Transient Receptor Potential Channels in Mechanosensing and Cell

Volume Regulation 183

Stine Falsig Pedersen and Bernd Nilius

1. Introduction 184

2. General Mechanisms of Mechano- or Osmosensing by Membrane Proteins 184

3. TRP Channels in Mechano- and Osmosensing 188

4. TRP Channels in Cell Volume Regulation 192

5. Experimental Procedures 193 Acknowledgments 201 References 201

11. Cell Volume Regulatory Ion Channels in Cell Proliferation and Cell Death 209

Florian Lang, Michael Foller, Karl Lang, Philipp Lang, Markus Ritter, Alexey Vereninov, Ildiko Szabo, Stephan M. Huber, and Erich Gulbins

1. Introduction 210

2. Anion Channels, Osmolyte Transport, and pH Regulation 211

3. Ca2+ and Unselective Cation Channels 212

4. K+ channels 213

5. Switching from Cell Proliferation to Suicidal Cell Death 215 Acknowledgments 216 References 216

12. Actin Cytoskeleton Architecture and Signaling in Osmosensing 227

Evangelia A. Papakonstanti and Christos Stournaras

1. Introduction 228

2. Morphological Analysis of Actin Cytoskeleton During

Cell Volume Changes 229

3. Quantitative Biochemical Analysis of Actin Cytoskeleton

Dynamics During Cell Volume Changes 230

4. Signaling Pathways Linking Actin Reorganization and Cell

Volume Regulation 231

5. Quantitative Assessment of Cellular Actin Cytoskeleton Dynamics 232

6. Quantification of Cellular Monomeric and Total Actin

Using the DNase I Inhibition Assay 233

7. DNase I Inhibition Assay Protocol 234

8. Quantification of Filamentous Actin Using Rhodamine-Phalloidin Fluorescence Measurements of Actin in Detergent Cell Extracts 235

9. Filamentous (F-) Actin Quantification Protocol 235

10. Quantitative Immunoblot Analysis of Triton X-100

Insoluble Cytoskeletal Pellets and Corresponding Supernatants 236

11. Triton Soluble/Insoluble Actin Ratio Determination 236 References 237

13. Osmotic Stress and DNA Damage 241

Natalia I. Dmitrieva and Maurice B. Burg

1. Introduction 242

2. Detection of DNA Damage by Comet Assay 242

3. Detection of DNA Breaks by In Situ End Labeling 246 References 250

14. Transcriptional Activator TonE-Binding Protein in Cellular Protection and Differentiation 253

Jeong Ah Kim, Un Sil Jeon, Min Seong Kwon, Sun Woo Lim, and H. Moo Kwon

1. Introduction 254

2. Immunological Detection of TonEBP 255

3. Functional Analyses of TonEBP 258

4. How to Identify TonEBP Target Genes 261

5. Biochemical Analysis of TonEBP 265 Acknowledgments 265 References 266

15. Desiccation Response of Mammalian Cells: Anhydrosignaling 269

Zebo Huang and Alan Tunnacliffe

1. Introduction 270

2. Cell Cultures 272

3. Desiccation 273

4. Cell Viability Test 273

5. Western Blotting 273

6. Relative Quantification of Gene Expression 275 Acknowledgments 276 References 276

16. Tonicity-Regulated Gene Expression 279

Joan D. Ferraris and Maurice B. Burg

1. Introduction 280

2. Quantitation of the Nuclear: Cytoplasmic Distribution

Ratio of the Transcription Factor TonEBP/OREBP in HEK293 Cells 286

3. Nonradioactive EMSA of Nuclear Extracts of HEK293 Cells for Binding of the Complex of TonEBP/OREBP and Associated Proteins to ORE Elements 288

