Cell-cell communication through connexin43 (Cx43)-based gap junction channels is rapidly inhibited upon activation of various G protein coupled receptors; however, the mechanism is unknown. We show that Cx43-based cell-cell communication is inhibited by depletion of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5] P-2) from the plasma membrane. Knockdown of phospholipase C beta 3 (PLC beta 3) inhibits PtdIns(4,5) P2 hydrolysis and keeps Cx43 channels open after receptor activation. Using a translocatable 5-phosphatase, we show that PtdIns(4,5) P2 depletion is sufficient to close Cx43 channels. When PtdIns(4,5) P2 is overproduced by PtdIns(4)P 5-kinase, Cx43 channel closure is impaired. We. nd that the Cx43 binding partner zona occludens 1 (ZO-1) interacts with PLC beta 3 via its third PDZ domain. ZO-1 is essential for PtdIns(4,5) P-2-hydrolyzing receptors to inhibit cell-cell communication, but not for receptor PLC coupling. Our results show that PtdIns(4,5) P-2 is a key regulator of Cx43 channel function, with no role for other second messengers, and suggest that ZO-1 assembles PLC beta 3 and Cx43 into a signaling complex to allow regulation of cell-cell communication by localized changes in PtdIns(4,5) P2.
