Studies on cell death and survival signals via connexin channels during cell injury

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Gap junctions are considered to play an important role in moderating cell death and survival. Genetic approaches to the connexins (Cxs) such as gene knockouts and RNA interference, which ultimately reduce Cx levels in the entire cell, have provided direct evidence that gap junctional intercellular communication is essential for cell survival. However, it is possible that one kind of Cx performs different functions depending on its location and/or a particular stage of temporal events. Such spatiotemporal differences in protein function are difficult to analyze with conventional genetic approaches. In this study, we have developed multiphoton excitation-evoked chromophore-assisted laser inactivation (MP-CALI),that specific gap junctions made of Cx-enhanced green fluorescent protein(EGFP)can be spatiotemporally inactivated by brief laser irradiation. Experiments were performed on a pair of HeLa cells connected via a small gap junction plaque (length, <1 μm)consisting of Cx43-EGFP. While … More junctional currents were measured using the dual whole-cell voltage-clamp -method, a single point of the Cx43-EGFP plaque was irradiated with 850-nm femtosecond laser light with a laser irradiance of 2.7 MW/cm^2for 380 ms. We found that during the laser irradiation, the gap junction current decreased "○80%. However the same irradiation did not change gap junctional currents between HeLa cell pairs expressing Cx43 tagged with monomeric red fluorescent protein(mRFP),indicating that MP-CALI at a wavelength of 850 nm specifically inactivates gap junctions made of Cx-EGFP. We also found that MPCALI can inhibit dye coupling between specifically selected cell pairs. MP-CALI of Cx43-EGFP showed a laser power dependent decrease of gap junction current i.e., the threshold laser power for current reduction is between 1.0 and 1.3 MW/cm2. Reactive oxygen species (ROS) scavengers, edaravone and sodium azide, impaired the MP-CALI response, indicating involvement of ROS in MP-CALI. Thus, MP-CALI could be useful for the elucidation of new Cx functions. Less