Gap-junctional single-channel permeability for fluorescent tracers in mammalian cell cultures.
We have developed a simple dye transfer method that allows quantification of the gap-junction permeability of small cultured cells. Fluorescent dyes (calcein and Lucifer yellow) were perfused into one cell of an isolated cell pair using a patch-type micropipette in the tight-seal whole cell configuration. Dye spreading into the neighboring cells was monitored using a low-light charge-coupled device camera. Permeation rates for calcein and Lucifer yellow were then estimated by fitting the time course of the fluorescence intensities in both cells. For curve fitting, we used a set of model equations derived from a compartment model of dye distribution. The permeation rates were correlated to the total ionic conductance of the gap junction measured immediately after the perfusion experiment. Assuming that dye permeation is through a unit-conductance channel, we were then able to calculate the single-channel permeance for each tracer dye. We have applied this technique to HeLa cells stably transfected with rat-Cx46 and Cx43, and to BICR/M1R(k) cells, a rat mammary tumor cell line that has very high dye coupling through endogenous Cx43 channels. Scatter plots of permeation rates versus junctional conductance did not show a strictly linear correlation of ionic versus dye permeance, as would have been expected for a simple pore. Instead, we found that the data scatter within a wide range of different single-channel permeances. In BICR/M1R(k) cells, the lower limiting single-channel permeance is 2.2 +/- 2.0 x 10(-12) mm3/s and the upper limit is 50 x 10(-12) mm3/s for calcein and 6.8 +/- 2.8 x 10(-12) mm3/s and 150 x 10(-12) mm3/s for Lucifer yellow, respectively. In HeLa-Cx43 transfectants we found 2.0 +/- 2.4 x 10(-12) mm3/s and 95 x 10(-12) mm3/s for calcein and 2.1 +/- 6.8 x 10(-12) mm3/s and 80 x 10(-12) mm3/s for Lucifer yellow, and in HeLa-Cx46 transfectants 1.7 +/- 0.3 x 10(-12) mm3/s and 120 x 10(-12) mm3/s for calcein and 1.3 +/- 1.1 x 10(-12) mm3/s and 34 x 10(-12) mm3/s for Lucifer yellow, respectively. This variability is most likely due to a yet unknown mechanism that differentially regulates single-channel permeability for larger molecules and for small inorganic ions.
Eckert
Reiner
R
Abteilung Biophysik, Biologisches Institut, Universität Stuttgart, Stuttgart, Germany. reiner.eckert@bio.uni-stuttgart.de
D000818:Animals; D001692:Biological Transport; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D018031:Connexin 43 / Q000502:physiology*; D017630:Connexins / Q000502:physiology*; D005452:Fluoresceins / Q000737:chemistry; D017629:Gap Junctions / Q000502:physiology*; D006801:Humans; D007546:Isoquinolines / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D018031:Connexin 43; D017630:Connexins; D005452:Fluoresceins; C487370:Gja3 protein, rat; D007546:Isoquinolines; D011994:Recombinant Proteins; C017475:lucifer yellow; C007740:fluorexon