Genomes and Genes
Structure and Dynamics of Gap Junction Channels
Principal Investigator: MARK JAY YEAGER
Abstract: DESCRIPTION (provided by applicant): Gap junctions are specialized regions of cell-to-cell contact in which hexameric oligomers, called connexons, dock end-to-end across a narrow extracellular gap, allowing the intercellular exchange of nutrients, metabolites, ions and small molecules. Previously, this research program focused on the 43 kDa connexin (Cx43) channel, which mediates ionic conduction between cardiac myocytes, thereby regulating the normal heartbeat, but also mediating potentially fatal cardiac arrhythmias. We have now expanded our research to include Cx26, mutations in which are the predominant cause of inherited, nonsyndromic deafness, and Cx40, which forms high conductance channels in the specialized conducting tissue of the heart. For the next cycle, we will pursue 3 specific aims: (1) Our major aim, to which we will devote 70% effort, is to use X-ray crystallography to determine an atomic resolution structure of one or more hexameric connexons, which includes improving upon the resolution of a recent 3.5 E structure of Cx26 (Maeda et al., 2009), as well as testing our cryoEM-based C1 model for the assignment and packing of the transmembrane ?-helices. (2) Our second aim, to which we will devote 20% effort, is to use electron cryocrystallography of 2D crystalline gap junction plaques isolated from cells to examine the higher resolution structure of the extracellular loops, in order to understand the molecular basis for isoform selectivity and how the packing of the loops forms a tight molecular seal to exclude the extracellular environment. (3) A third minor aim, to which we will devote 10% effort, is to use cryoEM and single particle image analysis of connexons to explore gating and regulation of a number of Cx26 truncation mutants. Of particular interest is the recent discovery that the N-tail of Cx26 forms a central gating plug (Oshima et al., 2007). We will perform a structure/function analysis by correlating the structure of truncation mutants with functional studies of channels incorporated into liposomes. Our research is enriched by several key collaborations: Dr. Andrew Harris uses a liposome assay to test the function of our Cx mutants;Dr. Ray Stevens is an expert membrane protein X-ray crystallographer;Dr. Qinghai Zhang has synthesized custom detergents for channel stabilization to enable 3D crystallization trials;and Dr. Anchi Chang is an expert in electron cryocrystallography. By this integrated approach, we will continue our quest to visualize an atomic structure of a gap junction channel. PUBLIC HEALTH RELEVANCE: The cells within all tissues of our body have to communicate with one another in order to coordinate their metabolic activities. This is accomplished by a unique set of molecular pores called gap junction channels, which are assembled from protein subunits called connexins. Gap junction channels are formed by the coupling of two half channels called connexons, which are themselves formed by a ring of 6 connexin subunits. A cylindrical connexon in the surface membrane of one cell is coupled to a corresponding connexon in the adjacent cell. This molecular conduit mediates the passage of ions and small molecules and thereby coordinates the metabolic activity of the tissue. Much of our effort is now focused on a particular connexin, Cx26, mutations in which are a major cause of hereditary deafness. We have generated substantial quantities of the protein so that we can use biophysical techniques such as electron and X-ray crystallography to determine an atomic structure of the channel. Such a detailed molecular picture will provide insight into how these channels are involved in such diverse processes as regulating the heartbeat and hereditary deafness.
Funding Period: 1994-04-01 - 2015-03-31
more information: NIH RePORT
- In vivo functional assay of a recombinant aquaporin in Pichia pastorisMark J Daniels
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Appl Environ Microbiol 72:1507-14. 2006..The simplicity of this assay that is specific for water channel activity should enable rapid screening for compounds that modulate water channel activity...
- Steroid-based facial amphiphiles for stabilization and crystallization of membrane proteinsSung Chang Lee
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
Proc Natl Acad Sci U S A 110:E1203-11. 2013....
- Lipid monolayer and sparse matrix screening for growing two-dimensional crystals for electron crystallography: methods and examplesMark Yeager
Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA, USA
Methods Mol Biol 955:527-37. 2013..Likewise, the sparse matrix method uses screening conditions for three-dimensional crystallization and is therefore of broad applicability...
- Unusual arginine formations in protein function and assembly: rings, strings, and stacksMarco A C Neves
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA
J Phys Chem B 116:7006-13. 2012..Control of the stability of arginine clusters may play an important role in protein-protein oligomerization, molecular recognition, and ligand binding...