4. Quantitation of Transcriptional and Transactivational Activities of TonEBP/OREBP Using Transient or Stable

Transfection of HEK293 Cells 290

Acknowledgment 293

References 293

17. Hyperosmotic Induction of Mitogen-Activated Protein

Kinase Scaffolding 297

Thomas L. Hilder, Michael H. Malone, and Gary L. Johnson

1. Introduction 298

2. RNA Interference 300

3. In Vitro Kinase Assays 302

4. Measuring Dynamic Interactions Between MAPK Scaffold

Members Using FRET 303

References 310

18. Osmoregulation of Bile Formation 313

Ralf Kubitz and Dieter Haussinger

1. Networks of Transport Systems 314

2. Osmoregulation of Canalicular Secretion 315

3. Osmosensing and Osmosignaling Pathways Toward

Canalicular Secretion 317

4. Osmoregulation of Sinusoidal Bile Acid Uptake 318

5. Methods Used to Study Osmoregulated Transporter Insertion/Retrieval 318

6. Concluding Remarks 320 Acknowledgments 321 References 322

19. Osmosignaling and Volume Regulation in Intestinal Epithelial Cells 325

Christina H. Lim, Alice G. M. Bot, Hugo R. de )onge, and Ben C. Tilly

1. Introduction 326

2. Properties of Ion Channels and Transporters Activated

During RVD in Intestinal Epithelial Cells 326

3. Osmosensing and Signaling 328

4. Model Systems 330

5. Measuring Ionic Responses 331

6. Concluding Remarks 338 References 338

20. Osmotic Regulation of Cellular Glucose Uptake 343

Philippe Gual, Teresa Gonzalez, Thierry Gremeaux, Yannick Le Marchand-Brustel, and Jean-François Tanti

1. Introduction 344

2. Hyperosmolarity and Glucose Transport 347

3. Hyperosmolarity and Membrane Ruffling 349

4. Hyperosmolarity and Signaling Pathways 350

5. Hyperosmolarity and Phosphatidylinositol 3-Kinase Activity 351

6. Conclusion 353 Acknowledgments 353 References 354

21. Effects of Osmolytes on Protein Folding and

Aggregation in Cells 355

Zoya Ignatova and Lila M. Gierasch

1. Introduction 356

2. Analysis of Effects of Osmolytes In Vivo 358

3. Comparison of the Influence of Osmolytes In Vivo with That In Vitro 367

4. Conclusions 370 Acknowledgments 370 References 370

22. Simulations of Macromolecules in Protective and Denaturing Osmolytes: Properties of Mixed Solvent Systems and Their

Effects on Water and Protein Structure and Dynamics 373

David A. C. Beck, Brian J. Bennion, Darwin O. V. Alonso, and Valerie Daggett

1. Introduction 374

2. Methods 375

3. Results 379

4. Discussion 389

5. Conclusions 392 Acknowledgments 393 References 393

23. Application of the Transfer Model to Understand

How Naturally Occurring Osmolytes Affect Protein Stability 397

Matthew Auton and D. Wayne Bolen

1. Introduction 398

2. Initial Observations of Osmolyte Effects on Proteins 398

3. The Transfer-Free Energy Model 401

4. How Transfer-Free Energies Are Measured 403

5. The Issue of Activity Coefficients: Solution Nonideality at the Solubility Limit 406

6. Additive, Concentration Scale-Independent and Model Compound-Independent Transfer-Free Energies of the Peptide Backbone Unit 408

7. The Transfer-Free Energy of Amino Acid Side Chains 412

8. Solvent-Accessible Surface Areas of Native and Denatured

States: Calculating Aa{ 413

9. Putting It All Together: Predicting m Values for the Energetics of Osmolyte-Induced Protein Folding/Unfolding 414

10. Summary 415

Acknowledgments 416

References 416

24. Mechanisms of High Salinity Tolerance in Plants 419

Narendra Tuteja

1. Introduction 420

2. Adverse Effect of Salinity Stress 421

3. Generic Pathway for Plant Response to Stress 422

4. Ion Pumps, Calcium, and SOS Pathways in Relation to Salinity Stress 424

5. Abscisic Acid and Transcription Factors in Salinity Stress Tolerance 427

6. Mitogen-Activated Protein Kinases and Salinity Stress 430

7. Glycine Betaine and Proline in Salinity Stress 430

8. Reactive Oxygen Species in Salinity Stress 431

9. DEAD-Box Helicases in Salinity Stress Tolerance 432

10. Cross-Tolerances Between Stresses 434

11. Future Prospective 435 Acknowledgments 436 References 436

25. Phenotype of the Taurine Transporter Knockout Mouse 439

Ulrich Warskulat, Birgit Heller-Stilb, Evelyn Oermann, Karl Zilles, Helmut Haas, Florian Lang, and Dieter Haussinger

1. Targeted Disruption of the taut Gene 440

2. Reduced Taurine Levels Lead to Various Diseases in taut—/— Mice 441

3. Reduced Exercise Capacity in taut—/— Mice 442

4. Pathophysiological Changes in Brain, Retina, Olfactory Bulb, and Inner Ear of taut—/— Mice 444

7. Available Organic Osmolyte Transporter Transgene and

Knockout Mice 452

References 452

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