NHERF mediated associations with actin cytoskeleton are involved in activation and regulation of TRPC4 channel

Receptor-stimulated activation of phospholipase C is accompanied with the release of Ca2+ from internal stores and the influx of Ca2+ from extracellular space. Ca2+ influx is mediated by store-operated channels (SOCs), which are thought to become activated when the internal Ca2+ stores are emptied. However, the exact mechanism that regulates the activity of the SOCs is not well understood. A secretion-like coupling mechanism involving actin-mediated insertion of new channels onto plasma membrane has been proposed for SOC activation. Transient receptor potential canonical proteins (TRPC) are postulated to form SOC. My research focuses on examining the secretion-like coupling hypothesis using murine TRPC4 as an example. In in vitro binding studies I showed that TRPC4 as well as phospholipase C (PLC) interacts with the first PDZ domain of Na+/H+ exchanger regulatory factor (NHERF) through C-terminal T/(S)XL motif. NHERF is known to interact with actin-binding ezrin-radixin-moesin (ERM) proteins. Using HEK-293 cell lines stably expressing mouse TRPC4, I showed by coimmunoprecipitation that wild type but not the C-terminal TRL motif deleted mutant TRPC4 is associated with ERM proteins and with actin. Stimulation of PLC by a muscarinic receptor agonist, carbachol, enhanced the association of TRPC4 with ERMs and with actin by 100%. Using immunofluorescence labeling and western blotting analysis of externally epitope-tagged TRPC4, I showed that there is about 2-fold increase in the amount of TRPC4 on the cell surface after the carbachol stimulation. Therefore, TRPC4 can form signaling complex with PLC via interactions with NHERF, which in turn links the lipase and the channel to the actin cytoskeleton. Furthermore, TRPC4 is trafficked and inserted into the plasma membrane through regulated association with actin and ERMs upon activation of PLC. My data provide the first physical evidence for the secretion-like coupling hypothesis.