The two-pore channel TPCN2 mediates NAADP-dependent Ca2+-release from lysosomal stores

Second messenger-induced Ca2+-release from intracellular stores plays a key role in a multitude of physiological processes. In addition to 1,4,5-inositol trisphosphate (IP3), Ca2+, and cyclic ADP ribose (cADPR) that trigger Ca2+-release from the endoplasmatic reticulum (ER), nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as a cellular metabolite that mediates Ca2+-release from lysosomal stores. While NAADP-induced Ca2+-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 Ca2+-release channels. TPCN2 transcripts are widely expressed in the body and encode a lysosomal protein forming homomers. TPCN2 mediates intracellular Ca2+-release after activation with low-nanomolar 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 Ca2+-release is almost completely abolished when the capacity of lysosomes for storing Ca2+ is pharmacologically blocked. By contrast, TPCN2-specific Ca2+-release is unaffected by emptying ER-based Ca2+ stores. In conclusion, these findings indicate that TPCN2 is a major component of the long-sought lysosomal NAADP-dependent Ca2+-release channel

In situ nanoscopy of single-grain nanomorphology and ultrafast carrier dynamics in metal halide perovskites

Designing next-generation light-harvesting devices requires a detailed understanding of the transport of photoexcited charge carriers. The record-breaking efficiencies of metal halide perovskite solar cells have been linked to effective charge-carrier diffusion, yet the exact nature of charge-carrier out-of-plane transport remains notoriously difficult to explain. The characteristic spatial inhomogeneity of perovskite films with nanograins and crystallographic disorder calls for the simultaneous and hitherto elusive in situ resolution of the chemical composition, the structural phase and the ultrafast dynamics of the local out-of-plane transport. Here we simultaneously probe the intrinsic out-of-plane charge-carrier diffusion and the nanoscale morphology by pushing depth-sensitive terahertz near-field nanospectroscopy to extreme subcycle timescales. In films of the organic–inorganic metal halide perovskite FA0.83Cs0.17Pb(I1−xClx)3 (where FA is formamidinium), domains of the cubic α-phase are clearly distinguished from the trigonal δ-phase and PbI2 nano-islands. By analysing deep-subcycle time shifts of the scattered terahertz waveform after photoexcitation, we access the vertical charge-carrier dynamics within single grains. At all of the measured locations, despite topographic irregularities, diffusion is surprisingly homogeneous on the 100 nm scale, although it varies between mesoscopic regions. Linking in situ carrier transport with nanoscale morphology and chemical composition could introduce a paradigm shift for the analysis and optimization of next-generation optoelectronics that are based on nanocrystalline materials

Comprehensive analysis of mitochondrial permeability transition pore activity in living cells using fluorescence-imaging-based techniques

Mitochondrial permeability transition (mPT) refers to a sudden increase in the permeability of the inner mitochondrial membrane. Long-term studies of mPT revealed that this phenomenon has a critical role in multiple pathophysiological processes. mPT is mediated by the opening of a complex termed the mPT pore (mPTP), which is responsible for the osmotic influx of water into the mitochondrial matrix, resulting in swelling of mitochondria and dissipation of the mitochondrial membrane potential. Here we provide three independent optimized protocols for monitoring mPT in living cells: (i) measurement using a calcein-cobalt technique, (ii) measurement of the mPTP-dependent alteration of the mitochondrial membrane potential, and (iii) measurement of mitochondrial swelling. These procedures can easily be modified and adapted to different cell types. Cell culture and preparation of the samples are estimated to take 1/41 d for methods (i) and (ii), and 1/43 d for method (iii). The entire experiment, including analyses, takes 1/42 h