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Two-pore channels and TRP mucolipins are ubiquitous endo-lysosomal cation channels of pathophysiological relevance. Both are Ca2+-permeable and regulated by phosphoinositides, principally PI(3,5)P2. Accumulating evidence has uncovered synergistic channel activation by PI(3,5)P2 and endogenous metabolites such as the Ca2+ mobilizing messenger NAADP, synthetic agonists including approved drugs and physical cues such as voltage and osmotic pressure. Here, we provide an overview of this coordination.

Two-pore cation channel, TPC1, is ubiquitous in the vacuolar membrane of terrestrial plants and mediates the long distance signaling upon biotic and abiotic stresses. It possesses a wide pore, which transports small mono- and divalent cations. K+ is transported more than 10-fold faster than Ca2+, which binds with a higher affinity within the pore. Key pore residues, responsible for Ca2+ binding, have been recently identified. There is also a substantial progress in the mechanistic and structural understanding of the plant TPC1 gating by membrane voltage and cytosolic and luminal Ca2+. Collectively, these gating factors at resting conditions strongly reduce the potentially lethal Ca2+ leak from the vacuole. Such tight control is impressive, bearing in mind high unitary conductance of the TPC1 and its abundance, with thousands of active channel copies per vacuole. But it remains a mystery how this high threshold is overcome upon signaling, and what type of signal is emitted by TPC1, whether it is Ca2+ or electrical one, or a transduction via protein conformational change, independent on ion conductance. Here we discuss non-exclusive scenarios for the TPC1 integration into Ca2+, ROS and electrical signaling.

When challenged by similar environmental conditions, phylogenetically distant taxa often independently evolve similar traits (convergent evolution). Meanwhile, adaptation to extreme habitats might lead to divergence between taxa that are otherwise closely related. These processes have long existed in the conceptual sphere, yet molecular evidence, especially for woody perennials, is scarce. The karst endemic Platycarya longipes, and its only congeneric species, P. strobilacea, which is widely distributed in the mountains in East Asia, provide an ideal model for examining the molecular basis of both convergent evolution and speciation. Using chromosome-level genome assemblies of both species, and whole genome resequencing data from 207 individuals spanning their entire distribution range, we demonstrate that P. longipes and P. strobilacea form two species-specific clades, which diverged around 2.09 million years ago. We find an excess of genomic regions exhibiting extreme interspecific differentiation, potentially due to long-term selection in P. longipes, likely contributing to the incipient speciation of the genus Platycarya. Interestingly, our results unveil underlying karst adaptation in both copies of the calcium influx channel gene TPC1 in P. longipes. TPC1 has previously been identified as a selective target in certain karst-endemic herbs, indicating a convergent adaptation to high calcium stress among karst-endemic species. Our study reveals the genic convergence of TPC1 among karst endemics, and the driving forces underneath the incipient speciation of the two Platycarya lineages.

When challenged by similar environmental conditions, phylogenetically distant taxa often independently evolve similar traits (convergent evolution). Meanwhile, adaptation to extreme habitats might lead to divergence between taxa that are otherwise closely related. These processes have long existed in the conceptual sphere, yet molecular evidence, especially for woody perennials, is scarce. The karst endemic Platycarya longipes and its only congeneric species, Platycarya strobilacea, which is widely distributed in the mountains in East Asia, provide an ideal model for examining the molecular basis of both convergent evolution and speciation. Using chromosome-level genome assemblies of both species, and whole-genome resequencing data from 207 individuals spanning their entire distribution range, we demonstrate that P. longipes and P. strobilacea form two species-specific clades, which diverged around 2.09 million years ago. We find an excess of genomic regions exhibiting extreme interspecific differentiation, potentially due to long-term selection in P. longipes, likely contributing to the incipient speciation of the genus Platycarya. Interestingly, our results unveil underlying karst adaptation in both copies of the calcium influx channel gene TPC1 in P. longipes. TPC1 has previously been identified as a selective target in certain karst-endemic herbs, indicating a convergent adaptation to high calcium stress among karst-endemic species. Our study reveals the genic convergence of TPC1 among karst endemics and the driving forces underneath the incipient speciation of the two Platycarya lineages.

Across phyla, voltage-gated ion channels (VGICs) allow excitability. The vacuolar two-pore channel AtTPC1 from the tiny mustard plant Arabidopsis thaliana has emerged as a paradigm for deciphering the role of voltage and calcium signals in membrane excitation. Among the numerous experimentally determined structures of VGICs, AtTPC1 was the first to be revealed in a closed and resting state, fueling speculation about structural rearrangements during channel activation. Two independent reports on the structure of a partially opened AtTPC1 channel protein have led to working models that offer promising insights into the molecular switches associated with the gating process. We review new structure-function models and also discuss the evolutionary impact of two-pore channels (TPCs) on K+ homeostasis and vacuolar excitability.

