Reconstituted human TPC1 is a proton-permeable ion channel and is activated by NAADP or Ca2+.
NAADP potently triggers Ca2+ release from acidic lysosomal and endolysosomal Ca2+ stores. Human two-pore channels (TPC1 and TPC2), which are located on these stores, are involved in this process, but there is controversy over whether TPC1 and TPC2 constitute the Ca2+ release channels. We therefore examined the single-channel properties of human TPC1 after reconstitution into bilayers of controlled composition. We found that TPC1 was permeable not only to Ca2+ but also to monovalent cations and that permeability to protons was the highest (relative permeability sequence: H+ >> K+ > Na(+) ≥ Ca2+). NAADP or Ca2+ activated TPC1, and the presence of one of these ligands was required for channel activation. The endolysosome-located lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] had no effect on TPC1 open probability but significantly increased the relative permeability of Na+ to Ca2+ and of H+ to Ca2+. Furthermore, our data showed that, although both TPC1 and TPC2 are stimulated by NAADP, these channels differ in ion selectivity and modulation by Ca2+ and pH. We propose that NAADP triggers H+ release from lysosomes and endolysomes through activation of TPC1, but that the Ca2+ -releasing ability of TPC1 will depend on the ionic composition of the acidic stores and may be influenced by other regulators that affect TPC1 ion permeation.
School of Medicine, University of St Andrews, St Andrews KY16 9TF, UK.
Samantha J
SJ
Pitt
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK.
Andy K M
AK
Lam
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK.
Katja
K
Rietdorf
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK.
Antony
A
Galione
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK. rebecca.sitsapesan@pharm.ox.ac.uk.
Rebecca
R
Sitsapesan
D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport / Q000502:physiology; D008247:Lysosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010539:Permeability; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011522:Protons; D000097903:Two-Pore Channels
D002118:Calcium; D015220:Calcium Channels; D009249:NADP; D018129:Phosphatidylinositol Phosphates; D011522:Protons; C407357:TPCN1 protein, human; D000097903:Two-Pore Channels; C106336:phosphatidylinositol 3,5-diphosphate; C024376:NAADP