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

1. Mammalian, Drosophila and C. elegans TRP channels
2. Practical protocols for functional studies of TRP channels, including TRPC, TRPV, TRPA, TRPM and the intracellularly localized TRPML channels
3. ThermoTRPs, including the new fast temperature jump apparatus and the high throughput random mutagenesis method for screening critical motifs involved in TRP channel regulation
4. Cell-based high-throughput screening assays for TRP channels and their applications in drug discoveries
5. 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 Assays
  Melissa Miller, Meng Wu, Jia Xu, David Weaver, Min Li, and Michael X. Zhu.
  • 1.1. INTRODUCTION
  • 1.2. ESTABLISHMENT OF STABLE CELL LINES
  • 1.3. ASSAY DEVELOPMENT
  • 1.5. VALIDATION AND OPTIMIZATION
  • 1.6. CONCLUDING REMARKS
  • ACKNOWLEDGMENTS
  • REFERENCES
• 2. Methods to Study TRPC Channel Regulation by Interacting Proteins
  Kirill Kiselyov, Kyu Pil Lee, Joseph P. Yuan, and Shmuel Muallem.
  • 2.1. INTRODUCTION
  • 2.2. CONTEXT-SPECIFIC REGULATION OF TRPC CHANNELS
  • 2.3. TRPC INTERACTION WITH AUXILIARY PROTEINS
  • 2.4. EXPLORATION TECHNIQUES
  • 2.5. PERSPECTIVES
  • ACKNOWLEDGMENTS
  • REFERENCES
• 3. Activation of TRP Channels in Mammalian Systems
  Tomohiro 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 Channels
  Michael Poteser and Klaus Groschner.
  • 4.1. INTRODUCTION
  • 4.2. CLASSICAL METHODS, THEIR ADVANTAGES AND LIMITATIONS
  • 4.3. COMPLEMENTARY AND ALTERNATIVE APPROACHES
  • 4.4. PERSPECTIVES
  • ACKNOWLEDGMENT
  • REFERENCES
• 5. Proteomic Analysis of TRPC Channels
  Timothy Lockwich, Anthony Makusky, Jeffrey A. Kowalak, Sanford P. Markey, and Indu S. Ambudkar.
  • 5.1. INTRODUCTION
  • 5.2. PROTEOMIC ANALYSIS OF TRPC-ASSOCIATED PROTEIN COMPLEX
  • 5.3. OVERVIEW OF TRPC PROTEOMIC ANALYSIS
  • 5.4. VALIDATION OF PROTEOMIC DATA
  • 5.5. QUANTITATIVE PROTEOMIC TECHNIQUES
  • 5.6. CONCLUSION
  • REFERENCES
• 6. Lessons of Studying TRP Channels with Antibodies
  Marcel 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 Antibodies
  Shang-Zhong Xu.
  • 7.1. INTRODUCTION
  • 7.2. PORE-BLOCKING ANTIBODY AND GENERATION
  • 7.3. ASSESSING TRPC CHANNEL FUNCTION USING PORE-BLOCKING ANTIBODIES
  • 7.4. ADVANTAGES AND DISADVANTAGES
  • 7.5. ENDOGENOUS FUNCTIONAL ANTIBODIES AND CLINICAL IMPLICATIONS
  • 7.6. CONCLUSION
  • ACKNOWLEDGMENT
  • REFERENCES
• 8. Strategies and Protocols to Generate Mouse Models with Targeted Mutations to Analyze TRP Channel Functions
  Marc Freichel, Ulrich Kriebs, Dominik Vogt, Stefanie Mannebach, and Petra Weißgerber.
  • 8.1. APPROACHES TO STUDYING THE FUNCTION OF TRP PROTEINS IN NATIVE SYSTEMS
  • 8.2. GENERATION OF TRANSGENIC ANIMALS BY GENE TARGETING STRATEGIES
  • 8.3. PROTOCOLS FOR GENE TARGETING EXPERIMENTS
  • ACKNOWLEDGMENT
  • REFERENCES
• 9. Studying Endogenous TRP Channels in Visceral and Vascular Smooth Muscles
  Alexander V. Zholos.
  • 9.1. INTRODUCTION
  • 9.2. ELECTROPHYSIOLOGICAL ANALYSIS OF NATIVE TRP CURRENTS
  • 9.3. ANALYSIS OF SIGNAL TRANSDUCTION PATHWAYS USING PATCH-CLAMP TECHNIQUES
  • 9.4. DATA ANALYSIS AND INTERPRETATION
  • 9.5. CONCLUSION
  • ACKNOWLEDGMENTS
  • REFERENCES
• 10. TRPC Channels in Neuronal Survival
  Junbo Huang, Wanlu Du, Hailan Yao, and Yizheng Wang.
  • 10.1. INTRODUCTION
  • 10.2. PROPERTIES OF TRPC CHANNELS
  • 10.3. DISTRIBUTION OF TRPCs IN THE NERVOUS SYSTEM
  • 10.4. CA²⁺ AND NEURONAL SURVIVAL
  • 10.5. TRPC CHANNELS IN NEURONAL SURVIVAL
  • 10.6. TOOLS TO STUDY TRPC CHANNELS
  • 10.7. PERSPECTIVES
  • ACKNOWLEDGMENT
  • REFERENCES
• 11. Electrophysiological Methods for the Study of TRP Channels
  Karel Talavera and Bernd Nilius.