- Design, synthesis, and properties of branch-chained maltoside detergents for stabilization and crystallization of integral membrane proteins: human connexin 26Wen Xu Hong
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA
Langmuir 26:8690-6. 2010..We showed that the branch-chained maltosides with optimized micellar properties performed as well as or better than the straight-chained analogues and enabled crystallization in different space groups...
- Damped-dynamics flexible fittingJulio A Kovacs
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA
Biophys J 95:3192-207. 2008..Applications to the computation of transition pathways between known conformers, homology and loop modeling, as well as protein docking, are also discussed...
- Single particle image reconstruction of the human recombinant Kv2.1 channelBrian Adair
The Scripps Research Institute, Department of Cell Biology, La Jolla, California 92037, USA
Biophys J 94:2106-14. 2008..1 may also represent a closed conformation. Substantial cytoplasmic density is closely associated with the T1 tetramerization domain and is ascribed to the approximately 184 kDa C-terminal regulatory domains within each tetramer...
- Gap junction channel structure in the early 21st century: facts and fantasiesMark Yeager
Department of Cell Biology, The Scripps Research Institute, 10550 N Torrey Pines Road, La Jolla, CA 92037, USA
Curr Opin Cell Biol 19:521-8. 2007..An experimentally determined structure at atomic resolution will be essential to confirm and resolve these concepts...
- Profiling of membrane protein variants in a baculovirus system by coupling cell-surface detection with small-scale parallel expressionMichael A Hanson
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Protein Expr Purif 56:85-92. 2007..This approach expedites the in vitro evaluation of a large number of mammalian membrane protein variants...
- Electron microscopy of integrinsBrian D Adair
Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA
Methods Enzymol 426:337-73. 2007....
- Molecular modeling and mutagenesis of gap junction channelsJulio A Kovacs
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
Prog Biophys Mol Biol 94:15-28. 2007..In spite of this substantial progress in understanding the structural biology of gap junction channels, an experimentally determined structure at atomic resolution will be essential to confirm these concepts...
- Computational prediction of atomic structures of helical membrane proteins aided by EM mapsJulio A Kovacs
Department of Molecular Biology, Department of Cell Biology, The Scripps Research Institute, La Jolla, CA, USA
Biophys J 93:1950-9. 2007..In addition, our method implements a penalty term through a so-called tethering map, derived from the EM map, which restrains the positions of the alpha-helices. The protocol was validated on three test cases: GpA, KcsA, and MscL...
- Bootstrap resampling for voxel-wise variance analysis of three-dimensional density maps derived by image analysis of two-dimensional crystalsAnchi Cheng
The Scripps Research Institute, Department of Cell Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
J Struct Biol 158:19-32. 2007....
- The second extracellular loop of the adenosine A1 receptor mediates activity of allosteric enhancersDylan P Kennedy
Department of Pharmacology D P K, Department of Molecular Physiology and Biological Physics S A L, M P, H F, M C, R F, M Y, Cardiovascular Research Center M A M, R F, M Y, Center for Membrane Biology M Y, and Department of Medicine, Division of Cardiovascular Medicine M Y, University of Virginia School of Medicine, Charlottesville, Virginia the Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California F M M, R A and the La Jolla Institute for Allergy and Immunology J L, La Jolla, California
Mol Pharmacol 85:301-9. 2014..This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists. ..
- Connexin Distribution in Physiological Versus Pathological Cardiac HypertrophyMichael R Zile; Fiscal Year: 2013..pathological hypertrophy, with ex- tensively characterized cytoskeletal properties in each setting. ..
- Jules Stein Eye Institute Core Grant for Vision ResearchWayne L Hubbell; Fiscal Year: 2013..Support in the form of the Core grant is requested to maintain these Modules through instrument service contracts, and to provide necessary personnel support to assist and train users and provide routine maintenance. ..
- STRUCTURE AND DYNAMICS OF CONNEXIN26 GAP JUNCTIONSGina E Sosinsky; Fiscal Year: 2013..The proposed research is significant because results will be useful in defining better drugs and other therapeutics that potentially ameliorate connexin-related diseases. ..
- EINSTEIN AGING STUDYRichard B Lipton; Fiscal Year: 2013..Together, these Projects will help disentangle the multifactorial processes that lead to cognitive and locomotor decline and dementia. ..