Two-pore channels (TPCs) are novel intracellular cation channels, which play a key role in numerous (patho-)physiological and immunological processes. In this chapter, we focus on their function in immune cells and immune reactions. Therefore, we first give an overview of the cellular immune response and the partaking immune cells. Second, we concentrate on ion channels which in the past have been shown to play an important role in the regulation of immune cells. The main focus is then directed to TPCs, which are primarily located in the membranes of acidic organelles, such as lysosomes or endolysosomes but also certain other vesicles. They regulate Ca2+ homeostasis and thus Ca2+ signaling in immune cells. Due to this important functional role, TPCs are enjoying increasing attention within the field of immunology in the last few decades but are also becoming more pertinent as pharmacological targets for the treatment of pro-inflammatory diseases such as allergic hypersensitivity. However, to uncover the precise molecular mechanism of TPCs in immune cell responses, further molecular, genetic, and ultrastructural investigations on TPCs are necessary, which then may pave the way to develop novel therapeutic strategies to treat diseases such as anaphylaxis more specifically.

Two-pore channels (TPCs) constitute a small family of cation channels expressed in endo-lysosomal compartments. TPCs have been characterized as critical elements controlling Ca2+ -mediated vesicular membrane fusion and thereby regulating endo-lysosomal vesicle trafficking. Exo- and endocytotic trafficking and lysosomal degradation are major mechanisms of adaption of epithelial transport. A prime example of highly regulated epithelial transport is the tubular system of the kidney. We therefore studied the localization of TPC protein 1 (TPC1) in the kidney and its functional role in the dynamic regulation of tubular transport.
Immunohistochemistry in combination with tubular markers were used to investigate TPC1 expression in proximal and distal tubules. The excretion of phosphate and ammonium, as well as urine volume and pH were studied in vivo, in response to dynamic challenges induced by bolus injection of parathyroid hormone or acid-base transitions via consecutive infusion of NaCl, Na2 CO3 , and NH4 Cl.
In TPC1-deficient mice, the PTH-induced rise in phosphate excretion was prolonged and exaggerated, and its recovery delayed in comparison with wildtype littermates. In the acid-base transition experiment, TPC1-deficient mice showed an identical rise in phosphate excretion in response to Na2 CO3 compared with wildtypes, but a delayed NH4Cl-induced recovery. Ammonium-excretion decreased with Na2 CO3 , and increased with NH4 Cl, but without differences between genotypes.
We conclude that TPC1 is expressed subapically in the proximal but not distal tubule and plays an important role in the dynamic adaptation of proximal tubular phosphate reabsorption towards enhanced, but not reduced absorption.

Two-pore channels (TPCs) are members of the superfamily of ligand-gated and voltage-sensitive ion channels in the membranes of intracellular organelles of eukaryotic cells. The evolution of ordinary plant TPC1 essentially followed a very conservative pattern, with no changes in the characteristic structural footprints of these channels, such as the cytosolic and luminal regions involved in Ca2+ sensing. In contrast, the genomes of mosses and liverworts encode also TPC1-like channels with larger variations at these sites (TPC1b channels). In the genome of the model plant Physcomitrium patens we identified nine non-redundant sequences belonging to the TPC1 channel family, two ordinary TPC1-type, and seven TPC1b-type channels. The latter show variations in critical amino acids in their EF-hands essential for Ca2+ sensing. To investigate the impact of these differences between TPC1 and TPC1b channels, we generated structural models of the EF-hands of PpTPC1 and PpTPC1b channels. These models were used in molecular dynamics simulations to determine the frequency with which calcium ions were present in a coordination site and also to estimate the average distance of the ions from the center of this site. Our analyses indicate that the EF-hand domains of PpTPC1b-type channels have a lower capacity to coordinate calcium ions compared with those of common TPC1-like channels.

NAADP (nicotinic acid adenine dinucleotide phosphate) is a second messenger, releasing Ca2+ from acidic calcium stores such as endosomes and lysosomes. PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate) is a phospho-inositide, residing on endolysosomal membranes and likewise releasing Ca2+ from endosomes and lysosomes. Both compounds have been shown to activate endolysosomal two-pore channels (TPCs) in mammalian cells. However, their effects on ion permeability as demonstrated specifically for TPC2 differ. While PI(3,5)P2 elicits predominantly Na+-selective currents, NAADP increases the Ca2+ permeability of the channel. What happens when both compounds are applied simultaneously was unclear until recently.