Calcium-dependent regulation of Rab activation and vesicle fusion by an intracellular P2X ion channel

Abstract

Rab GTPases play key roles in the delivery, docking and fusion of intracellular vesicles. However, the mechanism by which spatial and temporal regulation of Rab GTPase activity is controlled is poorly understood. Here we describe a mechanism by which localized calcium release through a vesicular ion channel controls Rab GTPase activity. We show that activation of P2XA, an intracellular ion channel localized to the Dictyostelium discoideum contractile vacuole system, results in calcium efflux required for downregulation of Rab11a activity and efficient vacuole fusion. Vacuole fusion and Rab11a downregulation require the activity of CnrF, an EF hand containing Rab GAP found in a complex with Rab11a and 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.

The Two-pore channel (TPC) interactome unmasks isoform-specific roles for TPCs in endolysosomal morphology and cell pigmentation

“…Phosphoinositides regulate TPC activation and recruitment of trafficking effectors (10,49,50). Localized ion fluxes triggered on TPC activation may support fusogenic events (10): Fusion of endosomes and lysosomes in vitro requires a Ca 2+ flux downstream of Rab activity (51), and reciprocally Ca 2+ fluxes regulate Rab cycling (52). Although each regulator acts as a discrete component in the endolysosomal trafficking machinery, demonstration that this triumvirate of regulators converges upon a single molecular entity (TPCs) highlights the important cellular role of these ancient channel complexes in endolysosomal trafficking.…”

Inhibition of Transient Receptor Potential Channel Mucolipin-1 (TRPML1) by Lysosomal Adenosine Involved in Severe Combined Immunodeficiency Diseases

“…Because TRPML1 acts as a Ca 2ϩ -permeable channel (31,32), to further evaluate the inhibitory effect of adenosine, TRPML1-4A activity was measured by a Fura-2-based Ca 2ϩ imaging assay (29,32). Consistent with another report (29), activation of TRPML1-4A by ML-SA1 induced a rapid increase in Fura-2 signal followed by a quick rundown.…”

An organelle K+ channel is required for osmoregulation in Chlamydomonas reinhardtii

“…During diastole of the contractile vacuole cycle, liquid-filled vesicles continue to fuse with the contractile vacuole Komsic-Buchmann et al, 2012;Luykx et al, 1997;Parkinson et al, 2014). Vesicle fusion requires the function of the exocyst because contractile vacuole formation is compromised in a sec6 mutant in C. reinhardtii (Komsic-Buchmann et al, 2012).…”

“…Vesicle fusion requires the function of the exocyst because contractile vacuole formation is compromised in a sec6 mutant in C. reinhardtii (Komsic-Buchmann et al, 2012). In D. discoideum, an ATP-sensitive P2X receptor ion channel that is localized to the contractile vacuole regulates organellar Ca 2+ homeostasis and vesicle fusion during contractile vacuole formation (Fountain et al, 2007;Parkinson et al, 2014). However, vesicle fusion alone would be expected to result in a flaccid vacuole, and additional transport of water into contractile vacuole driven by osmosis is therefore required.…”