capture the full value of its novel libraries by screening these unique molecules with several complementary approaches including target-based screens, target-class screens, and phenotypic and pathway screens. A key component toward carrying out this approach is a precise, efficient, and highly flexible screening platform. The presentation will include: an overview of Infinity's automated screening platform; utilization of the system's flexibility and capabilities to support Infinity's screening approach; integration of the Analyst GT and Discovery-1 into the system for primary screening; and use of the Discovery-1 for cell based secondary assays in downstream drug discovery projects.

• Development of a fully-automated angiogenesis assay for primary high-throughput screening (HTS)
  Brian Healy, Serono

  Currently, the use of High-Content Screening (HCS) in drug discovery has largely been focused on the areas of target validation, secondary assay and functional assay testing. Recently, there has been increasing effort on applying HCS for primary small molecule HTS. One area where HCS can have a significant impact on HTS is in phenotypic screening. Phenotypic screens, which measure a change in cell physiology in the presence of compounds, have been difficult to develop using traditional HTS methodology but have been successful using high throughput cellular imaging. BD Biosciences has developed an endothelial cell tube formation assay system that allows for the screening of angiogenesis inhibitors. At Serono, we have adapted this assay for automated screening on a Polara track-based cellular screening system with the Discovery-1 high content imaging system (Molecular Devices Corp.) for quantitation of total tube length. Topics to be discussed include: assay validation, screening performance, image algorithm development using MetaMorph, an overview of the automated screening system.

automated electrophysiology: safety profiling and other applications of IonWorks™ HT

• Impact of high throughput electrophysiology in new drug discovery: role of the IonWorks HT System in drug safety testing and functional assay development
  Arturo Picones, Ph.D., Exelixis, Inc.

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  Expanding the rigor and wealth of information content of patch-clamp electrophysiology technology to the high throughput realm has been a critically pursued goal within the new drug development industry targeting ion channels with therapeutical and/or safety liability purposes. IonWorks HT became the first commercial materialization along that route.

  The present report documents the validation process of this HT electrophysiological system at Exelixis, focusing on its implementation for pharmacological safety testing against HERG channels within the context of lead discovery and optimization programs. Refinements and improvements in the recording methodology of ion channel activity, its integration with other required automation procedures and the incorporation of acquired results into activity databases is presented. The projection and functionality of the IonWorks instrument as a powerful R&D tool for functional assay development comprising the biophysical analysis of a diversity of ion channels and cellular expression systems, sub-clone functional selection and pharmacological profiling are to be illustrated. Finally the degree of correspondence with classical patch-clamp results and the complementary coexistence of these two approaches targeting ion channel functioning are assessed.

• Rapid evaluation of ion channel drug targets using IonWorks
  Victor A. Panchenko, Ph.D., Vertex Pharmaceuticals

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  Traditionally, a bottleneck for drug discovery process on ion channel modulators has been limited by the low throughput of manual patch-clamp recordings. The development of fluorescent voltage-sensitive dyes improved the throughput from a few tests per day to tens of thousands of data points per day, greatly enabling drug discovery for various types of ligand- and voltage-gated ion channels. However, these techniques are limited by the inability to control the membrane potential and thus provide less information about channel activity and conductance than traditional patch-clamp techniques. Recently, the IonWorks system was introduced to provide a higher throughput assay, while retaining the benefits of traditional patch clamp recordings. Here we describe how the IonWorks system has helped advance our drug discovery efforts with an emphasis on its role in the evaluation of cardiovascular-related and other ion channel targets, including Nav1.5, and hERG.

• Applications of IonWorks HT for drug discovery: functional characterization of stable expressing hERG cell lines and as a screening tool for evaluating the cardiac liability of preclinical drug candidates
  Heather Guthrie, Hoffmann-La Roche Inc.

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  We used the IonWorks HT automated electrophysiology instrument as a means to systematically assess ~30 hERG channel expressing cell lines based on functional performance with planar chip array technology. The IonWorks reproducibly revealed candidate hERG cell lines that were characterized by large K+ conductance and stable recordings. We compared ion channel protein expression and pharmacology by Western blot and standard electrophysiology, respectively. Using this matrix approach, we were able to determine an optimal hERG channel cell line to be used for a subsequent screening assay. The utility of IonWorks was further demonstrated when used to carry out a single point concentration screen with 300 compounds to identify candidates with > 50 % hERG current inhibition at 30 µM in a single day. Approximately 160 compounds were re-screened over a one-week period using an eight-point concentration response curve with the IonWorks to establish IC50 values. Thus, the IonWorks is a valuable screening tool that can be involved in multiple aspects of ion channel drug discovery, from development of the cellular expression system to identifying novel compounds for safety assessment.

