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Full Description
Every cell in our bodies contains a great variety and number of permeability pathways for various organic and inorganic ions, water, metabolites, nutrients, and signalling molecules. Maintenance and precise control of gating within these pathways are fundamental principles of life as these underlie basic cellular functions such as communication, contractility, and metabolism. In Ion Channels: Methods and Protocols, Second Edition, experts in the field contribute chapters that focus on the strategies, approaches, methods, and protocols for studying a large family of proteins that form ionic channels in the plasma membrane and intracellular membranes of cells. Using practical examples from the cutting-edge current research, this volume will take a look back at the major methods and approaches that aided the progress toward the current understanding of ion channel function, structural design, and biological roles. The volume also aims to look forward and identify approaches that will lead us to future discoveries. Written in the highly successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, Ion Channels: Methods and Protocols, Second Edition will greatly assist researchers searching for specific methodology in studying ion channels on the path toward a greater understanding of these key biological features.
Contents
Approaches to Cloning of Pain-Related Ion Channel Genes.- Mammalian Expression Systems and Transfection Techniques.- Use of Escherichia coli for the Production and Purification of Membrane Proteins.- Transient Overexpression of Genes in Neurons Using Nucleofection.- Viral Gene Delivery: Optimized Protocol for Production of High Titre Lentiviral Vectors.- Two-Electrode Voltage Clamp.- Conventional Micropipette-Based Patch Clamp Techniques.- Recording of Ion Channel Activity in Planar Lipid Bilayers Experiments.- Recording Macroscopic Currents in Large Patches from Xenopus Oocytes.- Combined Single-Channel and Macroscopic Recording Techniques to Analyze Gating Mechanisms of the Large Conductance Ca2+ and Voltage Activated (BK) Potassium Channel.- Perforated Whole Cell Patch Clamp Recording.- Piezo-Electrically Driven Mechanical Stimulation of Sensory Neurons.- Automated Planar Patch-Clamp.- Recording Single-Channel Currents Using "Smart Patch-Clamp" Technique.- Using Total Internal Reflection Fluorescence Microscopy to Observe Ion channel Trafficking and Assembly.- Förster Resonance Energy Transfer-Based Imaging at the Cell Surface of Live Cells.- The Use of Dansyl-Calmodulin to Study Interactions with Channels and Other Proteins.- Imaging and Quantification of Recycled KATP Channels.- Generation of Antibodies that Are Externally-Acting Isoform-Specific Inhibitors of Ion Channels.- Site-Directed Mutagenesis to Study the Structure-Function Relationships of Ion Channels.- Cysteine-Based Cross-Linking Approach to Study Inter-Domain Interactions in Ion Channel Functions.- Analysis of Ca2+-Binding Sites in the MthK RCK Domain by X-RayCrystallography.- Isotope Labeling Strategies for Analysis of an Ion Channel Cytoplasmic Domain by NMR Spectroscopy.- Recording Dendritic Ion Channel Properties and Function from Cortical Neurons.- M Current Recording from Acute DRG Slices.- Studying Ion Channels in Human Erythrocytes by Direct and Indirect Means.- Recording Ion Channels in Isolated, Split-Opened Tubules.- Single Channel Analysis of TRPC Channels in the Podocytes of Freshly Isolated Glomeruli.- Ca2+ Imaging as a Tool to Assess TRP Channel Function in Murine Distal Nephrons.- Patch Clamping Drosophila Sensory Neurons.- Production and Validation of Recombinant Adeno-Associated Virus for Channelrhodopsin Expression in Neurons.- Optical Control of Ligand-Gated Ion Channels.
