Connexin Methods and Protocols

Direct cell-cell communication is a common property of multicellular organisms that is achieved through membrane channels which are organized in gap junctions. The protein subunits of these intercellular channels, the connexins, form a multigene family that has been investigated in great detail in recent years. It has now become clear that, in different tissues, connexins speak several languages that control specific cellular functions. This progress has been made possible by the availability of new molecular tools and the improvement of basic techniques for the study of membrane channels, as well as by the use of genetic approaches to study protein function in vivo. More important, connexins have gained visibility because mutations in some connexin genes have been found to be linked to human genetic disorders. Connexin Methods and Protocols presents in detail a collection of te- niques currently used to study the cellular and molecular biology of connexins and their physiological properties. The field of gap junctions and connexin research has always been characterized by a multidisciplinary approach c- bining morphology, biochemistry, biophysics, and cellular and molecular biology. This book provides a series of cutting-edge protocols and includes a large spectrum of practical methods that are available to investigate the fu- tion of connexin channels. Connexin Methods and Protocols is divided into three main parts.

Tools to Study Connexins. - Investigation of Connexin Gene Expression Patterns by In Situ Hybridization Techniques. - Sodium Dodecyl Sulfate-Freeze-Fracture Immunolabeling of Gap Junctions. - Purification of Gap Junctions. - Culturing of Mammalian Cells Expressing Recombinant Connexins and Two-Dimensional Crystallization of the Isolated Gap Junctions. - Connexins/Connexons. - Biochemical Analysis of Connexon Assembly. - Expression and Imaging of Connexin-GFP Chimeras in Live Mammalian Cells. - Analysis of Connexin Expression in Brain Slices by Single-Cell Reverse Transcriptase Polymerase Chain Reaction. - Use of Retroviruses to Express Connexins. - Spatiotemporal Depletion of Connexins Using Antisense Oligonucleotides. - Transfection and Expression of Exogenous Connexins in Mammalian Cells. - Assays For Function. - Assaying the Molecular Permeability of Connexin Channels. - Applying the Xenopus Oocyte Expression System to the Analysis of Gap Junction Proteins. - Mutagenesis to Study Channel Structure. - Dual Patch Clamp. - Determining Ionic Permeabilities of Gap Junction Channels. - Fluorescence Recovery After Photobleaching. - Capture of Transjunctional Metabolites. - Physiology and Biology of Connexins. - The Study of Connexin Hemichannels (Connexons) in Xenopus Oocytes. - Exploring Hemichannel Permeability In Vitro. - Inducing De Novo Formation of Gap Juntion Channels. - Recording and Analysis of Putative Direct Electrical Interactions in the Mammalian Brain. - Intercellular Calcium Signaling and Flash Photolysis of Caged Compounds. - Biochemical Analysis of Connexin Phosphorylation. - How to Close a Gap Junction Channel.