Nature Methods2, 55 - 62 (2005)
Published online: 21 December 2004; | doi:10.1038/nmeth730
LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling
Kenneth Dakin, YuRui Zhao
& Wen-Hong Li
Departments of Cell Biology and of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390, USA.
Using a new class of photo-activatible fluorophores, we have developed a new imaging technique for measuring molecular transfer rates across gap junction connexin channels in intact living cells. This technique, named LAMP, involves local activation of a molecular fluorescent probe, NPE-HCCC2/AM, to optically label a cell. Subsequent dye transfer through gap junctions from labeled to unlabeled cells was quantified by fluorescence microscopy. Additional uncagings after prior dye transfers reached equilibrium enabled multiple measurements of dye transfer rates in the same coupled cell pair. Measurements in the same cell pair minimized variation due to differences in cell volume and number of gap junctions, allowing us to track acute changes in gap junction permeability. We applied the technique to study the regulation of gap junction coupling by intracellular Ca2+ ([Ca2+]i). Although agonist or ionomycin exposure can raise bulk [Ca2+]i to levels higher than those caused by capacitative Ca2+ influx, the LAMP assay revealed that only Ca2+ influx through the plasma membrane store-operated Ca2+ channels strongly reduced gap junction coupling. The noninvasive and quantitative nature of this imaging technique should facilitate future investigations of the dynamic regulation of gap junction communication.
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