Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, C135
Two pore channels (TPC) are Ca2+-permeable, NAADP-activated ion-channels with marked sensitivity to luminal Ca2+
S. J. Pitt1, T. Funnell2, M. X. Zhu3, M. Sitsapesan1, E. Venturi1, M. Ruas2, J. Parrington2, G. Churchill2, A. Galione2, R. Sitsapesan1
1. Department of Physiology and Pharmacology, NSQI and Bristol Heart Institute, University of Bristol, Bristol, United Kingdom. 2. Department of Pharmacology, University of Oxford, Oxford, United Kingdom. 3. Department of Neuroscience, Biochemistry and Center for Molecular Neurobiology, The Ohio State University, Columbus, Ohio, United States.
Recent evidence links nicotinic acid adenine dinucleotide phosphate (NAADP)-induced mobilisation of Ca2+ from acidic organelles with two-pore domain channels (TPCs) (1), however, there has been no direct demonstration that TPCs can act as Ca2+-release channels. The hypothesis has also been challenged by the report that the skeletal isoform of the ryanodine receptor (RyR1) can be activated by NAADP (2). We have therefore reconstituted recombinant purified human TPC2 into planar phosphatidylethanolamine lipid bilayers under voltage-clamp conditions to investigate the putative single-channel properties of this protein. Using previously described techniques (3) we have compared the ability of NAADP to activate native rabbit skeletal RyR1, sheep cardiac RyR2 and purified human TPC2 channels. Students t-test was used to assess the difference between mean values. We show for the first time that TPC2 behaves as an ion-channel that is permeable to both monovalent (300±14pS; symmetrical 210 mM K+; S.D; n=3) and divalent cations (15±2pS; 10µM cis/50 mM trans Ca2+; S.D; n=5). A KCl gradient across the bilayer (210 mM trans: 510 mM cis) identified that the TPC2 reversal potential coincides with the calculated value for a channel ideally selective for cations. In all experiments, addition of trans NAADP had no effect on TPC2, but cis application induced marked channel activation (n=87) suggesting that TPC2 incorporates into the membrane in a fixed orientation with the cis chamber corresponding to the cytosolic face of the channel and the trans chamber corresponding to the lumen of the acidic stores. In symmetrical 210mM K+ and 10µM Ca2+, NAADP activated TPC2 channels with an EC50 of 500nM. Addition of 200µM luminal Ca2+ significantly increased the sensitivity of TPC2 to NAADP shifting the EC50 to 5nM. It has been previously demonstrated (4) that ligand-activation of RyR channels is also sensitive to luminal Ca2+ and therefore we have investigated how NAADP affects RyR1 and RyR2 in the presence of sensitising levels of luminal Ca2+. Addition of NAADP (≤1µM) had no significant effect on the Po of either RyR2 (0.14±0.10 to 0.14±0.08; n=3; S.D; P>0.05) or RyR1 (0.022±0.035 to 0.106±0.147; n=5; S.D; P>0.05). In contrast, large increases in TPC2 Po (0.001±0.002 to 0.4±0.2; n=3; S.D; P<0.05) could be elicited with much lower [NAADP] (10nM). Our results provide the first demonstration that TPC2 can function as an NAADP-sensitive ion-channel with the expected biophysical characteristics of an intracellular Ca2+-release channel. In contrast, we find no evidence to support the idea that NAADP activates RyR1 or RyR2. Our experiments also indicate that the luminal [Ca2+] of acidic stores may play an important physiological role in controlling NAADP-induced intracellular Ca2+-release.
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