| The definitive actions of 8-br-cAMP on connexin trafficking in a mouse mammary tumor cell line, MMT22, have not been fully characterized. In the current study, connexin/fluorescent fusion proteins (utilizing Cx26, Cx32, and Cx43 fused to EGFP or RFP) were assembled to investigate the reaction of several connexins to increased levels of 8-br-cAMP. The second messenger, cAMP, has been postulated to pass through and regulate gap junctions, and to be important in the control of gap functional permeability. In a number of instances, an increase in cAMP has been correlated with an increase in functional permeability. It has been suggested cAMP promotes increases in gap functional permeability by altering intracellular distributions of connexin, increasing the population of connexin at plasma membrane gap junction plaques while decreasing cytoplasmic pools. In the present study, transient transfection of connexin/fluorescent protein fusion proteins and subsequent fluorescent microscopy enabled characterization of the ability of MMT22 cells to biosynthesize full-length fusion proteins. Fusion protein expression was observed with Cx26/EGFP, while Cx32/EGFP and Cx43/RFP expression were not observed. Indirect immunofluorescence and fluorescent microscopy were utilized to determine Cx26/EGFP partially co-localized with endogenous Cx43, in the absence and presence of 8-br-cAMP. A 300μM concentration was sufficient for alteration of the intracellular distribution of Cx43, whereas a 600μM concentration was needed for Cx26/EGFP. Transient transfection and fluorescence microscopy also were employed to examine the ability of Cx26/EGFP to be biosynthesized by a connexin-deficient, communication-incompetent cell line, MCF7. The ability of the Cx26/EGFP fusion protein to traffic to cell-cell interfaces and assemble into functional gap junctions was demonstrated. The results of the current study supported the hypothesis that introducing the connexin/fluorescent protein fusion proteins into MMT22 cells enabled the identification of the gap functional responses stimulated by 8-br-cAMP, and showed they react in a manner similar to endogenous Cx43 to 8-br-cAMP. |
