Two-pore channels (TPCs) are intracellular voltage- and ligand-gated cation channels in eukaryotic cells [1]. TPCs play a special role in numerous immunological and (patho-)physiological processes ^[2][3][4][2,3,4] TPCs have two functional members in the human genome (TPC1 and TPC2) and belong to a novel class of non-selective cation channels [5]. TPCs have two subunits and each subunit consists of 12 transmembrane helices with two pore domains between the 5th and 6th segment or the 11th and 12th segment, respectively. The formation of dimers creates an asymmetrical pore, which is formed by neutral amino acids; thus allowing Na⁺, Ca²⁺, and H⁺ to penetrate the pore ^[6][7][8][6,7,8]. 

TPCs are subdivided into TPC1, TPC2, and TPC3. Compared to TPC1 and TPC2, TPC3 was not found in human, primate, or rodent cells ^[9][10]. However, TPC3 was found in the plasma membrane of cortical granules of starfish oocytes ^[10][11]. The protein structure of TPC1 was the first to be resolved in Arabidopsis thaliana by means of X-ray crystallography ^[1][11][1,12]. Only recently, the 3D structure of TPC1 and TPC2 was shown for Mus musculus and Homo sapiens, respectively, using cryo-electron microscopy.

Mast cells and basophils are innate leukocytes that both develop from hematopoietic stem cells. Mast cells can be found in almost all organs and are often located near neurons, blood vessels, and lymphatic vessels to transmit local inflammatory signals ^[12][17]. The intracellular structure of mast cells is unique as it contains a large number of granules, which are filled with multi-potent hormones, such as histamine, heparin, leukotrienes, many cyto- and chemokines, and growth factors ^[13][14][15][18,19,20]. Mast cells represent an important part of the innate immune system, which is essential for the defense against pathogens, such as viruses and bacteria, but also for wound healing. They are key effector cells in allergies and anaphylactic responses [2]. Activation takes place via the binding of immunoglobulin E (IgE) to the Fcε-receptor (FcεR) and subsequent high affinity binding to the respective allergen, resulting in crosslinking of two or more FcεR molecules ^[15][16][20,21]. During this process, inflammatory mediators, such as histamine, are released through degranulation. Numerous factors that favor an excessive release of histamine and heparin from the mast cell are the triggers of allergies, such as allergic asthma ^[17][18][22,23]. However, mast cells serve far more physiological functions than the well-known release of histamine ^[19][20][21][24,25,26]. In addition to mast cells, basophil granulocytes are also of great importance during anaphylaxis and allergic reactions, and consequently for the therapy of, for example, asthma ^[22][27]. In fact, there are some properties of basophils that are also found in mast cells. For example, like mast cells, basophils also contain characteristic granules in the cytoplasm ^[23][28]. Furthermore, basophils can also release mediators, such as histamine and cysteinyl leukotrienes ^[24][25][29,30]. Unlike mast cells, basophils primarily reside in the blood ^[25][30].

Allergen exposure and binding to IgE pre-coupled to FcεR in the plasma membrane of a mast cell activates phospholipase C (PLC); thus leading to the release of inositol 1,4,5-triphosphate and diacylglycerol (DAG). IP₃ then binds to the IP₃ receptor (IP₃R), which is located in the endoplasmic reticulum (ER) membrane. The ER is one of the most important organelles for the regulation of intracellular Ca²⁺ and the largest Ca²⁺ store in the cell ^[26][31]. The binding of IP₃ to IP₃R results in a release of Ca²⁺ from the ER, which, in turn, triggers a regulated exocytosis of secretory vesicles and the accompanying release of histamine from the cell. It was recently demonstrated that this process is fine-tuned by TPC1 in the endolysosomal membrane [2]. Therefore, the endolysosome has to be in close proximity to the ER. The Ca²⁺-permeable TPC1 can regulate the level of ER-Ca²⁺ by facilitating the release and uptake (likely indirectly) of Ca²⁺ from and into the endolysosome, respectively, and thus support the regulated exocytosis of histamine containing vesicles. The endolysosomal compartment itself has a homeostatic function, tightly regulating the ER Ca²⁺ concentration, and thereby the reactivity of mast cells. This suggested homeostatic crosstalk is dependent on TPC1 function in that TPC1 activity triggers a feed-forward loop causing the opening of IP₃-receptors. The local increase of Ca²⁺ is then triggering its uptake into endolysosomes; however, the exact molecular mechanisms underlying this uptake process remain elusive and warrant further investigations. With the lack of crosstalk of these two storage compartments, the ER is “overly” filled with Ca²⁺. However, in t ihis context it is not yet fully understood how Ca²⁺ is actively taken up into the endolysosome and whether additional transporters are involved. If TPC1 is blocked genetically or through pharmacological inhibition by means of, for example, the plant alkaloid tetrandrine or by antagonizing its activation via nicotinic acid adenine dinucleotide phosphate (NAADP) using trans-Ned-19 ^[27][32], there is no regulation of intracellular Ca²⁺ by means of TPC1 in the endolysosome [2]. When the signal cascade is then activated by IP₃ due to repeated allergen exposure, there is an increased release of the previously augmented ER-Ca²⁺ due to the aforementioned inhibition of TPC1. This, in turn, results in enhanced exocytosis (right panel) and, consequently, in an anaphylactic reaction.

