Abstract
Two-pore channels (TPC) are intracellular endo-lysosomal proteins with only recently emerging roles in organellar signalling and involvement in severe human diseases. Here, we investigated the functional properties of human TPC1 expressed in TPC-free vacuoles from Arabidopsis thaliana cells. Large (20 pA/pF) TPC1 currents were elicited by cytosolic addition of the phosphoinositide phosphatidylinositol-(3,5)-bisphosphate (PI(3,5)P2) with an apparent binding constant of ~15 nM. The channel is voltage-dependent, activating at positive potentials with single exponential kinetics and currents are Na+ selective, with measurable but low permeability to Ca2+. Cytosolic Ca2+ modulated hTPC1 in dual way: low μM cytosolic Ca2+ increased activity by shifting the open probability towards negative voltages and by accelerating the time course of activation. This mechanism was well-described by an allosteric model. Higher levels of cytosolic Ca2+ induced a voltage-dependent decrease of the currents compatible with Ca2+ binding in the permeation pore. Conversely, an increase in luminal Ca2+ decreased hTPC1 activity. Our data point to a process in which Ca2+ permeation in hTPC1 has a positive feedback on channel activity while Na+ acts as a negative regulator. We speculate that the peculiar Ca2+ and Na+ dependence are key for the physiological roles of the channel in organellar homeostasis and signalling.
Highlights
The endolysosomal system is composed of a series of internal compartments fundamental for cellular homeostasis and is involved in a variety of different physiological processes from signaling to cell growth up to defence mechanisms, to cite only few[1,2]
PI(3,5)P2-evoked currents were observed in Human TPC1 (hTPC1)-Enhanced Green Fluorescent Protein (EGFP) expressing vacuoles but not in untransformed control vacuoles (Fig. 1d), strongly suggesting that PI(3,5)P2-responses are due to activation of hTPC1 channels
Bath application of 100 nM nicotinic acid adenine dinucleotide phosphate (NAADP) on PI(3,5)P2 responding vacuoles did not elicit an increase of membrane current (Fig. 1d); these data point to a direct interaction of PI(3,5)P2 but not of NAADP with hTPC1
Summary
The endolysosomal system is composed of a series of internal compartments fundamental for cellular homeostasis and is involved in a variety of different physiological processes from signaling to cell growth up to defence mechanisms, to cite only few[1,2]. The major problem in the functional characterization of these proteins is represented by their intracellular localization This renders very difficult the application of electrophysiological techniques because of the sub-micrometric dimension of animal endosomes and lysosomes. The vacuole has its cytosolic side facing the external bath solution, which is an ideal experimental situation to investigate cytosolic modulators[35] Using this system we have previously found that hTPC2 is insensitive to NAADP, activated by PI(3,5)P2, and highly Na+ selective[9], in agreement with Wang et al.[11]. We found that both cytosolic and luminal Ca2+ are powerful modulators of hTPC1
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