PROPERTIES AND REGULATION OF GAP JUNCTIONAL HEMICHANNELS IN THE PLASMA-MEMBRANES OF CULTURED-CELLS

During the assembly of gap junctions, a hemichannel in the plasma memb rane of one cell is thought to align and dock with another in an appos ed membrane to form a cell-to-cell channel, We report here on the exis tence and properties of nonjunctional, plasma membrane connexin-43 (Cx 43) hemichannels. The opening of the hemichannels was demonstrated by the cellular uptake of 5(6)-carboxyfluorescein from the culture medium when extracellular calcium levels were reduced, Dye uptake exhibited properties similar to those of gap junction channels. For example, usi ng different dyes, the levels of uptake were correlated with molecular size: 5(6)-carboxyfluorescein (similar to 32%), 7-hydroxycoumarin-3-c arboxylic acid (similar to 24%), fura-2 (similar to 11%), and fluoresc ein-dextran (similar to 0.4%). Octanol and heptanol also reduced dye u ptake by similar to 50%. Detailed analysis of one clone of Novikoff ce lls transfected with a Cx43 antisense expression vector revealed a re duction in dye uptake levels according to uptake assays and a correspo nding decrease in intercellular dye transfer rates in microinjection e xperiments, In addition, a more limited decrease in membrane resistanc e upon reduction of extracellular calcium was detected in electrophysi ological studies of antisense transfectants, in contrast to control ce lls. Studies of dye uptake in HeLa cells also demonstrated a large inc rease following transfection with Cx43. Together these observations in dicate that Cx43 is responsible for the hemichannel function in these cultured cells. Similar dye uptake results were obtained with normal r at kidney (NRK) cells, which express Cx43. Dye uptake can be dramatica lly inhibited by 12-O-tetradeconylphorbol-13-acetate-activated protein kinase C in these cell systems and by a temperature-sensitive tyrosin e protein kinase, pp60(v-src) in LA25-NRK cells. We conclude that Cx43 hemichannels are found in the plasma membrane, where they are regulat ed by multiple signaling pathways, and likely represent an important s tage in gap junction assembly.