Abstract
Whole-cell patch-clamp-based electrophysiological techniques are powerful tools for examining the biophysical and pharmacological properties of ion channels. However, the recent validation of ion channels as novel drug targets necessitates the development of a faster screening method for ion channels. Therefore, we have developed a rapid, reliable, and sensitive cell-based high throughput screening (HTS) assay for T-type Ca2+ channels. We had previously constructed HEK293/α1G/Kir2.1 cell lines that stably expressed α1G and Kir2.1 subunits [1] and found that α1G T-type channel- sensitive Ca2+ signals were detected by the application of high concentrations of KCl under fura-2-based single cell measurements of intracellular Ca2+ concentration ([Ca2+]i). In the present study, we applied HEK293/??1G/Kir2.1 cells to the FDSS6000 (Functional Drug Screening System) to develop a fast and reliable cell-based HTS method for α1G T-type Ca2+ channels. After detecting 70 mM KCl-induced [Ca2+]i increases using the FDSS6000 system, we verified this new α1G channel HTS system by examining two T-type Ca2+ channel blockers, Ni2+ and mibefradil, and measuring the Z- factor (Z factor = 0.66) in 96-well plates. Furthermore, we assayed selected 3,4-dihydroquinazolin derivatives using this FDSS6000-based α1G channel HTS system at the level of IC50 values and compared the results with those obtained from whole-cell patch-clamp recordings. Taken together, our results suggest that the FDSS6000-based α1G channel HTS system is a fast and feasible assay for α1G T-type Ca2+ channels. This assay can be utilized as a primary screening method for Ttype Ca2+ channel-targeted chemicals and for the development of HTS systems for other types of ion channels.
Keywords: α1G T-type Ca2+ channel, HEK293, mibefradil, Fluo-3-AM, FDSS6000, patch-clamp
Combinatorial Chemistry & High Throughput Screening
Title: High Throughput Screening Assay of α1G T-type Ca2+ Channels and Comparison with Patch-Clamp Studies
Volume: 12 Issue: 3
Author(s): Yoonjee Kim, Soonja Kang, Jae Yeol Lee and Hyewhon Rhim
Affiliation:
Keywords: α1G T-type Ca2+ channel, HEK293, mibefradil, Fluo-3-AM, FDSS6000, patch-clamp
Abstract: Whole-cell patch-clamp-based electrophysiological techniques are powerful tools for examining the biophysical and pharmacological properties of ion channels. However, the recent validation of ion channels as novel drug targets necessitates the development of a faster screening method for ion channels. Therefore, we have developed a rapid, reliable, and sensitive cell-based high throughput screening (HTS) assay for T-type Ca2+ channels. We had previously constructed HEK293/α1G/Kir2.1 cell lines that stably expressed α1G and Kir2.1 subunits [1] and found that α1G T-type channel- sensitive Ca2+ signals were detected by the application of high concentrations of KCl under fura-2-based single cell measurements of intracellular Ca2+ concentration ([Ca2+]i). In the present study, we applied HEK293/??1G/Kir2.1 cells to the FDSS6000 (Functional Drug Screening System) to develop a fast and reliable cell-based HTS method for α1G T-type Ca2+ channels. After detecting 70 mM KCl-induced [Ca2+]i increases using the FDSS6000 system, we verified this new α1G channel HTS system by examining two T-type Ca2+ channel blockers, Ni2+ and mibefradil, and measuring the Z- factor (Z factor = 0.66) in 96-well plates. Furthermore, we assayed selected 3,4-dihydroquinazolin derivatives using this FDSS6000-based α1G channel HTS system at the level of IC50 values and compared the results with those obtained from whole-cell patch-clamp recordings. Taken together, our results suggest that the FDSS6000-based α1G channel HTS system is a fast and feasible assay for α1G T-type Ca2+ channels. This assay can be utilized as a primary screening method for Ttype Ca2+ channel-targeted chemicals and for the development of HTS systems for other types of ion channels.
