Phospholipid Minds the Gap (Junction)

Abstract

Gap junction channels allow communication in the form of small molecules that flow between adjacent cells. Conductance through gap junctions formed of the connexin43 (Cx43) protein is decreased after stimulation of G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) at the cell surface, but the actual mechanism of channel regulation has been unclear. van Zeijl et al . report that in Rat-1 fibroblasts, one way gap junction conductance can be regulated is through changes in the abundance of the phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ]. The authors confirmed decreased conductance of gap junctions formed from Cx43 in cells transfected with an active form of the G protein alpha subunit Gα q . Gα q activates phospholipase C-β (PLC-β), which catalyzes hydrolysis of PtdIns(4,5)P 2 , forming the second messengers inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol, and experiments with a fluorescently labeled probe containing a pleckstrin homology domain that binds PtdIns(4,5)P 2 revealed that PtdIns(4,5)P 2 was depleted from the plasma membrane in cells expressing the active Gα q protein. Depletion of PLC-β3 with RNAi prevented depletion of PtdIns(4,5)P 2 from the plasma membrane in cells treated with GPCR agonists. The communication with the gap junction appeared to require PtdIns(4,5)P 2 itself, rather than IP 3 or DAG, and channel inhibition could be reproduced by depletion of PtdIns(4,5)P 2 with an engineered version of phosphoinositide 5-phosphatase targeted to the plasma membrane. Similarly, overexpression of PtdIns(4)P 5-kinase [to maintain the abundance of PtdIns(4,5)P 2 at the membrane] also prevented receptor-mediated decreases in gap junctional communication. Immunoprecipitation experiments showed that PLC-β3 associated with the scaffold protein ZO-1 (zona occludens 1), and depletion of ZO-1 with RNAi also reduced receptor-mediated regulation of gap junction channels. Thus, the authors propose that ZO-1 helps assemble complexes containing PLC-β3 and Cx43. It’s not altered second messenger production that shuts down the channel but rather the action of PLC-β3 to deplete membrane concentrations of PtdIns(4,5)P 2 , which appears to directly modulate conductance of gap junctions as it does other ion channels and transporters. L. van Zeijl, B. Ponsioen, B. N. G. Giepmans, A. Ariaens, F. R. Postma , P. Várnai, T. Balla, N. Divecha, K. Jalink, W. H. Moolenaar, Regulation of connexin43 gap junctional communication by phosphatidylinositol 4,5-bisphosphate. J. Cell Biol. 177 , 881-891 (2007). [Abstract] [Full Text]