The human two-pore channel 1 is modulated by cytosolic and luminal calcium.

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.

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