Abstract
ABSTRACTATP and ADP are ancient extra-cellular signalling molecules that in Dictyostelium amoebae cause rapid, transient increases in cytosolic calcium due to an influx through the plasma membrane. This response is independent of hetero-trimeric G-proteins, the putative IP3 receptor IplA and all P2X channels. We show, unexpectedly, that it is abolished in mutants of the polycystin-type transient receptor potential channel, TrpP. Responses to the chemoattractants cyclic-AMP and folic acid are unaffected in TrpP mutants. We report that the DIF morphogens, cyclic-di-GMP, GABA, glutamate and adenosine all induce strong cytoplasmic calcium responses, likewise independently of TrpP. Thus, TrpP is dedicated to purinergic signalling. ATP treatment causes cell blebbing within seconds but this does not require TrpP, implicating a separate purinergic receptor. We could detect no effect of ATP on chemotaxis and TrpP mutants grow, chemotax and develop almost normally in standard conditions. No gating ligand is known for the human homologue of TrpP, polycystin-2, which causes polycystic kidney disease. Our results now show that TrpP mediates purinergic signalling in Dictyostelium and is directly or indirectly gated by ATP.
Highlights
ATP and other purines are ancient signalling molecules used widely in animals as neuro-transmitters and by protozoa and plants for diverse purposes (Burnstock and Verkhratsky, 2009)
We found that stimulation with a uniform concentration of cyclic AMP (cAMP) causes a transient increase in cytosolic Ca2+ levels after a delay of 6.0±1.0 s, with a peak at 22.1±4.3 s (n=7) and return to baseline by 58.1±3.2 s (n=7) (Fig. 1D)
The major advance described in this paper is the discovery that this response is mediated by the transient receptor potential (Trp) channel TrpP, since in TrpP null mutants the fast calcium response is totally abolished, yet can be restored when the protein is re-expressed
Summary
ATP and other purines are ancient signalling molecules used widely in animals as neuro-transmitters and by protozoa and plants for diverse purposes (Burnstock and Verkhratsky, 2009). Calcium signalling has ancient origins and it is likely that ancestral single-celled eukaryotes were able to produce Ca2+ gradients across their plasma membrane using calcium pumps and transporters, and activate calcium entry into the cytoplasm through regulated channels in the plasma membrane and the membranes of MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB1 0QH, UK Present day microbes use calcium signalling in a wide variety of ways and have recognizable homologues in their genomes to many components of calcium signalling found in mammalian cells (Martinac et al, 2008; Collins and Meyer, 2011; Plattner and Verkhratsky, 2015) These ancient signalling processes can be combined so that ATP causes a cytoplasmic calcium increase
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