4. TPC Regulation by NAADP and PI(3,5)P₂

It is well known that TPCs can be activated by NAADP, a Ca²⁺ mobilizing agent ^[28][29][30][33,34,35]. For cytotoxic T cells it has been shown that NAADP application triggers the release of Ca²⁺ from the (endo-)lysosome, which subsequently provokes a secretion of perforin and granzyme from the secretory granules into the extracellular space ^[31][32][36,37]. In previous studies, binding sites of NAADP were examined ^[33][34][38,39]. There, it was found that NAADP most likely cannot bind directly to TPCs, but indirectly via accessory proteins. Such an accessory protein was recently identified ^[35][36][40,41]. The Jupiter microtubule associated homolog 2 (JPT2), also known as Sm-like protein LSM12, was suggested to be such an interaction partner for NAADP and TPCs. Previously, it was also described that TPCs can be activated via PI(3,5)P₂ and in this context were considered as voltage-dependent Na⁺ channels ^{[8][37][38][39]}[8,13,42,43]. This has been assumed because activation of TPCs via PI(3,5)P₂ rather leads to a Na⁺ than a Ca²⁺ flux ^{[7][37][38][40]}[7,13,42,44]. In t ihis context it is vital to mention a recently published work by Gerndt et al. [7], who found that ion selectivity of the TPC2 in primary murine macrophages depends on the activating ligand, thereby differentially promoting lysosomal function. Therein, rthesearche authors show that activation of TPC2 by means of PI(3,5)P₂ leads to an increase in lysosomal exocytosis, whereas activation by means of NAADP decreases lysosomal exocytosis due to a change in vesicular pH. This has been previously described for NAADP and can probably be attributed to the agonist-specific effect on proton permeability ^[41][42][45,46]. However, on the same subject, but in this case using mast cells as an exocytotic model system, it was found by Arlt et al. [2] that activation of TPC1 by either NAADP or PI(3,5)P₂ alone does not lead to an enhanced degranulation of mast cells. Neither TPC1-deficient nor wild-type mast cells showed a change in their membrane surface area upon perfusion. However, upon simultaneous activation of G-proteins by the nonhydrolyzable analogue of GTP, GTPγS, degranulation was even reduced, suggesting that the timely release of local Ca²⁺ from the endo-lysosomes is key for regulated exocytosis. Consequently, when TPC1 was inhibited with the plant alkaloid tetrandrine or with the NAADP antagonist trans Ned-19, exocytosis was enhanced due to an increase in cytosolic Ca²⁺ released from the ER. The fact that the debate about endogenous ligands of TPCs, namely NAADP and PI(3,5)P₂, is relatively controversial was previously taken up in several articles ^{[38][43][44][45][46]}[42,47,48,49,50]. In Ruas et al. ^[44][48] it was suggested that TPCs are important for the conduction of both cations (Ca²⁺ and Na⁺) and that activation is possible via both NAADP and PI(3,5)P₂. It was assumed that this could not always be clearly demonstrated in the past due to inadequate mouse models in which the TPC function was sometimes unintentionally preserved ^[43][44][47,48]. When distinguishing between plant and mammalian TPC channels, the latter have been suggested as potential receptors for NAADP ^[28][29][47][33,34,51]. However, there is also increasing evidence in recent studies that TPCs are Na⁺ selective channels and are also activated by PI(3,5)P₂ instead of NAADP ^[38][46][42,50]. A decisive step forward in this matter came from the work of Guo et al. [8], who performed an electrophysiological characterization and comparison of the ion selectivity properties of plant AtTPC1 (Arabidopsis thaliana) and mammalian HsTPC2 (Homo sapiens). By the implementation of structure-directed mutagenesis, the nonselective AtTPC1 was transformed into a Na⁺ selective channel similar to HsTPC2. This identified the most important filter residues that are central to the definition of ion selectivity of TPCs. Ultimately, however, there are still some gaps in this context that must be filled with future experiments, especially since PI(3,5)P₂ can regulate TPC1 in addition to NAADP and this more likely leads to a Na⁺-specific current. The recent discovery of two promising agonists for TPC2, which seem to selectively trigger Na⁺- or Ca²⁺-conductance, might help to shed light onto this conundrum [7]. The first agonist, named TPC2-A1-P, seems to be responsible for selective Na⁺ currents. The second agonist, termed TPC2-A1-N, in turn triggers an increased Ca²⁺ signal. Based on these results, it has already been speculated that TPC2-A1-N could be an imitator of NAADP, whereas TPC2-A1-P probably mimics PI(3,5)P₂ [7]. However, the question then arises as to why, in the case of TPC-A1-N, no accessory proteins, such as JPT2/LSM12, are required, as it has been suggested for NAADP ^[35][36][40,41]. Therefore, future ultrastructural experiments are necessary, which may provide further insights into the exact molecular mechanism of channel activation.