Quantifying Gap Junction Permeability

Parental (untransfected) HeLa cells and HeLa cells stably transfected with wild-type Cx50, Cx50R33E, Cx50E162R or Cx50R33E,E162R were grown on glass coverslips until they reached 90‒95% confluence. Then, one cell within a cluster was microinjected with a solution containing 9% Neurobiotin (charge: + 1; MW: 287.2; Vector Laboratories, Burlingame, CA, USA) and 5% Lucifer yellow (charge − 2; MW: 444.4; Sigma-Aldrich, St. Louis, MO, USA) for 1 min using a picospritzer (model PLI-188; Nikon Instruments Inc., Melville, NY, USA) [63 (link)]. After allowing the microinjected gap junction tracers to diffuse to neighboring cells for 10 min, cells were fixed in 4% paraformaldehyde for 15 min and incubated with Cy3-streptavidin conjugate (Sigma-Aldrich) to detect Neurobiotin by fluorescence microscopy [63 (link)]. Lucifer yellow facilitated identification of the injected cell, because human Cx50 has limited permeability to this dye and did not spread [64 (link)]. The extent of Neurobiotin intercellular transfer was determined by counting the number of adjacent cells containing the tracer. The number of microinjections ranged from 10 to 24 for cells expressing the different constructs. Data are presented as mean ± S.E.M. Statistical analysis was performed using Student’s t-test. Graphs were generated in SigmaPlot 10 (Systat Software, Inc., Palo Alto, CA, USA).