Export Options
About this article
Cite this article as:
Kim Yoonjee, Kang Soonja, Lee Yeol Jae and Rhim Hyewhon, High Throughput Screening Assay of α1G T-type Ca2+ Channels and Comparison with Patch-Clamp Studies, Combinatorial Chemistry & High Throughput Screening 2009; 12 (3) . https://dx.doi.org/10.2174/138620709787581710
DOI https://dx.doi.org/10.2174/138620709787581710 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
Call for Papers in Thematic Issues
Artificial Intelligence Methods for Biomedical, Biochemical and Bioinformatics Problems
Recently, a large number of technologies based on artificial intelligence have been developed and applied to solve a diverse range of problems in the areas of biomedical, biochemical and bioinformatics problems. By utilizing powerful computing resources and massive amounts of data, methods based on artificial intelligence can significantly improve the ...read more
Eco-friendly Agents for Biological Control of Pathogenic Diseases
The discovery of an alternative biological approach to disease management includes work on medicinal products derived from natural sources as a starting point for the development of eco-friendly agents for these diseases and the injuries they cause, as well as reducing human contact with hazardous chemicals and their residues. We ...read more
Emerging trends in diseases mechanisms, noble drug targets and therapeutic strategies: focus on immunological and inflammatory disorders
Recently infectious and inflammatory diseases have been a key concern worldwide due to tremendous morbidity and mortality world Wide. Recent, nCOVID-9 pandemic is a good example for the emerging infectious disease outbreak. The world is facing many emerging and re-emerging diseases out breaks at present however, there is huge lack ...read more
Exploring Spectral Graph Theory in Combinatorial Chemistry
Scope of the Thematic Issue: Combinatorial chemistry involves the synthesis and analysis of a large number of diverse compounds simultaneously. Traditional methods rely on brute force experimentation, which can be time-consuming and resource-intensive. Spectral Graph Theory, a branch of mathematics dealing with the properties of graphs in relation to the ...read more
- Author Guidelines
- Graphical Abstracts
- Fabricating and Stating False Information
- Research Misconduct
- Post Publication Discussions and Corrections
- Publishing Ethics and Rectitude
- Increase Visibility of Your Article
- Archiving Policies
- Peer Review Workflow
- Order Your Article Before Print
- Promote Your Article
- Manuscript Transfer Facility
- Editorial Policies
- Allegations from Whistleblowers
Related Articles
-
The Current WHO Classification of Tumours of the Central Nervous System: Histopathology and Additional Diagnostic Methods
Current Medical Imaging Patent Selections:
Recent Patents on Regenerative Medicine Valproate and Neuroendocrine Changes in Relation to Women Treated for Epilepsy and Bipolar Disorder: A Review
Current Medicinal Chemistry A Review of Pharmacokinetic Parameters of Metabolites and Prodrugs
Drug Metabolism Letters Clinical Pharmacogenetics and Potential Application in Personalized Medicine
Current Drug Metabolism Pharmacology of Voltage-Gated Proton Channels
Current Pharmaceutical Design Anticonvulsant Activity Of Benzylamides Of Some Amino Acids And Heterocyclic Acids
Protein & Peptide Letters Anxiety: A Systematic Review of Neurobiology, Traditional Pharmaceuticals and Novel Alternatives from Medicinal Plants
CNS & Neurological Disorders - Drug Targets Neuropsychiatric Disturbances in Alzheimer’s Disease: What Have We Learned from Neuropathological Studies?
Current Alzheimer Research Targeting the Nogo-A Signalling Pathway to Promote Recovery Following Acute CNS Injury
Current Pharmaceutical Design Implications of Prion Protein Biology
Current Neurovascular Research The Potential Role of Pro-Inflammatory and Anti-Inflammatory Cytokines in Epilepsy Pathogenesis
Endocrine, Metabolic & Immune Disorders - Drug Targets Scorpion Extracts Inhibit Tumor Growth in Esophageal Cancer and Lung Adenocarcinoma
Current Signal Transduction Therapy High-throughput Genotyping Methods for Pharmacogenomic Studies
Current Pharmacogenomics Nutrition and Bone Health: Its Relationship to Osteoporosis
Current Nutrition & Food Science Migraine and Central Sensitization: Clinical Features, Main Comorbidities and Therapeutic Perspectives
Current Rheumatology Reviews Nanotherapeutics in Neuropathologies: Obstacles, Challenges and Recent Advancements in CNS Targeted Drug Delivery Systems
Current Neuropharmacology Synthesis, Antibacterial and Antifungal Activities of 3-Amino-5- methyl[1,1'-biphenyl]-2,4-dicarbonitrile Derivatives
Letters in Drug Design & Discovery Disrupted Functional Connectivity Related to Differential Degeneration of the Cingulum Bundle in Mild Cognitive Impairment Patients
Current Alzheimer Research Spontaneous Recurrent Seizures Mediated Cardiac Dysfunction via mTOR Pathway Upregulation: A Putative Target for SUDEP Management
CNS & Neurological Disorders - Drug Targets