Prior to delivery to the plasma membrane and assembly into gap junction channels, connexins are first assembled into hexameric hemichannels. Most assembly of oligomeric transmembrane proteins occurs in the ER and is a prerequisite for further transport along the secretory pathway. However, connexin hemichannel assembly appears to be more complex, since a number of intracellular compartments have been implicated in hemichannel assembly, including the endoplasmic reticulum (ER), ER-Golgi intermediate compartment (ERGIC) and trans Golgi network (TGN). Defining the intracellular compartments involved in connexin hemichannel assembly is further complicated by the potential for multiple connexin trafficking pathways and the possibility that different connexins, such as Cx32 and Cx43, may have the potential to oligomerize in different compartments.
The mechanisms which regulate the assembly of connexins into gap junction channels remain poorly understood. Unlike most transmembrane protein complexes which are assembled in the endoplasmic reticulum, gap junction assembly begins in distinct intracellular compartments, depending on the connexin examined. For instance, Cx32 assembly occurs in the ER/ERGIC (Model 1). In contrast, Cx43 and Cx46 assembly occur in the trans Golgi network (model 2). How the site of connexin oligomerization helps regulate the formation of gap junction channels is not known at present.
Cx43 and Cx46 are differentially sorted by osteoblasts and alveolar epithelial cells. The immunofluorescence image (left) shows osteoblasts, where Cx43 (red) is assembled into gap junction channels localized to the cell surface and Cx46 (green) is retained in the TGN as a monomer. The differential localization and oligomerization of these two connexins suggests that cells possess novel post-ER mechanisms for the control of protein assembly. A long term goal of the lab is to identify co-factors that participate in this process.
More recently we have begun to apply these tools and approaches to study assembly and trafficking of tight junction proteins known as claudins. Claudins are tetraspan transmembrane proteins that form the physical basis for tight junction and thus are key regulators of epithelial and endothelial barrier function. Although both claudins and connexins have some similarities in primary and secondary structure, our preliminary work suggests that claudin assembly, trafficking and compatibility are regulated differently than connexins.
Defining the mechanisms that regulate claudin assembly and turnover is another long term goal of the lab.
Projects:
We have designed ER-retained connexin chimeras which are differentially oligomerized in a connexin-dependent manner. ER-retained Cx43 chimeras are being used as "bait" for trapping, isolate and identify putative chaperones involved in post-ER quality control pathways.
Define subcellular compartments where other connexins oligomerize.
We are creating chimeras containing elements from different connexins to :
identify motifs that regulate connexin-connexin interactions.
identify sorting motifs that direct conneixn targeting in polarized cells.
Define intracellular compartments where claudins oligomerize.
Define claudin motifs which regulate heteromeric and heterotypic compatibility.
Screen for other multimeric protein complexes oligomerized in post-ER compartments.
Selected References
Mitchell, L. A., C. E. Overgaard, C. Ward, S. S. Margulies and M. Koval. 2011. Differential effects of claudin-3 and claudin-4 on alveolar epithelial barrier function, Amer. J. Physiol. Lung Cell. Mol. Biol., 301:L40-49 (PubMed)
Su, V., R. Nakagawa, M. Koval, A.F. Lau. 2010. Ubiquitin-independent proteasomal degradation of ER-localized connexin43 mediated by CIP75. J Biol Chem. 285:40979-40990 (PubMed)
Das, S., J. Das Sarma, J. D. Ritzenthaler, T. Smith, J. Maza, B. E. Kaplan, L. A. Cunningham, L. Suaud, R. C. Rubenstein, M. Koval. 2009. Regulation of connexin43 oligomerization and trafficking by ERp29, Mol. Biol. Cell, 20:2593-2604 (PubMed)
Das Sarma, J., B. E. Kaplan, D. Willemsen and M. Koval. 2008. Identification of rab20 as a potential regulator of connexin43 trafficking. Cell Commun. and Adhesion, 15:65-74.
(PubMed)
Mrsny, R. J., G. T. Brown, , K. Gerner-Smidt, A. G. Buret, J. B. Meddings, C. Quan, M. Koval and A. Nusrat. 2008. A key claudin extracellular loop domain is critical for epithelial barrier integrity. Amer. J. Path., 172:905-915 (PubMed)
Koval, M. 2008. Gap Junctions - Connexin Functions and Roles in Human Disease. in Cell Junctions: Adhesion, Development And Disease, Susan E. LaFlamme and Andrew Kowalczyk, editors, Wiley-VCH. pp 197-216.
Daugherty, B.L., C. Ward, T. Smith, J. D. Ritzenthaler, M. Koval. 2007. Regulation of heterotypic claudin compatibility, J. Biol. Chem. 282:30005-30013 (PubMed)
Das Sarma, J., S. Das and M. Koval. 2006. Regulation of connexin43 oligomerization is saturable. Cell Commun. and Adhesion 12:237-247 (PubMed).