• Voltage-gated calcium channel assay development and optimization on IonWorks HT
  Joseph G. McGivern, Amgen

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  Voltage-gated Ca²⁺ channels are integral membrane proteins that exist usually as multi-subunit complexes comprising of a pore-forming α1-subunit and one or more auxiliary subunits. Voltage-gated Ca²⁺ channels can be subdivided into two classes depending on biophysical properties. T-type Ca²⁺ channels are defined by the low voltages required for activation and usually they inactivate rapidly during maintained depolarization. In contrast, high-voltage-activated Ca²⁺ channels (L-, N-, P/ Q- and R-type) only open in response to strong membrane depolarization and they tend to be slowly or non-inactivating.

  We expressed a recombinant T-type Ca²⁺ channel α1-subunit in CHO cells. We used the whole-cell patch-clamp method to record voltage-gated Ca²⁺ currents in our cell line as a means to verify the functional expression and appropriate biophysical and pharmacological properties of the channel. Our T-type Ca²⁺ channel had a threshold of activation of -70 mV and the peak current amplitude was observed at -40 mV. The Ca²⁺ currents were inactivating in nature and were sensitive to inhibition by Ni²⁺.

  While conventional patch-clamp is a gold standard method for studying ion channel function and pharmacology, it suffers from the drawback of being limited in terms of throughput. Therefore, we used IonWorks HT to develop a higher throughput electrophysiological assay for T-type Ca²⁺ channels. In order to optimize the Ca²⁺ current assay, we investigated the effects of cell culture and preparation methods as well as recording buffer composition on seal resistance, peak current amplitude and current stability. Through careful analysis and selection of the appropriate experimental conditions, we have developed a high quality and high throughput Ca²⁺ current assay that can be used for screening focused sets of compounds and validating molecules identified using other high throughput screening platforms such as FLIPR.

• IonWorks HT: lead generation using high-throughput electrophysiology
  Jennings Worley, Ph.D., Amphora Discovery Corporation

  Significant developments in scientific understanding, technology and the implementation of lead generation best practices have generated renewed excitement in ion channel drug discovery. The recent introduction of several technologies designed to increase the throughput of electrophysiological recordings are poised to further impact ion channel lead generation. The IonWorksTM HT system has emerging as a valuable screening tool that can be involved in many phases of drug discovery, from development of cellular expression to late phase safety assessment. The coordinated use of IonWorks with other ion channel assay formats such as cell-based fluorescence measurements and traditional patch clamp are capable of improving data quality and supporting more aggressive chemistry. Amphora has reduced the IonWorks™ HT system to practice and the presentation will demonstrate our strategic use this technology in evolving the paradigm of lead generation for ion channels.

• A high-throughput electrophysiological assay for T-type Ca²⁺ channels on IonWorks HT
  Yi-xin Qian¹, Nick Callamaras², and Joseph G. McGivern^{1
  1}Amgen Inc. ²Molecular Devices Corporation.

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  Voltage-gated Ca²⁺ channels can be subdivided into two major classes. T-type Ca²⁺ channels are activated at low voltages and inactivate rapidly whereas L-, N-, P/ Q- and R-type Ca²⁺ channels open in response to large depolarization and are slowly or non-inactivating. We expressed a recombinant T-type Ca²⁺ channel α1-subunit in both HEK and CHO cells that had a threshold of activation of -70 mV, a peak current amplitude at -40 mV and inactivated during prolonged depolarizations, as determined by whole cell patch-clamp. Whole cell patch-clamp is the gold standard method for studying ion channel pharmacology but suffers from limited throughput. Therefore, we used IonWorks HT to develop a higher throughput electrophysiological assay for T-type Ca²⁺ channels. To optimize this Ca²⁺ current assay, we investigated the effects of cell lines, culture methods, recording buffer formulation, and assay materials on seal resistance, peak current amplitude, current stability, and pharmacology.