A method to determine the relative cAMP permeability of connexin channels.
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 Ca2+ concentration, which was monitored by Ca2+ 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 Ca2+ concentration in the neighboring cell. We developed a standardization procedure for the Ca2+ 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.
Institut für Genetik, Abt. Molekulargenetik, Universität Bonn, Römerstrasse 164, 53117 Bonn, Germany.
Peter
P
Bedner
Heiner
H
Niessen
Benjamin
B
Odermatt
Klaus
K
Willecke
Hartmann
H
Harz
D001681:Biological Assay / Q000379:methods; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002450:Cell Communication / Q000187:drug effects / Q000235:genetics; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000235:genetics; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels; D004058:Diffusion / Q000187:drug effects; D017629:Gap Junctions / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006651:Histocytochemistry / Q000379:methods; D006801:Humans; D007473:Ion Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D018408:Patch-Clamp Techniques / Q000379:methods; D014162:Transfection
D017630:Connexins; D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; C082920:connexin 45; D000242:Cyclic AMP; D002118:Calcium