Regulation of connexin43 gap junctional communication by phosphatidylinositol 4,5-bisphosphate

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]P₂) from the plasma membrane. Knockdown of phospholipase Cβ3 (PLCβ3) 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 find that the Cx43 binding partner zona occludens 1 (ZO-1) interacts with PLCβ3 via its third PDZ domain. ZO-1 is essential for PtdIns(4,5)P₂-hydrolyzing receptors to inhibit cell–cell communication, but not for receptor–PLC coupling. Our results show that PtdIns(4,5)P₂ is a key regulator of Cx43 channel function, with no role for other second messengers, and suggest that ZO-1 assembles PLCβ3 and Cx43 into a signaling complex to allow regulation of cell–cell communication by localized changes in PtdIns(4,5)P₂.