Preface |
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xi | |
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xiii | |
Color Plates |
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xvii | |
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Expression of Ion Channels in Xenopus Oocytes |
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1 | (26) |
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1 | (1) |
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Advantages and Disadvantages of Xenopus Oocytes |
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2 | (1) |
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Procedures for Using Oocytes |
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3 | (2) |
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5 | (12) |
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Electrophysiological Analysis |
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5 | (1) |
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Two-electrode Whole Cell Voltage-clamp |
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5 | (2) |
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Cut-open Oocyte Voltage-clamp |
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7 | (2) |
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9 | (2) |
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11 | (1) |
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12 | (1) |
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13 | (1) |
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Serial Recording Using the Roboocyte |
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14 | (2) |
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Parallel Recording Using the OpusXpress |
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16 | (1) |
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17 | (4) |
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Characterization of cDNA Clones for a Channel |
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17 | (1) |
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Structure-Function Correlations |
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18 | (1) |
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Studies of Human Disease Mutations |
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19 | (2) |
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21 | (6) |
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21 | (1) |
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21 | (6) |
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Molecular Biology Techniques for Structure -- Function Studies of Ion Channels |
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27 | (32) |
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27 | (1) |
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Methods for cDNA Subcloning |
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28 | (8) |
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Conventional Sub-cloning Using Restriction Enzymes and DNA Ligase |
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28 | (3) |
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PCR-based cDNA Sub-cloning |
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31 | (2) |
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Sub-cloning cDNA through Site-specific Recombination |
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33 | (3) |
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Generation of Chimeric Channel cDNAs |
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36 | (7) |
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Use of Restriction Enzymes to Generate Chimeric Channel cDNAs |
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36 | (3) |
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PCR-mediated Overlap Extension for Chimera Generation |
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39 | (4) |
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PCR-mediated Integration or Replacement of cDNA Fragments |
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43 | (1) |
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Site-directed Mutagenesis |
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43 | (7) |
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Examples of the Use of Site-directed Mutagenesis |
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45 | (5) |
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Modification of the QuikChange Method for the Replacement of cDNA Fragments |
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50 | (1) |
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Epitope-tagged Channels and Fusion Partners |
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50 | (2) |
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Channel Subunit Concatamers |
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52 | (1) |
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53 | (6) |
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54 | (5) |
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Unnatural Amino Acids as Probes of Ion Channel Structure -- Function and Pharmacology |
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59 | (20) |
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59 | (1) |
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Unnatural Amino Acid Mutagenesis Methodology |
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60 | (4) |
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Unnatural Amino Acid Mutagenesis for Ion Channel Studies |
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64 | (1) |
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Structure-Function Example Studies |
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65 | (7) |
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Nicotinic Acetylcholine Receptor |
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65 | (2) |
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Drug Interactions with the hERG Voltage-gated Potassium Ion Channel |
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67 | (5) |
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Other Uses of Unnatural Amino Acids as Probes of Protein Structure and Function |
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72 | (1) |
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73 | (6) |
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74 | (1) |
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74 | (5) |
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Functional Expression of Ion Channels in Mammalian Systems |
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79 | (32) |
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79 | (1) |
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cDNA Cloning and Manipulation |
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80 | (1) |
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Choice of Host Cell Background |
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81 | (4) |
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Post-translational Processing of Heterologous Expressed Ion Channels |
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85 | (5) |
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90 | (1) |
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Transient Expression Systems |
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91 | (5) |
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``Standard'' Transient Expression |
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91 | (1) |
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92 | (4) |
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Stable Expression of Ion Channels |
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96 | (7) |
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Bicistronic Expression Systems |
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96 | (4) |
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Stable Expression of Multiple Subunits |
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100 | (1) |
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101 | (2) |
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103 | (8) |
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103 | (1) |
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104 | (7) |
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Analysis of Electrophysiological Data |
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111 | (34) |
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111 | (1) |
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111 | (2) |
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Expression Systems and Related Recording Techniques |
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113 | (4) |
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Expression in Xenopus Oocytes |
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113 | (2) |
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Expression in Mammalian Cells |
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115 | (1) |
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Leak and Capacitance Subtraction |
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116 | (1) |
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117 | (19) |
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Analysis of Pore Properties -- Permeation |
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118 | (3) |
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Analysis of Fast Voltage-dependent Block -- the Woodhull Model |
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121 | (1) |
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Information on Gating Properties from Macroscopic Measurements |
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122 | (2) |
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Equilibrium Properties -- Voltage-gated Channels |
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124 | (2) |
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Equilibrium Properties -- Ligand Gated Channels |
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126 | (3) |
