Figure 4.
Detection of ATP influx through a single Cx43 hemichannel by luciferace/luciferine. A, An inside-out patch (DIC, p1) was excised from Cx43-expressing C6 cells. The pipette solution contained 130 mm Na2ATP. A baseline image was first obtained before the patch electrode was moved close to the puff pipette (DIC, p2) containing luciferase and luciferin (K salt, 1:3). A low pressure was applied to the puff pipette to eject the enzyme solution. When a voltage step of +80 mV (intracellular) was applied to the patch, channel openings were observed under voltage-clamp of the inside-out patch (traces over the images). Simultaneous imaging showed that light intensity increased with the channel activity. Channel openings declined after a few minutes for unknown reasons. B, A control inside-out patch without channel activity showed no light increase. C, The relationship between the increase in light intensity (ΔF/F, filled circles; F is the baseline) and the channel open probability (Po, filled squares) with patches containing channels. No light was emitted when the patch contained no active channels (open circles). The termination of channel activity preceded the termination of light increase, which reflects that flow of the solution takes time to remove released ATP. n = 6 and 11 patches for the channel and no channel groups, respectively. Error bars indicate SEM.