A method to determine the relative cAMP permeability of connexin channels

• Published in: Experimental cell research 2003-11, Vol.291 (1), p.25-35
• Main Authors: Bedner, Peter, Niessen, Heiner, Odermatt, Benjamin, Willecke, Klaus, Harz, Hartmann
• Format: Article
• Language: English
• Subjects: Biological Assay - methods; Caged cAMP; Calcium - metabolism; Calcium imaging; Calcium Signaling - drug effects; Calcium Signaling - physiology; Cell Communication - drug effects; Cell Communication - genetics; Cell Membrane Permeability - drug effects; Cell Membrane Permeability - genetics; CNG channel; Connexin45; Connexins - genetics; Connexins - metabolism; Cyclic AMP - metabolism; Cyclic AMP - pharmacology; Cyclic Nucleotide-Gated Cation Channels; Diffusion - drug effects; Gap junctions; Gap Junctions - drug effects; Gap Junctions - genetics; Gap Junctions - metabolism; HeLa Cells; Histocytochemistry - methods; Humans; Ion Channels - drug effects; Ion Channels - genetics; Ion Channels - metabolism; Membrane Potentials - drug effects; Membrane Potentials - genetics; Patch-Clamp Techniques - methods; Transfection
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1016/S0014-4827(03)00323-9

Here we present a method by which gap junction-mediated intercellular diffusion of adenosine 3′,5′-cyclic monophosphate (cAMP) molecules can be monitored in “real-time” and the cAMP permeability of different gap junction channels can be compared. Intercellular cAMP diffusion was investigated throughout this study in human HeLa cells coexpressing murine connexin45 and cyclic nucleotide-gated (CNG) ion channels. The CNG channels were used as cAMP sensors, since CNG channel activation led to an increase of the cytosolic Ca 2+ concentration, which was monitored by Ca 2+ imaging. A cAMP gradient was generated between two contacting cells by restricting the photolysis of caged cAMP to only one cell. The intercellular diffusion of cAMP was measured by the increase in Ca 2+ concentration in the neighboring cell. We developed a standardization procedure for the Ca 2+ signal which allowed estimation of the amount of cAMP that diffused from cell to cell. The number of gap junction channels between each cell pair investigated was determined by double whole-cell patch-clamp measurements. On the basis of these data we calculated how many gap junction channels contributed to the diffusion of a certain amount of cAMP. The new method can be used to compare the selective permeabilities of different gap junction channels for cAMP and for cGMP which also activates the CNG channel.