Ion Channels: Methods and Protocols

Part I: Cloning and Heterologous Expression of Ion Channel Genes

1. Approaches to Cloning of Pain-Related Ion Channel Genes

Armen N. Akopian

2. Mammalian Expression Systems and Transfection Techniques

Daunia Laurenti and Lezanne Ooi

3. Use of Escherichia coli for the Production and Purification of Membrane Proteins

Vincent G.L. Postis, Andrea E. Rawlings, Amelia Lesiuk, and Stephen A. Baldwin

4. Transient Overexpression of Genes in Neurons Using Nucleofection

Hannah M. Kirton, Louisa Pettinger, and Nikita Gamper

5. Viral Gene Delivery: Optimized Protocol for Production of High Titre Lentiviral Vectors

James Hewinson, Julian F.R. Paton, and Sergey Kasparov

Part II: Electrophysiological Methods to Study Ion Channel Functions

6. Two-Electrode Voltage Clamp

Bingcai Guan, Xingjuan Chen, and Hailin Zhang

7. Conventional Micropipette-Based Patch Clamp Techniques

Jonathan D. Lippiat and David C. Wrighton

8. Recording of Ion Channel Activity in Planar Lipid Bilayers Experiments

Eleonora Zakharian

9. Recording Macroscopic Currents in Large Patches from Xenopus Oocytes

Tibor Rohacs

10. Combined Single-Channel and Macroscopic Recording Techniques to Analyze Gating Mechanisms of the Large Conductance Ca²⁺ and Voltage Activated (BK) Potassium Channel

Nguyen V. Nguyen, Aleksandra Gruslova, Wojciech A. Kosiba, and Bin Wang

11. Perforated Whole Cell Patch Clamp Recording

John E. Linley

12. Piezo-Electrically Driven Mechanical Stimulation of Sensory Neurons

Jizhe Hao, Jérôme Ruel, Bertrand Coste, Yann Roudaut, Marcel Crest, and Patrick Delmas

13. Automated Planar Patch-Clamp

Carol J. Milligan and Clemens Möller

14. Recording Single-Channel Currents Using "Smart Patch-Clamp" Technique

Anamika Bhargava and Julia Gorelik

Part III: Imaging and Fluorescence Methods to Study Ion Channels

15. Using Total Internal Reflection Fluorescence Microscopy to Observe Ion channel Trafficking and Assembly

Sarah Schwarzer, Gregory I. Mashanov, Justin E. Molloy, and Andrew Tinker

16. Förster Resonance Energy Transfer-Based Imaging at the Cell Surface of Live Cells

Sonya M. Bierbower and Mark S. Shapiro

17. The Use of Dansyl-Calmodulin to Study Interactions with Channels and Other Proteins

Alessandro Alaimo, Covadonga Malo, Pilar Areso, Kerman Aloria, Oscar Millet, and Alvaro Villarroel

18. Imaging and Quantification of Recycled K_ATP Channels

Christopher J. Cockcroft

Part IV: Biochemical and Structure-Functional Approaches in Ion Channel Studies

19. Generation of Antibodies that Are Externally-Acting Isoform-Specific Inhibitors of Ion Channels

Jacqueline Naylor and David J. Beech

20. Site-Directed Mutagenesis to Study the Structure-Function Relationships of Ion Channels

Wei Yang and Lin-Hua Jiang

21. Cysteine-Based Cross-Linking Approach to Study Inter-Domain Interactions in Ion Channel Functions

Lin-Hua Jiang

22. Analysis of Ca²⁺-Binding Sites in the MthK RCK Domain by X-Ray Crystallography

Frank J. Smith and Brad S. Rothberg

23. Isotope Labeling Strategies for Analysis of an Ion Channel Cytoplasmic Domain by NMR Spectroscopy

Karin Abarca-Heidemann, Elke Duchardt-Ferner, Jens Woehnert, and Brad S. Rothberg

Part V: Studying Ion Channels in Native Tissues

24. Recording Dendritic Ion Channel Properties and Function from Cortical Neurons

Mala M. Shah

25. M Current Recording from Acute DRG Slices

Kirstin E. Rose, Sylvain Gigout, and Nikita Gamper

26. Studying Ion Channels in Human Erythrocytes by Direct and Indirect Means

Stephan M. Huber

27. Recording Ion Channels in Isolated, Split-Opened Tubules

Elena Mironova, Vladislav Bugay, Oleh Pochynyuk, Alexander Staruschenko, and James D. Stockand

28. Single Channel Analysis of TRPC Channels in the Podocytes of Freshly Isolated Glomeruli

Daria V. Ilatovskaya and Alexander Staruschenko

29. Ca²⁺ Imaging as a Tool to Assess TRP Channel Function in Murine Distal Nephrons

Mykola Mamenko, Oleg Zaika, Roger G. O'Neil, and Oleh Pochynyuk

30. Patch Clamping Drosophila Sensory Neurons

Volodymyr Kucher, Benjamin A. Eaton, James D. Stockand, and Nina Boiko

Part VI: Ion Channels as Research Tools

31. Production and Validation of Recombinant Adeno-Associated Virus for Channelrhodopsin Expression in Neurons

John Y. Lin

32. Optical Control of Ligand-Gated Ion Channels

Stephanie Szobota, Catherine McKenzie, and Harald Janovjak