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The rapid expansion of the TRP field has generated a large amount of excellent original work across many different research fields. However, investigators are not necessarily familiar with the pros and cons of the variety of methods used to study TRP channels. Because of functional and genetic diversity, as well as the different physiological roles they play, techniques used for studying TRP channels range from single molecular analysis to behavioral animal studies. Methods in multiple areas, such as molecular biology, fluorescence imaging, electrophysiology, cell biology, genetics, proteomics, pharmacology, system physiology, and behavioral assessment, are employed to investigate various aspects of these channels. Choosing among many possible topics in these broad areas was a daunting task.
A comprehensive review of the field, TRP Channels spans the information gap by providing broad coverage of current methods and techniques commonly used in TRP channel research, and detailed protocols with thorough discussions of the advantages and disadvantages across methods. Some topics covered include
- Mammalian, Drosophila and C. elegans TRP channels
- Practical protocols for functional studies of TRP channels, including TRPC, TRPV, TRPA, TRPM and the intracellularly localized TRPML channels
- ThermoTRPs, including the new fast temperature jump apparatus and the high throughput random mutagenesis method for screening critical motifs involved in TRP channel regulation
- Cell-based high-throughput screening assays for TRP channels and their applications in drug discoveries
- TRP channel functions in native cells, including smooth muscles, neurons, and cancers
Showcasing the current status of the field, TRP Channels covers the major techniques used in various areas of research. The majority of the chapters are protocol oriented, with the goal of providing clear directions for laboratory use. Because of the breadth of the TRP field, the applications of some methods are described in multiple chapters by experts working on a variety of channel types that serve different physiological functions, highlighting distinctive views on how the methodology can be utilized. Some chapters include discussion on the usefulness and pitfalls associated with the use of certain techniques. Together with chapters that offer comprehensive reviews on the functional regulation and other roles of TRP channels, students and investigators new to the field should find this book particularly informative.
Contents
- Series Preface
- Preface
- Editor
- Contributors
- 1. High-Throughput Screening of TRPC Channel Ligands Using Cell-Based AssaysMelissa Miller, Meng Wu, Jia Xu, David Weaver, Min Li, and Michael X. Zhu.
- 2. Methods to Study TRPC Channel Regulation by Interacting ProteinsKirill Kiselyov, Kyu Pil Lee, Joseph P. Yuan, and Shmuel Muallem.
- 3. Activation of TRP Channels in Mammalian SystemsTomohiro Numata, Shigeki Kiyonaka, Kenta Kato, Nobuaki Takahashi, and Yasuo Mori.
- 3.1. INTRODUCTION
- 3.2. PLC-COUPLED RECEPTORS AND DOWNSTREAM SIGNALING CASCADES
- 3.3. RECEPTOR-INDUCED PROTEIN TRANSLOCATION
- 3.4. CHANGES IN REDOX STATUS AND COVALENT CYSTEINE MODIFICATION
- 3.5. NONCOVALENT ACTION OF CHEMICALS AND SIGNALING MEDIATORS
- 3.6. PH
- 3.7. TEMPERATURE
- 3.8. MEMBRANE VOLTAGE
- 3.9. MECHANOSTIMULATION
- 3.10. PROTEIN–PROTEIN INTERACTION
- 3.11. CONCLUSION
- REFERENCES
- 4. Studying Subunit Interaction and Complex Assembly of TRP ChannelsMichael Poteser and Klaus Groschner.
- 5. Proteomic Analysis of TRPC ChannelsTimothy Lockwich, Anthony Makusky, Jeffrey A. Kowalak, Sanford P. Markey, and Indu S. Ambudkar.
- 6. Lessons of Studying TRP Channels with AntibodiesMarcel Meissner, Verena C. Obmann, Michael Hoschke, Sabine Link, Martin Jung, Gerhard Held, Stephan E. Philipp, Richard Zimmermann, and Veit Flockerzi.
