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L. Ebihara, L. Dong; Structural Basis for Differences in Fluorescent Dye Transfer Through Gap Junctional Channels Formed From the Lens Gap Junctional Proteins, Cx43 and Chicken Cx45.6 . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5420.
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Gap junctional channels formed from the lens connexins, chicken Cx45.6 and Cx43, show differences in voltage gating, unitary conductance and molecular permeability. We have previously shown that the unitary conductance of Cx45.6 is 202 pS, which is around two times higher than Cx43 (100 pS). To further characterize the permeability properties of these channels, we used anionic fluorescent dyes to examine quantitative differences in dye transfer between pairs of HeLa cells stably transfected with Cx45.6, Cx43 or a chimeric gap junctional protein,Cx45.6*43NT.
A series of constructs including chicken Cx45.6, Cx43 and a Cx45.6 chimera containing the N–terminus of Cx43 (Cx45.6*43NT) were expressed in HeLa cells. Protein expression was studied by immunofluorescence. The dual whole cell recording technique was used to measure gap junctional conductance. Dye transfer over time was monitored by fluorescence microscopy.
Our results showed that Cx45.6 gap junctional channels transfered the negatively charged dye, Lucifer Yellow (LY), much more slowly than Cx43 or Cx45.6*43NT. To investigate the mechanism for the difference in LY permeability, we examined the ability of the channels to transfer three Alexa dyes that have similar charge and molecular structure but range in size from MW 350 to 760. Our results showed that Cx45.6*43NT and wild–type Cx43 were permeable to all 3 dyes. However, Cx45.6 gap junctional channels were permeable only to the smallest dye, Alexa 350.
These results suggest that Cx45.6 gap junctional channels have a smaller size exclusion limit than Cx43 channels and that the N–terminus may a major determinant for this difference.
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