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129 | (3) |
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132 | (1) |
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Nonstationary Noise Analysis |
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133 | (2) |
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Gating Current Measurements in Voltage Gated Channels |
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135 | (1) |
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136 | (6) |
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Amplitude Histogram Analysis |
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136 | (2) |
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Kinetic Single Channel Analysis |
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138 | (4) |
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142 | (3) |
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142 | (1) |
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142 | (3) |
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Automated Planar Array Electrophysiology for Ion Channel Research |
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145 | (20) |
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145 | (1) |
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Overview of Planar Array Recording |
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145 | (2) |
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Experimental Methods and Design |
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147 | (11) |
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148 | (1) |
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Cell Sealing and Recording |
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149 | (3) |
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152 | (3) |
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Experimental Design and Data Analysis |
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155 | (3) |
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Overall Success Rates and Throughput |
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158 | (1) |
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159 | (3) |
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162 | (3) |
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162 | (1) |
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162 | (3) |
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Ion Flux and Ligand Binding Assays for Analysisof Ion Channels |
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165 | (22) |
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165 | (1) |
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166 | (9) |
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Radioactive Ion Flux Assays |
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167 | (1) |
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Nonradioactive Ion Flux Assays based on Atomic Absorption Spectrometry |
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168 | (1) |
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Nonradioactive Rubidium Efflux Assay |
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168 | (6) |
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Nonradioactive Lithium Influx Assay |
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174 | (1) |
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Nonradioactive Chloride Influx Assay |
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174 | (1) |
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174 | (1) |
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175 | (12) |
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Heterogeneous Binding Assays Employing Radioligands |
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177 | (1) |
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Homogeneous Binding Assays Employing Radioligands |
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178 | (2) |
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Homogeneous Binding Assays Employing Fluorescent-Labeled Ligands and Fluorescence Polarization |
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180 | (1) |
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181 | (1) |
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182 | (1) |
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182 | (5) |
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Ion Channel Assays Based on Ion and Voltage-sensitive Fluorescent Probes |
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187 | (26) |
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187 | (1) |
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Membrane Potential Probes |
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188 | (6) |
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188 | (2) |
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190 | (2) |
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Advantages and Limitations of Membrane Potential Probes |
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192 | (2) |
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Ion-sensitive Fluorescent Probes |
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194 | (2) |
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194 | (1) |
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195 | (1) |
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Fluorescence Assays for Ion Channels |
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196 | (9) |
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196 | (1) |
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Non-voltage-gated Calcium Permeable Channels |
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197 | (3) |
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200 | (1) |
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201 | (2) |
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203 | (2) |
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Assays for Monitoring Channel Trafficking |
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205 | (2) |
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207 | (6) |
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208 | (5) |
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Approaches for Ion Channel Structural Studies |
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213 | (28) |
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213 | (3) |
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Expression of Membrane Proteins for Structural Studies |
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216 | (3) |
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216 | (1) |
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217 | (1) |
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217 | (1) |
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218 | (1) |
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219 | (4) |
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223 | (4) |
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227 | (2) |
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Use of Antibody Fragments |
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229 | (1) |
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Generation of First Diffraction Datasets |
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230 | (2) |
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Selenomethionine Phasing of Membrane Proteins |
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232 | (1) |
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MAD Phasing and Edge Scanning |
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233 | (1) |
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Negative B- factor Application (Structure Factor Sharpening) |
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234 | (1) |
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235 | (6) |
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235 | (6) |
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Molecular Modeling and Simulations of Ion Channels: Applications to Potassium Channels |
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241 | (28) |
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241 | (1) |
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242 | (4) |
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246 | (8) |
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246 | (1) |
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247 | (1) |
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248 | (1) |
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248 | (2) |
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M2 Helices and Hinge Motion |
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250 | (1) |
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Intracellular Domain Dynamics |
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251 | (1) |
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Interactions with Ligands |
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251 | (2) |
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Towards an Integrated Gating Model |
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253 | (1) |
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254 | (7) |
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254 | (2) |
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S6 Helices, Hinges and Gating |
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256 | (1) |
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257 | (1) |
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The Nature of the Voltage Sensor |
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258 | (2) |
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260 | (1) |
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Summary and Future Directions |
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261 | (8) |
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262 | (1) |
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262 | (7) |
Subject Index |
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269 | |