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.

The zeta potential (which approximates the surface potential) of the acid resistant green alga Dunaliella acidophila (optimal growth at pH 1.0) and the salt resistant D. parva (grown at pH 7.6) were calculated from the electrophoretic mobility of cells as determined by means of free-flow electrophoresis. Dunaliella acidophila cells exhibit a positive zeta potential (-f 5 to +20mV) at acidic external pH values, whereas negative zeta potentials (-30mV) were measured at neutral pH values. Negative zeta potentials of the same order of magnitude were also measured for D. parva cells (pH 7.6). Low concentrations of La'"*' and AP"*" did not affect the positive zeta potential of D. acidophila at acidic pH values, whereas higher concentrations caused a shift to more positive potentials. However, at neutral pH these cations caused a significant decrease of the negative zeta potential. The impermeant polycation poly-L-lysine acted in a sitnilar manner to Al'"^ or La'"^. The effect of impermeant cations and anions on various physiological reactions also supports the existence of a positive zeta potential for D. acidophila and of a negative zeta potential for D. parva: polycations such as DEAE-dextran and poly-Llysine strongly inhibitied photosynthesis and rnobility of D. parva, but did not affect these reactions in D. acidophila. Comparable differential inhibitions were also observed for Al' "*" and La'^, Impermeant anions such as Dextran-sulfate exhibited effects in the opposite direction: inhibition was stronger with D. acidophila and weaker with D. parva. However, the differential inhibition by impermeant anions was much less pronounced in comparison with impenneant cations due to the relatively high pK^, values of anionic solutes and consequently relatively high protonation at pH 1.0. The physiological consequences of an asymmetrically charged plasma membrane (excess of positive charges outside, excess of negative charges on the cytoplasmic side) of D. acidophila are discussed in regard to the extreme acid resistance of this alga and its resistance to cationic toxic solutes in industrial wastes.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2þ from intracellular stores at low nanomolar concentrations. NAADP-evoked Ca2þ-release has been demonstrated in invertebrates and numerous mammalian cell types including pancreatic acinar and betacells, cardiac and smooth muscle cells, T-lymphocytes, platelets and neurons. Several studies using NAADP binding assays and Ca2þ imaging experiments linked members of the two-pore channel family (TPCN1-3) with NAADPinduced Ca2þ release from lysosome-like acidic organelles. However, there has been no direct demonstration that TPCNs can act as NAADP-sensitive Ca2þ release channels. Recently, we developed a highly efficient method to record ionic currents in single isolated lysosomes. These recordings are performed using an automated patch clamp approach that involves the immobilization of isolated lysosomes on a solid matrix planar glass chip. Using this method we have provided direct evidence that TPCN2 is a highly selective Ca2þ channel. Furthermore, we identified an amino acid residue in the putative pore region that is crucial for conferring high Ca2þ-selectivity. Here, we extend our biophysical characterization of TPCN2 and report a detailed analysis of the permeation properties of these channels. Overall, our study lays the groundwork for the understanding of TPCN2 function in lysosomal stores. Our glass chip based method will provide electrophysiological access not only to lysosomal TPCN channels but also to a broad range of other intracellular ion channels.

No abstract

No abstract

No abstract