- 6.1. INTRODUCTION
- 6.2. GENERATION OF POLYCLONAL ANTISERA FOR TRPM4 IN RABBITS USING SYNTHETIC PEPTIDES AS ANTIGENS
- 6.3. GENERATION OF POLYCLONAL ANTISERA FOR TRPC3 IN RABBITS USING RECOMBINANT PROTEIN FRAGMENTS AS ANTIGENS
- 6.4. GENERATION OF RECOMBINANT FRAGMENT ANTIGEN BINDING (FAB FRAGMENTS) FOR TRPC4 AND TRPM3 USING SYNTHETIC PEPTIDES AS ANTIGENS
- 6.5. GENERAL COMMENTS ON THE VALIDATION OF ANTIBODIES
- 6.6. COMPARING PROTEIN EXPRESSION DATA WITH RNA EXPRESSION DATA
- 6.7. CONCLUSION
- ACKNOWLEDGMENTS
- REFERENCES
- 7. Assessing TRPC Channel Function Using Pore-Blocking AntibodiesShang-Zhong Xu.
- 8. Strategies and Protocols to Generate Mouse Models with Targeted Mutations to Analyze TRP Channel FunctionsMarc Freichel, Ulrich Kriebs, Dominik Vogt, Stefanie Mannebach, and Petra Weißgerber.
- 9. Studying Endogenous TRP Channels in Visceral and Vascular Smooth MusclesAlexander V. Zholos.
- 10. TRPC Channels in Neuronal SurvivalJunbo Huang, Wanlu Du, Hailan Yao, and Yizheng Wang.
- 11. Electrophysiological Methods for the Study of TRP ChannelsKarel Talavera and Bernd Nilius.
- 12. High-Throughput Approaches to Studying Mechanisms of TRP Channel ActivationHongzhen Hu, Michael Bandell, Jorg Grandl, and Matt Petrus.
- 13. Methods Used for Studying TRP Channel Functions in Sensory NeuronsLouis S Premkumar.
- 13.1. INTRODUCTION
- 13.2. IN VITRO NEURONAL PREPARATIONS TO STUDY SENSORY NEURONS
- 13.3. EXPRESSION OF TRP CHANNELS IN DRG/TG NEURONS
- 13.4. FUNCTIONAL STUDIES OF TRP CHANNELS IN SENSORY NEURONS
- 13.5. EX VIVO PREPARATIONS TO STUDY TRP CHANNEL FUNCTION IN SENSORY NEURONS
- 13.6. IN VIVO BEHAVIORAL STUDIES TO EVALUATE THE FUNCTION OF DRG/TG NEURONS
- 13.7. STATISTICAL METHODS FOR DATA ANALYSIS
- 13.8. CONCLUDING REMARKS
- ACKNOWLEDGMENTS
- REFERENCES
- 14. Time-Resolved Activation of Thermal TRP Channels by Fast Temperature JumpsFeng Qin.
- 15. Fluorescence Microscopy in ThermoTRP Channel ResearchFan Yang and Jie Zheng.
- 16. Regulation of TRP Channels by Osmomechanical StressMin Jin, Jonathan Berrout, and Roger G. O’Neil.
- 17. Study of TRP Channels in Cancer CellsV′yacheslav Lehen′kyi and Natalia Prevarskaya.
- 17.1. INTRODUCTION
- 17.2. ROLE OF TRP CHANNEL ISOFORM EXPRESSION AND FUNCTION IN THE STUDY OF THE CANCER INITIATION AND PROGRESSION
- 17.3. TRP PROTEIN TRANSLATION FROM ALTERNATIVE START CODONS: AN UNKNOWN MECHANISM OF CANCER CELL-ENHANCED RESISTANCE AND SURVIVAL
- 17.4. TRP PROTEIN POSTTRANSLATIONAL MODIFICATIONS: DIRECT OR INDIRECT MODULATION OF THE CHANNEL ACTIVITY
- 17.5. CONCLUSIONS
- REFERENCES
- 18. In Vitro and in Vivo Assays for the Discovery of Analgesic Drugs Targeting TRP ChannelsJun Chen, Regina M. Reilly, Philip R. Kym, and Shailen Joshi.
- 19. Studying TRP Channels in Intracellular MembranesMohammad Samie and Haoxing Xu.
- 20. Methods for Studying Drosophila TRP ChannelsElkana Kohn and Baruch Minke.
- 21. Studying TRP Channels in Caenorhabditis elegansRui Xiao and X. Z. Shawn Xu.
- NLM CatalogRelated NLM Catalog Entries
- TRP ChannelsTRP Channels
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