Novel Chemical Tools to Study Ion Channel Biology (Advances in Experimental Medicine and Biology Book 869)

This volume describes chemical approaches to assess ion channel structure, function and pharmacology. Topics discussed include the use of engineered ionizable side chains to obtain information on permeation pathways and the local environment; the modification of engineered cysteine side chains, including cysteine scanning mutagenesis and the attachment of fluorescent probes and bio-reactive tethers; and the nascent use of genetic code expansion, evaluating its applications to ion channel and membrane proteins. This comprehensive text provides multifaceted perspectives on the great diversity of state-of-the-art methods which take advantage of the ever-expanding chemical toolbox to study ion channel biology. Capturing the contributions and innovations of renowned laboratory researchers in transmembrane protein study for the first time, this book is comprehensive in scope. It covers a wide array of experimental approaches: photochemistry, novel biological tools, and innovative spectroscopy, all combined with traditional techniques of electrophysiology and molecular biology. __Novel Chemical Tools to Study Ion Channel Biology__, part of the bestselling __Advances in Experimental Medicine and Biology__ series is ideal for researchers and advanced students interested in biochemistry, biophysics, fluorometry, electrophysiology, and chemical biology. .​ Contents 6 Contributors 7 Introduction 9 Applying Chemical Biology to Ion Channels 9 Engineered Ionizable Side Chains 13 1 Introduction 14 2 The Method: Rationale, Previous Results, and Practical Implementation 15 3 Concluding Remarks 30 References 30 Cysteine Modification: Probing Channel Structure, Function and Conformational Change 32 1 Introduction 33 2 Approach/Methods 35 2.1 Methanethiosulfonates 37 2.2 p-Chloromercuribenzenesulfonate and Other Mercurials. 39 2.3 Maleimides 40 2.4 “Silent” Reaction 41 3 Main Body 41 3.1 Ligand-Gated Channels—Cys-Loop Receptors 41 3.2 A Few Examples of SCAM in Other Channels 50 4 Conclusions 51 References 51 Functional Site-Directed Fluorometry 62 1 Introduction 63 2 Methods 65 3 Applications 69 3.1 Diversity of Membrane Proteins Explored with Functional Site-Directed Fluorometry 70 3.2 Functional Site-Directed Fluorometry Elucidates Voltage-Induced Conformational Changes 70 3.3 Functional Site-Directed Fluorometry Provides Insight Into Molecular Interactions 71 3.4 Functional Site-Directed Fluorometry in Ligand-Gated Ion Channels 73 3.5 Additional Applications of Functional Site-Directed Fluorometry 74 3.6 Limitations of Functional Site-Directed Fluorometry 75 4 Outlook 77 References 77 Bioreactive Tethers 84 1 Introduction 84 2 Approach 87 3 Applications 93 3.1 Photoswitchable Tethers 93 3.2 Molecular Rulers 97 3.3 High Affinity Biochemically-Reactive Tethers 100 References 103 Flipping the Photoswitch: Ion Channels Under Light Control 108 1 Introduction 109 2 Synthetic Photochromes for Biological Research 109 3 Design Principles of Photochromic Ion Channel Controllers 111 3.1 PCLs: One-Component Photopharmacology 111 3.2 PTLs: Ligands-on-a-Leash 113 3.3 PXs: Molecular Tweezers 113 4 Applications of PCLs and PTLs 113 4.1 PCLs of Ligand-Gated Ion Channels 113 4.2 PCLs of Voltage-Gated Ion Channels 115 4.3 PTLs of Ligand-Gated Ion Channels 116 4.4 PTLs of Voltage-Gated Ion Channels 117 5 Outlook 119 References 121 Incorporation of Non-Canonical Amino Acids 125 1 Introduction 125 2 Approaches of Non-Canonical Amino Acid Incorporation 128 2.1 In Vivo Nonsense Suppression in Xenopus Oocytes 128 2.2 Evolved Orthogonal tRNA and Aminoacyl-tRNA Synthetase Pairs 131 2.2.1 Genetically Encoding Non-Canonical Amino Acids in Prokaryotes 132 2.2.2 Genetically Encoding non-Canonical Amino Acids in Eukaryotes 133 2.2.3 Achaeal Pyrrolysyl-tRNA-Synthetase/tRNA Pairs Display Orthogonality in Prokaryotes as well as Eukaryotes 137 2.2.4 Optimization of ncAA Incorporation Efficiencies and Protein Yields 137 2.3 Semi-Synthetic Approaches 139 3 Applications of Genetic Code Expansion to ion Channels and Receptors 143 3.1 Applications of In Vivo Nonsense Suppression 143 3.2 Applications of Orthogonal ncAA-RS/tRNA Pairs 144 3.2.1 Early Adaptation and Application of ncAA-RS/tRNA Pairs 144 3.2.2 Promise and Potential Caveats with the Applications of Cross-Linkers 145 3.2.3 Recoding Voltage-Gated Enzymes and Potassium Channels: Optogenetics, Fluorophores and Structure-Function Insights 146 3.3 Applications of Protein Semisynthesis 147 3.3.1 D-Ala Substitution in the K + Selectivity Filter 148 3.3.2 Ester Substitutions in the K +Selectivity Filter 148 4 Outlook and Future Directions 149 References 150 Index 158 "This volume describes chemical approaches to assess ion channel structure, function and pharmacology. Topics discussed include the use of engineered ionizable side chains to obtain information on permeation pathways and the local environment; the modification of engineered cysteine side chains, including cysteine scanning mutagenesis and the attachment of fluorescent probes and bio-reactive tethers; and the nascent use of genetic code expansion, evaluating its applications to ion channel and membrane proteins. This comprehensive text provides multifaceted perspectives on the great diversity of state-of-the-art methods which take advantage of the ever-expanding chemical toolbox to study ion channel biology. Capturing the contributions and innovations of renowned laboratory researchers in transmembrane protein study for the first time, this book is comprehensive in scope. It covers a wide array of experimental approaches: photochemistry, novel biological tools, and innovative spectroscopy, all combined with traditional techniques of electrophysiology and molecular biology. Novel Chemical Tools to Study Ion Channel Biology, part of the bestselling Advances in Experimental Medicine and Biology series is ideal for researchers and advanced students interested in biochemistry, biophysics, fluorometry, electrophysiology, and chemical biology"--Back cover Front Matter....Pages i-viii Introduction....Pages 1-4 Engineered Ionizable Side Chains....Pages 5-23 Cysteine Modification: Probing Channel Structure, Function and Conformational Change....Pages 25-54 Functional Site-Directed Fluorometry....Pages 55-76 Bioreactive Tethers....Pages 77-100 Flipping the Photoswitch: Ion Channels Under Light Control....Pages 101-117 Incorporation of Non-Canonical Amino Acids....Pages 119-151 Back Matter....Pages 153-154