Characterization of the Structure and Intermolecular Interactions between the Connexin 32 Carboxyl-terminal Domain and the Protein Partners Synapse-associated Protein 97 and Calmodulin

Abstract

Background: SAP97 and CaM play a role in the regulation of connexin 32 (Cx32) gap junctions. Results: SAP97 and CaM affect the same Cx32CT residues, calmodulin induces Cx32CT ␣-helical structure, and Cx32CT mutations that cause X-linked Charcot-Marie-Tooth disease (CMTX) affect the binding of SAP97 and CaM. Conclusion: Cx32-protein partner interactions are important for channel regulation and myelin homeostasis. Significance: Cx32CT mutations may cause CMTX by disrupting the binding of SAP97 and CaM.

Regulation of Cellular Communication by Signaling Microdomains in the Blood Vessel Wall

“…Several studies now suggest that this may be regulated through binding of the calcium sensing subunit calmodulin. Calmodulin interacts with both Cx32 and Cx43 to maintain a closed gap junction channel, potentially by altering channel conformation or through physical interactions with calmodulin or calcium itself (Van Eldik et al, 1985;Torok et al, 1997;Sotkis et al, 2001;De Vuyst et al, 2006;Stauch et al, 2012;Xu et al, 2012). This interaction is not homologous between connexin isoforms: gap junction channels formed of Cx40 isoforms are insensitive to high levels of calcium, potentially because of a lack of the putative binding sites for calmodulin (Lurtz and Louis, 2007;Xu et al, 2012).…”

The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells

“…One possible explanation for these data is that Cx32-CT-TQ-Myr either sequesters a protein(s) at the cell surface that inhibits plaque growth and/or recruits additional proteins that facilitate growth. The latter possibility is supported by the Cx32-mediated recruitment of disc large protein, Dlg, to the cell surface in hepatocytes possibly through direct or indirect interaction (44,45). Lack of a robust effect of Cx32-CT-TQMyr alone in incrementing Cx32⌬220 plaque growth compared with that observed upon expressing Cx32-WT may be due the time required by Cx32-CT-Myr, which is delivered randomly to the cell surface, to arrive near the connexons or plaques as compared with when Cx32-CT is available as a part of the connexon itself.…”

“…In this regard it is noteworthy to mention that in contrast to ubiquitously expressed Cx43, only a few proteins have been shown to interact with the cytoplasmic tail of Cx32. These proteins included SAP97/Dlg (44,45), calmodulin (56,57), ZO-1 (58), caveolin-1 (59), and occludin (60). However, in LNCaP and BxPC3 cells, none of these proteins except caveolin-1 co-localized with Cx32 as assessed by immunocytochemical analysis, 3 and hence their involvement in regulating the assembly of Cx32 into gap junctions is less likely.…”

Tyrosine phosphatase TC-PTP directly interacts with connexin43 to regulate gap junction intercellular communication

“…Cells were scrape-loaded as described previously (Stauch et al, 2012). Briefly, LA-25 cells seeded on coverslips were transfected with TC-PTP 1-314 or pcDNA3.1 vector (mock group) for 24 h at 37˚C and then incubated at 40˚C for 1 h before the addition of the MAPK inhibitor (U0126, 50 mM) or PKC inhibitor (BIM, 0.1 mM) at 40˚C for another 30 min.…”

“…The result was confirmed by repeating the experiment three times and, for each trial, four side-by-side images were captured. The method to quantify dye transfer was as described previously (Stauch et al, 2012). Briefly, cells containing Lucifer Yellow were counted, and the cells with dextran, which indicated the initially loaded cells, were excluded from the count.…”