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G. P2XA. CnrF Rab GAP activity to Rab11a is enhanced by the presence of calcium and the EF-hand domain. 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. == Introduction == Regulation of intracellular vesicle traffic is fundamental for normal cell function and its mis-regulation is associated with congenital developmental disorders, cancer and neurological dysfunction1. Studies of vesicle traffic in different systems have revealed the evolutionarily conserved role played by Rab GTPases. Every organelle of both the endocytic and exocytic pathways expresses several Rab GTPases, which must be sequentially activated to allow precise delivery, docking and fusion of different membrane compartments2-6. Another regulator of vesicle fusion events is intracellular calcium. Transient and localized increases in calcium have been shown to facilitate some, but not all, vesicle fusion events7-19. However, it is currently unknown whether interplay between calcium and Rab GTPases, could coordinately regulate vesicle fusion. TheDictyostelium discoideumcontractile vacuole (CV) system is an intracellular vesicle required for osmoregulation. The CV cycle is a highly regulated process, orchestrated by Rab proteins, their regulators and their effectors20-27. The contractile vacuole is also an acidic calcium store (acidocalcisome)28, and therefore provides an excellent model system to study the coordinated regulation of vesicle trafficking by Rab proteins and calcium. Upon hypo-osmotic shock, water enters tubules of the CV system, a process accompanied by activation of Rab11a which is localized to CV membranes22,25. Drainin, a putative volume-sensing Rab11a-GTP binding protein, is subsequently recruited to maturing vacuoles20,22,29,30. Next, vacuoles are prepared for fusion with the plasma membrane through the recruitment of the Rab GAP, Disgorgin, and Rab8a22,23. Once tethered to the plasma membrane, the non-polarised CV becomes polarised and committed to pore formation23. This process is defined by a ring to patch transition, in which different proteins become concentrated at the front or back of the CV23. How the correct spatial and temporal regulation of these Rab proteins is achieved is poorly understood. Recently, we discovered that a homologue of mammalian P2X receptors, P2XA, is exclusively localized to theDictyosteliumCV system31. P2X receptors are calcium-permeable ion channels gated by ATP which function in diverse physiological processes32,33. However, the intracellular localization of P2XA inDictyosteliumcells is inconsistent with a role in regulating responses to extracellular ATP31. Instead, P2XA knockout cells exhibit defects in responses to hypo-osmotic shock31,34(Supplementary Figure 1). Under hypo-osmotic shock, knockout cells become rounded and the rate of CV discharge is much reduced31,34. This raises the possibility that P2XA may be an important conduit for vacuolar calcium release, and that this calcium is required for the correct regulation of vacuolar cycling35. However, major questions remain unanswered. Firstly, it is unknown whether the intracellular function of P2XA indeed requires ion channel activity and calcium flux. Secondly, it is unknown whether disruption of P2XA activity, and therefore calcium flux, affects vesicle fusion or some other event in the CV cycle, such as maturation or delivery. Finally, it is unknown how an ATP-gated ion channel could regulate a Rab-GTP dependent process in a calcium-dependent manner at the molecular level. == Results == == Intracellular P2XA function requires ion channel activity == To determine if P2XA ion channel activity is required for osmoregulation, mutations were generated that resulted in inactive (K67A/K289A), less active (R285K), or hyperactive (R63A) versions of P2XA when tested in HEK293 cells (Figure Momordin Ic 1A and B). Next, gene replacementDictyosteliumstrains were generated in which the endogenous gene was replaced with wild-type or point mutated versions and tested for osmotic shock defects. In each case mutated receptors P4HB still localized to the contractile vacuole (Supplementary figure 2). However, the gene replacement strains exhibited clear differences in their ability to respond to osmotic shock, with responsiveness correlating very well with ion channel activity (Figure 1 C and D). == Figure 1. P2XA mutants with altered ATP sensitivity show defects in osmoregulation. == A. Currents evoked by ATP (10 M – 3 mM) in HEK cells expressing wild type (WT) or mutated P2XA receptors. Each Momordin Ic panel shows superimposed current traces for the concentrations indicated. ATP application was 2 s (black bar). B. Concentration-response curves for wild type (black), R285K (blue), R63A (purple) and K67A/K289A (green) receptors. Error bars represent s.e.m. of responses from wild type (8 cells), R285K (4 cells), R63A (4 cells) and K67A/K289A (3 cells). Compared Momordin Ic to WTP2XA (black), concentration response curves for R63A (purple) are shifted left (p<0.001 in a tukey test), whilst R285K (blue) is shifted right (p<0.001 in a tukey test). K67A/K289A (green) is non-functional (p<0.0001 in a tukey test). Statistical source data for Fig 1B can be found inSupplementary Table 2. C. Bright-field.