Second messenger-induced Ca 2+ -release from intracellular stores plays a key role in a multitude of physiological processes. In addition to 1,4,5-inositol trisphosphate (IP 3 ), Ca 2+ , and cyclic ADP ribose (cADPR) that trigger Ca 2+ -release from the endoplasmatic reticulum (ER), nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as a cellular metabolite that mediates Ca 2+ -release from lysosomal stores. While NAADP-induced Ca 2+ -release has been found in many tissues and cell types, the molecular identity of the channel (s) conferring this release remained elusive so far. Here, we show that TPCN2, a novel member of the two-pore cation channel family, displays the basic properties of native NAADP-dependent Ca 2+ -release channels. TPCN2 transcripts are widely expressed in the body and encode a lysosomal protein forming homomers. TPCN2 mediates intracellular Ca 2+ -release after activation with lownanomolar concentrations of NAADP while it is desensitized by micromolar concentrations of this second messenger and is insensitive to the NAADP analog nicotinamide adenine dinucleotide phosphate (NADP). Furthermore, TPCN2-mediated Ca 2+ -release is almost completely abolished when the capacity of lysosomes for storing Ca 2+ is pharmacologically blocked. By contrast, TPCN2-specific Ca 2+ -release is unaffected by emptying ER-based Ca 2+ stores. In conclusion, these findings indicate that TPCN2 is a major component of the long-sought lysosomal NAADP-dependent Ca 2+ -release channel.

Two-pore channels (TPCNs) have been proposed to form lysosomal Ca 2؉ release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca 2؉ channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino acid residue in the putative pore region that is crucial for conferring high Ca 2؉ selectivity. Our glass chipbased method will provide electrophysiological access not only to lysosomal TPCN channels but also to a broad range of other intracellular ion channels.Nicotinic acid adenine dinucleotide phosphate (NAADP) 3 is a second messenger that releases Ca 2ϩ from intracellular stores at low nanomolar concentrations. NAADP-evoked Ca 2ϩ release has been demonstrated in invertebrates and numerous mammalian cell types including pancreatic acinar and ␤-cells, cardiac and smooth muscle cells, T-lymphocytes, platelets, and neurons (1). Recent studies using NAADP binding assays and Ca 2ϩ imaging experiments indicated that members of the twopore channel family (TPCN1-3) constitute the native NAADP receptor (2, 3). TPCNs share sequence homology with members of the transient receptor potential (TRP) cation channel family, suggesting that they may directly form the NAADPgated Ca 2ϩ conductance. However, direct proof of ion channel activity of TPCNs is still missing, leaving open the possibility that another ion channel protein assembled with TPCNs could underlie the Ca 2ϩ current. A major obstacle to address this key issue is that TPCNs are strictly localized in endolysosomal organelles, in particular acidic lysosomes that are not readily accessible to standard patch clamp measurements. Here we present a new method to record ionic currents in isolated lysosomes. Specifically, we provide direct evidence that TPCN2 is a highly selective Ca 2ϩ channel and identify an amino acid residue in the putative pore region that is crucial for conferring Ca 2ϩ selectivity. EXPERIMENTAL PROCEDURESGeneration and Culture of Stable Cell Lines-Stable cell lines for enhanced GFP-tagged murine wild type TPCN2 (3) and mutant TPCN2 were generated using the Flp-In TM system (Invitrogen) according to manufacturer's protocol. Mutations in the putative selectivity filter of murine TPCN2 (N257A and E643A) were introduced using the QuikChange site-directed mutagenesis kit (Stratagene, LA Jolla, CA). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 g/ml streptomycin, 100 g/ml hygromycin and kept at 37°C, 10% CO 2 .Preparation of Lysosomes-To increase the size of the lysosomes (usually Ͻ0.5 M), cells were treated with 1 M vacuolin for 2 h. This small compound is known to selectively increase the size of endosomes and lysosomes (4, 5). Lysosomes were prepared according to Schenkman and Ci...

Since its launch in the early 1980s, the patch clamp method has been extensively used to study ion channels in the plasma membrane, but its application to the study of intracellular ion channels has been limited. Unlike the plasma membrane, intracellular membranes are usually not stable enough to withstand mechanical manipulation by glass electrodes during seal formation and rupturing of the membrane. To circumvent these problems, we developed a method involving the immobilization of isolated organelles on a solid matrix planar glass chip. This glass chip contains a microstructured hole that supports the formation of gigaseals and subsequent electrophysiological recordings despite the high fragility of intracellular membranes. Here, we report the experimental details of this method using lysosomes, which are the smallest cellular organelles, as a model system. We demonstrate that we can record endogenous ionic currents from wild-type lysosomes, as well as from lysosomes overexpressing ion channels, and expect that this method will provide electrophysiological access to a broad range of intracellular ion channels.