The spatiotemporal relationship between local Ca2+ signaling and P2X2R-activated membrane blebbing

Abstract

Mammalian P2X receptors (P2XRs), a family of seven ionotropic purinergic receptors, function as ion channels modulating diverse cellular processes such as secretion, apoptosis and proliferation in response to extracellular ATP. Previously, it was shown that upon ATP stimulus, the P2X7 receptor (a member of P2XR family) triggers plasma membrane (PM) blebbing in HEK293 cells. In this study, we demonstrate that this phenomenon extends to another member of the P2XR family–P2X2 receptor (P2X2R). Similar to P2X7 receptor, P2X2R blebbing is dependent on Ca2+–calmodulin and ROCK-I. To elucidate the spatiotemporal relationship between Ca2+ signaling and blebbing, protein biosensors and switches were used to image and generate Ca2+ signals, respectively, while observing PM blebbing in cells. Blebbing cannot be initiated by Ca2+ influx from the endoplasmic reticulum or by Ca2+ transport across the PM by other Ca2+ channels. To trigger blebbing, it is necessary for Ca2+ to enter specifically through the P2X2R. Lastly, a local Ca2+ signal near a fragment that encodes the intracellular P2X2R C-terminus tail is sufficient to trigger blebbing.