  • 11.1. INTRODUCTION
  • 11.2. PATCH-CLAMP TECHNIQUE
  • 11.3. STUDY OF PORE PROPERTIES
  • 11.4. GATING PROPERTIES
  • 11.5. GATING MODIFIERS
  • REFERENCES
• 12. High-Throughput Approaches to Studying Mechanisms of TRP Channel Activation
  Hongzhen Hu, Michael Bandell, Jorg Grandl, and Matt Petrus.
  • 12.1. INTRODUCTION
  • 12.2. HIGH-THROUGHPUT MUTAGENESIS APPROACH
  • 12.3. HIGH-THROUGHPUT FUNCTIONAL ASSAYS
  • 12.4. FOLLOW-UP STUDIES
  • 12.5. CONCLUSIONS
  • ACKNOWLEDGMENTS
  • REFERENCES
• 13. Methods Used for Studying TRP Channel Functions in Sensory Neurons
  Louis 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 Jumps
  Feng Qin.
  • 14.1. INTRODUCTION
  • 14.2. THERMODYNAMIC PRINCIPLES OF TEMPERATURE GATING
  • 14.3. MEASUREMENT OF TEMPERATURE DEPENDENCE
  • 14.4. FAST TEMPERATURE JUMPS
  • 14.5. A SUBMILLISECOND TEMPERATURE JUMP SYSTEM
  • 14.6. APPLICATIONS OF FAST TEMPERATURE JUMPS
  • 14.7. SUMMARY
  • REFERENCES
• 15. Fluorescence Microscopy in ThermoTRP Channel Research
  Fan Yang and Jie Zheng.
  • 15.1. A BRIEF REVIEW OF FLUORESCENCE MICROSCOPY
  • 15.2. DISCOVERY OF THERMOTRP CHANNELS
  • 15.3. SUBUNIT ASSEMBLY OF THERMOTRP CHANNELS
  • 15.4. TRAFFICKING OF THERMOTRP CHANNELS
  • 15.5. STRUCTURE–FUNCTION RELATIONSHIP IN THERMOTRP CHANNELS
  • 15.6. EMERGING OPTICAL TECHNIQUES
  • 15.7. CONCLUSION
  • REFERENCES
• 16. Regulation of TRP Channels by Osmomechanical Stress
  Min Jin, Jonathan Berrout, and Roger G. O’Neil.
  • 16.1. INTRODUCTION
  • 16.2. OSMOMECHANICAL REGULATION OF TRP CHANNELS
  • 16.3. APPLICATION OF OSMOMECHANICAL STRESSES: PRACTICAL CONSIDERATIONS
  • 16.4. CONCLUSIONS
  • ACKNOWLEDGMENTS
  • REFERENCES
• 17. Study of TRP Channels in Cancer Cells
  V′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 Channels
  Jun Chen, Regina M. Reilly, Philip R. Kym, and Shailen Joshi.
  • 18.1. INTRODUCTION
  • 18.2. EXPRESSION SYSTEMS
  • 18.3. IN VITRO ASSAYS FOR TRP CHANNELS
  • 18.4. PRECLINICAL PAIN AND SIDE EFFECT MODELS
  • 18.5. CONCLUSION
  • REFERENCE
• 19. Studying TRP Channels in Intracellular Membranes
  Mohammad Samie and Haoxing Xu.
  • 19.1. INTRODUCTION
  • 19.2. STUDYING INTRACELLULAR LOCALIZATION OF TRP CHANNELS
  • 19.3. STUDYING CHANNEL PROPERTIES OF INTRACELLULAR TRPS
  • 19.4. CONCLUSION
  • ACKNOWLEDGMENTS
  • REFERENCES
• 20. Methods for Studying Drosophila TRP Channels
  Elkana Kohn and Baruch Minke.
  • 20.1. INTRODUCTION
  • 20.2. GENETICS
  • 20.3. EXTRACELLULAR AND INTRACELLULAR VOLTAGE RECORDINGS
  • 20.4. WHOLE-CELL PATCH-CLAMP CURRENT RECORDINGS FROM ISOLATED OMMATIDIA
  • 20.5. PHOTORECEPTOR IMAGING
  • 20.6. ELECTRON MICROSCOPY
  • ACKNOWLEDGMENTS
  • REFERENCES
• 21. Studying TRP Channels in Caenorhabditis elegans
  Rui Xiao and X. Z. Shawn Xu.
  • 21.1. INTRODUCTION
  • 21.2. IDENTIFICATION OF TRP CHANNELS IN C. ELEGANS
  • 21.3. GENETIC CHARACTERIZATION OF TRP CHANNELS IN C. ELEGANS
  • 21.4. BEHAVIORAL CHARACTERIZATION OF C. ELEGANS TRP CHANNELS
  • 21.5. FUNCTIONAL CHARACTERIZATION OF TRP CHANNELS IN C. ELEGANS
  • 21.6. CONCLUSION
  • ACKNOWLEDGMENTS
  • REFERENCES