The long term goal of this project is to understand how cells regulate the transport and assembly of gap junctions. Newly synthesized connexins are assembled into hexamers in an intracellular compartment prior to the formation of gap junction channels. We found that ROS cells differentially assemble and sort two endogenously expressed gap junction proteins, CX43 and CX46. ROS cells assemble CX43 into hexamers which are transported to the plasma membrane. In contrast, CX46 remains a monomer and is restricted to the trans Golgi network (TGN). The goal of this proposal is to understand how CX43 and CX46 are differentially regulated by these cells. To identify domains that control connexin targeting, we will make epitope tagged connexin chimeras containing domains from CX43 and CX46. These will be transfected into cell lines and then analyzed by immunofluorescence microscopy for transport to the plasma membrane and/or TGN. These constructs will then be analyzed by sucrose gradient fractionation of Triton X-100 solubilized cell extracts to determine whether all TGN localized connexins are retained as monomers. We will then determine whether Cx43 and Cx46 can form hybrid gap junction channels. This is a key point-the compatibility of these connexins to form hybrid channels would imply that there is an active mechanism to keep them from co-assembling in ROS cells. Hybrid CX43+CX46 channels formed during in vitro translation or by Hela cells where both proteins are transported to the plasma membrane will be solubilized as intact hexamers and identified by co-immunoisolation. Finally, we will determine whether Cx46 monomers co-exist with Cx43 hexamers in the same intracellular compartments. Cells will be treated to arrest connexin transport at different steps, homogenized and intact vesicles containing CX46 will be immunoisolated and then analyzed for the presence of CX43 hexamers. Understanding the fundamentals of differential CX43 and CX46 sorting and assembly will provide insights into the regulation of gap junctional communication and is likely to reflect general principles of protein assembly in post-Golgi compartments.
| Das, Shamie; Smith, Tekla D; Sarma, Jayasri Das et al. (2009) ERp29 restricts Connexin43 oligomerization in the endoplasmic reticulum. Mol Biol Cell 20:2593-604 |
| Das Sarma, Jayasri; Iacono, Kathryn; Gard, Lilli et al. (2008) Demyelinating and nondemyelinating strains of mouse hepatitis virus differ in their neural cell tropism. J Virol 82:5519-26 |
| Das Sarma, Jayasri; Kaplan, Benjamin E; Willemsen, Dounia et al. (2008) Identification of rab20 as a potential regulator of connexin 43 trafficking. Cell Commun Adhes 15:65-74 |
| Muro, Silvia; Mateescu, Madalina; Gajewski, Christine et al. (2006) Control of intracellular trafficking of ICAM-1-targeted nanocarriers by endothelial Na+/H+ exchanger proteins. Am J Physiol Lung Cell Mol Physiol 290:L809-17 |
| Koval, Michael (2006) Pathways and control of connexin oligomerization. Trends Cell Biol 16:159-66 |
| Koval, Michael (2006) Claudins--key pieces in the tight junction puzzle. Cell Commun Adhes 13:127-38 |
| Muro, Silvia; Gajewski, Christine; Koval, Michael et al. (2005) ICAM-1 recycling in endothelial cells: a novel pathway for sustained intracellular delivery and prolonged effects of drugs. Blood 105:650-8 |
| Das Sarma, Jayasri; Das, Shamie; Koval, Michael (2005) Regulation of connexin43 oligomerization is saturable. Cell Commun Adhes 12:237-47 |
| Patel, Anand S; Reigada, David; Mitchell, Claire H et al. (2005) Paracrine stimulation of surfactant secretion by extracellular ATP in response to mechanical deformation. Am J Physiol Lung Cell Mol Physiol 289:L489-96 |
| Maza, Jose; Das Sarma, Jayasri; Koval, Michael (2005) Defining a minimal motif required to prevent connexin oligomerization in the endoplasmic reticulum. J Biol Chem 280:21115-21 |
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