title: The human two-pore channel 1 is modulated by cytosolic and luminal calcium.
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.
authors.affiliation: Institute of Biophysics, National Research Council, Via De Marini 6, 16149 Genova, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Lagostena
authors.affiliation: Department of Biosciences, University of Milano, Via Celoria, 26, 20133 Milano, Italy.
authors.forename: Margherita
authors.initials: M
authors.lastname: Festa
authors.affiliation: Institute of Biophysics, National Research Council, Via De Marini 6, 16149 Genova, Italy.
authors.forename: Michael
authors.initials: M
authors.lastname: Pusch
authors.affiliation: Institute of Biophysics, National Research Council, Via De Marini 6, 16149 Genova, Italy.
authors.forename: Armando
authors.initials: A
authors.lastname: Carpaneto
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008564:Membrane Potentials; D018408:Patch-Clamp Techniques; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D000097903:Two-Pore Channels
chemical_list: D002118:Calcium; D015220:Calcium Channels; D018129:Phosphatidylinositol Phosphates; D011994:Recombinant Proteins; D012964:Sodium; C407357:TPCN1 protein, human; D000097903:Two-Pore Channels; C106336:phosphatidylinositol 3,5-diphosphate