Koval, M. 2006. Claudins: key pieces in the tight junction puzzle. Cell Commun. Adhesion, 13:127-138 (PubMed)
Koval, M. 2006. Pathways and control of connexin oligomerization. Trends Cell Biol. 16:159-166 (PubMed)
Laing J.G., M. Koval, and T. H. Steinberg. 2005. Association with ZO-1 Correlates with Plasma Membrane Partitioning in Truncated Connexin45 Mutants. J Membr. Biol. 207:45-53 (PubMed).
Maza, J., J. Das Sarma and M. Koval. 2005. Defining a minimal motif required to prevent connexin oligomerization in the endoplasmic reticulum. J Biol Chem., 280:21115-21121 (PubMed).
Daugherty, B., M. Mateescu, A. S. Patel, K. Wade, S. Kimura, L. W. Gonzales, P. L. Ballard and M. Koval. 2004. Developmental regulation of claudin localization by fetal alveolar epithelial cells, Amer. J. Physiol. Lung Cell. Mol. Biol., 287:L1266-1273 (PubMed).
Lin, G.C., J.K. Rurangirwa, M. Koval, and T.H. Steinberg. 2004. Gap junctional communication modulates agonist-induced calcium oscillations in transfected HeLa cells. J Cell Sci. 117:881-7 (PubMed).
Maza, J., M. Mateescu, J.D. Sarma, and M. Koval. 2003. Differential oligomerization of endoplasmic reticulum-retained connexin43/connexin32 chimeras. Cell Commun Adhes. 10:319-22 (PubMed).
Muro, S., X. Cui, C. Gajewski, J.C. Murciano, V.R. Muzykantov, and M. Koval. 2003. Slow intracellular trafficking of catalase nanoparticles targeted to ICAM-1 protects endothelial cells from oxidative stress. Am J Physiol Cell Physiol. 285:C1339-47 (PubMed).
Wang, F., B. Daugherty, L. L. Keise, Z. Wei, J. P. Foley, R. C. Savani, and M. Koval. 2003. Heterogeneous claudin expression by alveolar epithelial cells. 2003. Amer. J. Respir. Cell Mol. Biol., 29:62-70. DOI 10.1165/rcmb.2002-0180OC(PubMed)
Das Sarma, J. F. Wang, and M. Koval. 2002. Targeted gap junction protein constructs reveal connexin-specific differences in oligomerization. J. Biol. Chem., 277:20911-20918. DOI 10.1074/jbc.M111498200 (PubMed)
Das Sarma, J., R. I. Meyer, F. Wang, V. Abraham, C. W. Lo and M. Koval. 2001. Multimeric connexin interactions prior to the trans Golgi apparatus. J. Cell Sci., 114:4013-4024 (PubMed)
Laing, J. G., R. N. Manley-Markowski, M. Koval, R. Civitelli, and T. H. Steinberg. 2001. Connexin45 interacts with Zonula Occludens-1 and Connexin43 in Osteoblastic Cells. J. Biol. Chem, 276:23051-23055.
(PubMed)
Koval, M., E. Hick, J. Harley, and T. H. Steinberg. 1997. Connexin46 is retained as monomers in a trans-Golgi compartment of osteoblastic cells. J. Cell Biol., 137:847-857.
(PubMed)
Koval, M., S. T. Geist, E. M. Westphale, A. E. Kemendy, R. Civitelli, E. C. Beyer and T. H. Steinberg. 1995. Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43. J. Cell Biol., 130:987-995.
(PubMed)
Prior to delivery to the plasma membrane and assembly into gap junction channels, connexins are first assembled into hexameric hemichannels. Most assembly of oligomeric transmembrane proteins occurs in the ER and is a prerequisite for further transport along the secretory pathway. However, connexin hemichannel assembly appears to be more complex, since a number of intracellular compartments have been implicated in hemichannel assembly, including the endoplasmic reticulum (ER), ER-Golgi intermediate compartment (ERGIC) and trans Golgi network (TGN). Defining the intracellular compartments involved in connexin hemichannel assembly is further complicated by the potential for multiple connexin trafficking pathways and the possibility that different connexins, such as Cx32 and Cx43, may have the potential to oligomerize in different compartments.
Cx43 and Cx46 are differentially sorted by osteoblasts and alveolar epithelial cells. The immunofluorescence image (left) shows osteoblasts, where Cx43 (red) is assembled into gap junction channels localized to the cell surface and Cx46 (green) is retained in the TGN as a monomer. The differential localization and oligomerization of these two connexins suggests that cells possess novel post-ER mechanisms for the control of protein assembly. A long term goal of the lab is to identify co-factors that participate in this process.