ATP is central to energy production in cells, but even though ATP was also shown to be released from cells in the 1950s, our knowledge of its extracellular roles has subsequently progressed slowly. Receptors for ATP of the ion channel (P2X; seven subunits) and G protein-coupled (P2Y; eight receptors) types were identified in the early 1990s. In mammals, the extracellular signalling physiology of nucleotides at P2X receptors is being worked out. Key roles are in taste sensation, bladder emptying, oxygen sensing, inflammation and pain.
Ion channels are attractive drug targets and clinical trials are currently in progress to test the therapeutic value of antagonists at P2X receptors. Despite this, relative to other ion channel proteins, we know very little of the molecular structure, interacting partners, membrane trafficking, and physiological roles of P2X receptors. Our research seeks to redress such deficits.
Our approach is stimulated by a recent and surprising finding that the genome of the social amoeba Dictyostelium discoideum contains five sequences that are weakly related (10-15% sequence identity) to vertebrate P2X receptors (Figure 1). Alignments reveal the presence of some key residues, but complete absence of others. It was therefore difficult to be sure if any of these predicted proteins functioned as ATP gated ion channels. We therefore tested this idea for one sequence, now named DdP2XA.
Several lines of evidence showed that DdP2XA encodes a ligand-gated ion channel in which key structure/function relationships were conserved with vertebrate P2X receptors. When DdP2XA was expressed in HEK293 cells, exogenous ATP resulted in robust inward currents. Mutation of several key residues thought to be required for P
2X receptor function, strongly reduced these responses. DdP2XA was localized to the plasma membrane of HEK293 cells, and DdP2XA formed a trimer under non-denaturing gel conditions (Figure 1).
We were therefore surprised to discover that DdP2XA-GFP is not found at the cell surface, but is instead concentrated on the contractile vacuole, an intracellular organelle required for responses to osmotic shock. Consistent with its localization, P2XA null mutant cells showed impaired response to hypotonic shock (Figure 2).
Recently, we have discovered that activation of P2XA, results in calcium efflux required for downregulation of Rab11a activity and efficient vacuole fusion. Rab GTPases play key roles in the delivery, docking and fusion of different intracellular vesicles. However, the mechanism by which spatial and temporal regulation of Rab GTPase
activity is controlled is poorly understood. Our findings suggest a novel mechanism by which localized calcium release through a vesicular ion channel controls Rab GTPase activity. Furthermore, we have shown that vacuole fusion and Rab11a downregulation require the activity of CnrF, a novel EF hand containing Rab GAP protein found in a complex with Rab11a and P2XA. CnrF exhibits Rab GAP activity to Rab11a, which is greatly enhanced by the presence of calcium and the EF-hand domain in vivo and in vitro. These findings suggest that P2XA activation results in vacuolar calcium release, which triggers activation of CnrF Rab GAP activity and subsequent downregulation of Rab11a to allow vacuole fusion. Given that P2X channels and this novel class of calcium dependent Rab GAPs are widely conserved, this work provides fundamental insights into Rab GTPase regulation in vesicular trafficking.
Currently, we are employing genetic, biochemical and cell biological approaches to further define the role of Dictyostelium P2X genes and to characterize the factors that regulate P2X receptor function in Dictyostelium and mammalian cells.