Communication between cells in a tissue takes place in two different ways: release of molecules such as neurotransmitters, hormones or calcium ions; formation of intercellular channels in the plasma membrane enveloping the cell that connect directly the cytoplasm of two adjacent cells. Multicellular organisms have evolved to use different types of intercellular channels, called gap junctions. In vertebrates, a gap junction channel is formed by the apposition of two hexamers of gap junction proteins, one from each adjacent cell. Gap junctions are found in essentially all tissues suggesting an enormous diversity of function. These intercellular channels are critical for integrating and regulating basic cell processes such as metabolic cooperation, ionic transmission, differentiation and hormonal regulation. Breakdown in gap junctional communication can lead to developmental defects, abnormal cell proliferation such as cancer and the failure of tissues to function in their normal capability. Examples of the latter come from the study of patients with hereditary gap junction diseases such as the dysfunction of peripherial nerves (X-linked Charcot-Marie-Tooth Disease) and certain forms of malformation of the heart, deafness, cataracts and skin diseases. We focus on the structure and function of Connexin26 (Cx26), the second smallest of the gap junction protein family. Mutations in the DNA of Cx26 account for about one half of cases of prelingual inherited deafness in Caucasian populations. We have isolated preparations of Cx26 gap junctions and hemichannels in sufficient quantities for biochemical and structural studies by electron microscopy (EM) and atomic force microscopy (AFM).
In Specific Aim 1, we will determine the structure of the Cx26 channel beyond 8 E using state of the art cryo-electron crystallography.
In Specific Aim 2, we continue our lower resolution analyses of mutant Cx26 channels and hemichannels in order to determine differences in pore structure and connexon stability.
In Specific Aim 3, a 3D EM analysis of several isoforms will determine if differences in pore size and shape are related to their physiological properties. Finally, Specific Aim 4 is focused on both imaging under in vitro physiological conditions and on protein unfolding of gap junctions using AFM and these results will be correlated with our EM structures. Each of these aims is intended to complement the others leading to improved structural and physiological models of Cx26 channels germane to the entire connexin family. ? Gap junction channels directly regulate cell-cell activities by passing metabolites, ions and signaling molecules. This project investigates the structure-function basis of intercellular communication between cells in tissues and organs by focusing on how gap junction intercellular membrane channels are put together from their constituent proteins, the connexins, and how specific parts of the connexin are involved in gating, docking or regulation of the channel. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM065937-05
Application #
7374024
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Shapiro, Bert I
Project Start
2003-08-01
Project End
2012-01-31
Budget Start
2008-02-01
Budget End
2009-01-31
Support Year
5
Fiscal Year
2008
Total Cost
$310,068
Indirect Cost
Name
University of California San Diego
Department
Neurosciences
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Ambrosi, Cinzia; Ren, Cynthia; Spagnol, Gaelle et al. (2016) Connexin43 Forms Supramolecular Complexes through Non-Overlapping Binding Sites for Drebrin, Tubulin, and ZO-1. PLoS One 11:e0157073
Meckes, Brian; Ambrosi, Cinzia; Barnard, Heather et al. (2014) Atomic force microscopy shows connexin26 hemichannel clustering in purified membrane fragments. Biochemistry 53:7407-14
Cone, Angela C; Cavin, Gabriel; Ambrosi, Cinzia et al. (2014) Protein kinase C?-mediated phosphorylation of Connexin43 gap junction channels causes movement within gap junctions followed by vesicle internalization and protein degradation. J Biol Chem 289:8781-98
Wang, Junjie; Ambrosi, Cinzia; Qiu, Feng et al. (2014) The membrane protein Pannexin1 forms two open-channel conformations depending on the mode of activation. Sci Signal 7:ra69
Ambrosi, Cinzia; Walker, Amy E; Depriest, Adam D et al. (2013) Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix. PLoS One 8:e70916
Cone, Angela C; Ambrosi, Cinzia; Scemes, Eliana et al. (2013) A comparative antibody analysis of pannexin1 expression in four rat brain regions reveals varying subcellular localizations. Front Pharmacol 4:6
Martell, Jeffrey D; Deerinck, Thomas J; Sancak, Yasemin et al. (2012) Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat Biotechnol 30:1143-8
Ellisman, Mark H; Deerinck, Thomas J; Shu, Xiaokun et al. (2012) Picking faces out of a crowd: genetic labels for identification of proteins in correlated light and electron microscopy imaging. Methods Cell Biol 111:139-55
Dolmatova, Elena; Spagnol, Gaelle; Boassa, Daniela et al. (2012) Cardiomyocyte ATP release through pannexin 1 aids in early fibroblast activation. Am J Physiol Heart Circ Physiol 303:H1208-18
Yu, Yong-Chun; He, Shuijin; Chen, She et al. (2012) Preferential electrical coupling regulates neocortical lineage-dependent microcircuit assembly. Nature 486:113-7

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