1. Introduction 270

1.1 Overview of nucleosome structure 271

2. Relative equilibrium stability (affinity) of histone–DNA interactions in nucleosomes 272

2.1 Relative affinity equals relative equilibrium stability 272

2.2 Competition assays for relative free-energy measurements 273

2.3 Technical issues in relative free-energy measurements 275

2.4 Range of affinities 278

3. Relation of nucleosome stability to nucleosome positioning 279

3.1 Translational nucleosome positioning 279

3.2 Rotational positioning 280

3.3 Unfavorable positioning 281

3.4 Experiments 281

4. Physical basis of DNA sequence preferences 282

4.1 Free-energy cost of DNA bending 283

4.2 Molecular mechanics of DNA bending and bendability 284

4.3 Bent and bendable DNA sequences 286

4.4 Parameter sets for prediction of DNA bending and bendability 288

4.5 DNA twisting 290

4.6 Energetics of nucleosomal DNA packaging 291

5. DNA sequence motifs for nucleosome packaging 292

5.1 Natural and designed nucleosomal DNAs 293

5.2 New rules and reagents from physical selection studies 294

5.3 Molecular basis of DNA sequence preferences 299

5.4 Special properties of the TA step 300

5.5 Unfavorable sequences 302

5.6 Natural genomes 303

5.7 Evolutionary approach toward an optimal sequence 305

5.8 Optimization by design 305

6. Dynamic nucleosome instability 308

6.1 Site-exposure equilibria 308

6.2 DNA sequence-dependence to site-exposure equilibria 312

6.3 Nucleosome translocation 315

6.4 Action of processive enzymes 319

7. Conclusions 319

8. Acknowledgements 320

9. References 320

The nucleosome core particle is the fundamental repeating subunit of chromatin. It consists of two molecules each of the four ‘core histone’ proteins, H2A, H2B, H3 and H4, and a 147 bp stretch of DNA. The lowest level of chromatin organization consists of a repeated array of nucleosome core particles separated by variable lengths of ‘linker DNA’. In many, but not all, cases, each core particle plus its linker DNA is associated with one molecule of a fifth ‘linker’ histone protein, H1. The complex of the core particle plus its linker DNA and H1 (when present) is called a ‘nucleosome’.

1. Introduction 326

1.1 What? Terminology and general properties 327

1.2 Why? Reasons for biophysical study 329

1.3 How? Special issues for study of connexin channels 330

2. Molecular and structural context 331

2.1 Biochemical features 331

2.2 Structures 334

2.2.1 Junctional channels 335

2.2.2 Hemichannels 338

2.2.3 Heteromeric channels 342

2.2.4 Junctional plaques 347

3. Experimental approaches and issues specific to study of connexin channel physiology 349

3.1 Macroscopic currents 349

3.1.1 Junctional channels 349

3.1.2 Hemichannels 354

3.2 Single-channel currents 355

3.2.1 Junctional channels 355

3.2.2 Hemichannels 358

3.3 Molecular permeability 361

3.3.1 A selection of tracers 361

3.3.2 Junctional channels 362

3.3.3 Hemichannels 366

3.4 Other 367

4. Structural issues 368

4.1 What lines the pore? 368

4.2 Docking between hemichannels 373

4.2.1 Structural and molecular basis 374

4.2.2 Determinants of specificity of interaction 380

5. Permeability and selectivity 381

5.1 Among the usual ions 383

5.1.1 Unitary conductance 383

5.1.2 Selectivity 384

5.1.3 Nonlinear single-channel I–V relations and their molecular determinants 386

5.2 Among large permeants 391

5.2.1 Uncharged molecules 392

5.2.2 Charged molecules 393

5.2.3 Cytoplasmic/signaling molecules 396

6. Voltage sensitivity 399

6.1 Macroscopic transjunctional voltage sensitivity 404

6.2 Microscopic voltage sensitivity – Vj-gating 407

6.2.1 Molecular basis – voltage sensor 407

6.2.2 Molecular basis – transduction and/or state stability 409

6.3 Microscopic voltage sensitivity – loop gating 412

6.4 Vm-gating 414

7. Direct chemical modulation 415

7.1 Phosphorylation 417

7.2 Cytoplasmic pH and aminosulfonates 419

7.3 Calcium ion 424

7.4 Lipophiles 424

7.4.1 Long chain n-alkyl alcohols 425

7.4.2 Fatty acids and fatty acid amides 426

7.4.3 Halothane 426

7.5 Glycyrrhetinic acid and derivatives 427

7.6 Cyclic nucleotides 428

7.7 Other candidates 430

8. Connexinopathies 431

9. Summary 435

10. Acknowledgements 438

11. References 438

Connexins are the proteins that form the intercellular channels that compose gap junctions in vertebrates. Connexin channels mediate electrotonic coupling between cells and serve important functions as mediators of intercellular molecular signaling. Convincing demonstration of the latter function has been elusive, as have the experimental tools required for detailed functional study of the channels. Recently, substantial progress has been made on both fronts. Connexin channels are now known to be dynamic, multifunctional channels intimately involved in development, physiology and pathology, and amenable to study by state-of-the-art approaches. A host of developmental and physiological defects are caused by defects in connexin channels, and therefore in the intercellular molecular movement they mediate. The channel structure has been determined to 7·5 Å resolution within the plane of the membrane. Experimental paradigms have been developed that enable application of the tools of modern channel biophysics to study connexin channel structure–function. As a result, the biophysical mechanisms and biological functions of connexin channels now enjoy a vigorous and expanding experimental interest. This article focuses on the former, but with attention to issues likely to have biological consequences.