TPC2 is a novel NAADP-sensitive Ca2+ release channel, operating as a dual sensor of luminal pH and Ca2+

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca2+ required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca2+ from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca2+-release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca2+ that will enable it to act as a Ca2+ release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca2+] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca2+ release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca2+ release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μM but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.Publisher PDFPeer reviewe

Synthesis of phosphorothioamidites derived from 3′-thio-3′-deoxythymidine and 3′-thio-2′,3′-dideoxycytidine and the automated synthesis of oligodeoxynucleotides containing a 3′-S-phosphorothiolate linkage

The synthesis of N4-benzoyl-5′-O-dimethoxytrityl-2′,3′-dideoxy-3′-thiocytidine and its phosphorothioamidite is described for the first time, together with a shortened procedure for the preparation of 5′-O-dimethoxytrityl-3′-deoxy-3′-thiothymidine and its corresponding phosphorothioamidite. The first fully automated coupling procedure for the incorporation of a phosphorothioamidite into a synthetic oligodeoxynucleotide has been developed, which conveniently uses routine activators and reagents. Coupling yields using this protocol were in the range of 85–90% and good yields of singularly modified oligonucleotides were obtained. Coupling yields were also equally good when performed on either a 0.2 or 1 µmol reaction column, thus facilitating large scale syntheses required for mechanistic studies

¹H NMR (400 MHz, DMSO-D₆) (top) and ¹³C NMR (100 MHz, DMSO-D₆) (bottom) spectra of 2,6-difluoro-<i>N</i>-(1<i>H</i>-pyrazol-3-yl)benzamide (21).

1H NMR (400 MHz, DMSO-D6) (top) and 13C NMR (100 MHz, DMSO-D6) (bottom) spectra of 2,6-difluoro-N-(1H-pyrazol-3-yl)benzamide (21).</p

Chemical structures of the small molecule SOCE inhibitors examined in this study and their common names.

Chemical structures of the small molecule SOCE inhibitors examined in this study and their common names.</p

Synthetic route to 7-azaindole (12).

Calcium (Ca2+) is a key second messenger in eukaryotes, with store-operated Ca2+ entry (SOCE) being the main source of Ca2+ influx into non-excitable cells. ORAI1 is a highly Ca2+-selective plasma membrane channel that encodes SOCE. It is ubiquitously expressed in mammals and has been implicated in numerous diseases, including cardiovascular disease and cancer. A number of small molecules have been identified as inhibitors of SOCE with a variety of potential therapeutic uses proposed and validated in vitro and in vivo. These encompass both nonselective Ca2+ channel inhibitors and targeted selective inhibitors of SOCE. Inhibition of SOCE can be quantified both directly and indirectly with a variety of assay setups, making an accurate comparison of the activity of different SOCE inhibitors challenging. We have used a fluorescence based Ca2+ addback assay in native HEK293 cells to generate dose-response data for many published SOCE inhibitors. We were able to directly compare potency. Most compounds were validated with only minor and expected variations in potency, but some were not. This could be due to differences in assay setup relating to the mechanism of action of the inhibitors and highlights the value of a singular approach to compare these compounds, as well as the general need for biorthogonal validation of novel bioactive compounds. The compounds observed to be the most potent against SOCE in our study were: 7-azaindole 14d (12), JPIII (17), Synta-66 (6), Pyr 3 (5), GSK5503A (8), CM4620 (14) and RO2959 (7). These represent the most promising candidates for future development of SOCE inhibitors for therapeutic use.</div

Raw data used to calculate IC₅₀ values.

Baseline corrected Δ340/380 ratios from three independent biological replicates (Plate A, B and C) which each included three technical replicates for each condition tested. (XLSX)</p

¹H NMR (400 MHz, CDCl₃) (top) and ¹³C NMR (100 MHz, CDCl₃) (bottom) spectra of 4,6-dimethoxypyridin-3-yl)boronic acid (30).

1H NMR (400 MHz, CDCl3) (top) and 13C NMR (100 MHz, CDCl3) (bottom) spectra of 4,6-dimethoxypyridin-3-yl)boronic acid (30).</p