Gap junctions play dynamic roles in cellular processes, however, there is a fundamental knowledge gap in understanding how gap junction proteins, the connexins, are regulated and gated based on a structure that has rigid and flexible domains. Connexin expression and function are highly regulated and the sequence of each isoform imparts specificity (permselectivity) to which molecules pass through the pore. The connexin hexamer (connexon or hemichannel) have three domains defined by the lipid bilayer. Two hemichannels pair at their extracellular domains to form an intercellular channel. The conserved transmembrane and extracellular domains are fairly rigid while the cytoplasmic domain is flexible. The sequence variability in the cytoplasmic domains, particularly in the C-terminus, allows for binding of partner proteins unique to each isoform. Within the context of this compartmentalized structure, our central hypothesis is that the monomer is tightly packed in its rigid domains, but flexibility in the cytoplasmic domains permit supra-molecular complexes to be formed in cells as well as binding of proteins controlling phosphorylation and gating. Connexin-opathies, hereditary human diseases, are often caused by mutations that often disrupt packing or partner interactions. For example, Cx26 mutations account for ~1/2 of cases of pre-lingual non-syndromic deafness in Caucasian populations but cases are found in populations across all continents. The proposed studies explore this hypothesis with three specific aims. (1) To investigate the stability of the transmembrane region of the Cx26 hexamer using mutations known to cause heredity deafness. These experiments will be correlated with ones probing channel function and structure. (2) To determine the 3D structure by cryo-electron microscopy (cryo-EM) and single particle reconstruction of Cx50 hemichannels. Cx50 intercellular channels serve critical functions in lens and its dysfunction leads to cataracts. It has extensive less ordered cytoplasmic domains typically not resolvable by crystallography. In this aim, single particle reconstruction is the best technique to obtain a structure of the large, full-length Cx50 hemichannel. (3) To create electron tomographic volumes of genetically labeled Cx43 intercellular channels and cytoskeletal and scaffolding proteins in situ to better understand the cytoplasmic architecture interacting with a gap junction. Cx43 contains binding domains for cytoskeletal components and the scaffolding protein, ZO-1. It is widespread through most organ systems with particularly important roles in vasculature and heart. The long-term goal is to obtain a more complete depiction of full-length connexins at the highest resolution obtainable. The approach is innovative because it uses a multi-resolution imaging strategy coordinated with biochemical and functional analyses of channels and hemichannels. The proposed research is significant because results will be useful in defining better drugs and other therapeutics that potentially ameliorate connexin-related diseases.

Public Health Relevance

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 #
4R01GM065937-12
Application #
8976261
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Flicker, Paula F
Project Start
2003-08-01
Project End
2016-08-31
Budget Start
2015-12-01
Budget End
2016-08-31
Support Year
12
Fiscal Year
2016
Total Cost
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
Cone, Angela C; Cavin, Gabriel; Ambrosi, Cinzia et al. (2014) Protein kinase Cδ-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
Meckes, Brian; Ambrosi, Cinzia; Barnard, Heather et al. (2014) Atomic force microscopy shows connexin26 hemichannel clustering in purified membrane fragments. Biochemistry 53:7407-14
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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