Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca2+ waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca2+]i associated with Ca2+waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca2+-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca2+ waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca2+]i changes were characterized by initiating Ca2+ puffs associated with the perinuclear ER. By contrast, in Cx–GFP-transfected cells and in the presence of apyrase, [Ca2+]i changes were propagated without initiating perinuclear Ca2+ puffs and were communicated between cells at the sites of the Cx–GFP gap junctions. The efficiency of Cx expression determined the extent of Ca2+ wave propagation. These results demonstrate that intercellular Ca2+ waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other