Since its development by Neher and Sakmann (1976), the patch clamp technique has been successfully applied to study ion channels in the plasma membrane. In the 1980s and 1990s ion channels in inner and outer membranes of mitochondria (Sorgato et al., 1987), ion channels in chloroplast envelope membranes (Pottosin et al., 1993; Heiber et al., 1995), and in thylakoid membranes (Pottosin and Schönknecht, 1995) have been patch clamped successfully. Just recently, it became possible to also patch-clamp ion channels in endolysosomal membranes. Such endolysosomal ion channels comprise mucolipins (TRPML channels; Dong et al., 2010; Shen et al., 2012; Samie et al., 2013; Cheng et al., 2014; Sakurai et al., 2015; Li et al., 2016), P2X4 (Qureshi et al., 2007; Huang et al., 2014; Zhong et al., 2016), TMEM175 (Cang et al., 2015), BK (Cao et al., 2015), CLCs (Jentsch et al., 2015; Zifarelli, 2015), TRPM2 (Lange et al., 2009), and Two-pore channels (TPCs; Wang et al., 2012; Cang et al., 2013, 2014; Grimm et al., 2014). With this novel, modified patch clamp technique endolysosomal ion channels can be studied directly in their native membranes. This has undoubtedly contributed to a better understanding of the function and physiology of endolysosomal ion channels such as the TPCs. TPCs derive their name from the two pore domains that are found on each subunit. TPC subunits consist of 12 transmembrane domains (TMDs) with the putative pore loops between TMD5/6 and TMD11/12, respectively. Two subunits dimerize to form one functional pore. With this topology, the TPCs are residing between the six TMD containing cation channels such as, e.g., the TRP channels including the endolysosomal TRPML channels and the voltage-gated cation channels (Ca_vs and Na_vs) with their 24 TMDs. Humans have two isoforms, TPC1 and TPC2. Using a combination of knockout mouse and knockdown models, cell biological methods, imaging technologies and the endolysosomal patch clamp technique, the functions of these channels have been explored in more detail in recent years.

In 2009 several groups provided evidence that TPCs are activated by NAADP (nicotinic acid adenine dinucleotide phosphate), a Ca²⁺-mobilizing second messenger which had been discovered in Clapper et al. (1987) together with cADPR (cyclic ADP ribose; Brailoiu et al., 2009; Calcraft et al., 2009; Zong et al., 2009). This finding, that TPCs are NAADP-activated calcium release channels on endolysosomal membranes was challenged by Wang et al. (2012) as well as Cang et al. (2013) who claimed that TPCs are Na⁺ selective channels activated by PI(3,5)P₂ (phosphatidylinositol 3,5-bisphosphate) and not by NAADP. Although there is a consensus that TPCs can, under various conditions and recording configurations, be activated by PI(3,5)P₂, it is unclear up to now why NAADP activation of TPCs is less consistently observed. A number of recent reviews, e.g., by Jentsch et al. (2015), Marchant and Patel (2015), or Ruas et al. (2015b) have summarized the current debate in detail. Nevertheless, a few critical points shall be repeated here. First, Jha et al. (2014) found that in the absence of Mg²⁺, NAADP-evoked TPC2-mediated Na⁺ currents are readily detectable using the same recording configuration as Wang et al. (2012). This suggests that Mg²⁺ may have impacted the detection of NAADP currents in the previous studies. Secondly, NAADP activation of TPCs may require an auxiliary protein (Lin-Moshier et al., 2012; Walseth et al., 2012; Morgan et al., 2015; Ruas et al., 2015b) which may be missing or may be diluted under certain experimental conditions. Finally, concern about the validity of TPC knockout mouse models used to show that NAADP-responses are still present in the absence of TPCs was raised (Ruas et al., 2015b). Recently, Ruas et al. (2015a) generated a novel TPC-null mouse line with demonstrable absence of both TPC1 and TPC2. In this study Ruas et al. (2015a) show that the loss of endogenous TPCs abolished NAADP-dependent Ca²⁺ responses as assessed by single-cell Ca²⁺ imaging or endolysosomal patch-clamp. NAADP-sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca²⁺-permeability. Ruas et al. (2015a) further reported that the mouse line formerly described as TPC-null (Wang et al., 2012; Cang et al., 2013) expresses truncated TPCs but that these truncated TPCs still support NAADP-induced Ca²⁺ release.

Nevertheless, it will be necessary in the future to determine in more detail under which conditions NAADP responses are detectable and under which conditions they are not, what influence technical characteristics (e.g., the size of measured endolysosomes) may have and what the identity of the putative intermediator protein may be. In addition, it remains to be clarified what the upstream signals are that lead to the formation of either NAADP or PI(3,5)P₂ prior to TPC activation.

One fundamental difference between TPC1 and TPC2 is their voltage dependence. Cang et al. (2014) reported that TPC1 is a voltage-gated, non-inactivating Na⁺ channel, which they called lysoNaV and which is formed only by TPC1 but not by TPC2. Luminal alkalization was found to also open TPC1 by shifting the channel’s voltage dependence of activation toward hyperpolarization. The authors concluded that endolysosomes sense both voltage and pH changes with lysoNaV (Cang et al., 2014).

Interestingly, in other studies it was found that luminal alkalization inhibits NAADP evoked TPC2 Ca²⁺ currents while PI(3,5)P₂ evoked TPC2 Na⁺ currents are not inhibited (Schieder et al., 2010; Wang et al., 2012; Jha et al., 2014). Taken together these findings suggest that TPC1 and TPC2, despite significant similarities in sequence and function may differ substantially in their overall activation mechanisms.

Studies with TPC knockout and knockdown models have revealed that TPCs participate in the regulation of multiple endolysosomal trafficking pathways which when dysregulated can lead to or influence the development of a range of different diseases such as lysosomal storage, metabolic, infectious diseases, or cancer. Thus, it was found that loss of TPCs can lead to or accelerate the development of hypercholesterolemia and fatty liver hepatitis under certain dietary conditions (Grimm et al., 2014), can slow-down intracellular virus and bacterial toxin trafficking (Ruas et al., 2010, 2014; Sakurai et al., 2015), can affect VEGF-induced neoangiogenesis (Favia et al., 2014), autophagy (Lu et al., 2013a,b; Fernández et al., 2016), and human hair pigmentation (Sulem et al., 2008; Ambrosio et al., 2016; Bellono et al., 2016), and the acrosome reaction in sperm (Arndt et al., 2014). Moreover, physiological roles of TPCs in cardiomyocytes (Capel et al., 2015; Davidson et al., 2015) and pancreatic β cells (Arredouani et al., 2015) have been postulated. Most of these proposed physiological roles and functions have in common that endolysosomal trafficking processes and/or endolysosomal cation homeostasis are disrupted or disturbed to a certain degree. Importantly, however, TPC1 as well as TPC2 knockout mice have no reduced life span or an obvious reduction in quality of life in contrast to mice in which the endolysosomal cation channel TRPML1 is knocked out (Grishchuk et al., 2014; Figure 2). This suggests that in contrast to TRPML1, the system is able to somehow compensate for the loss of one or both TPCs. Hence, TPCs may be infact more relevant under stress and under challenging environmental conditions than under basal conditions.

A role for TPCs in promoting endolysosomal trafficking appears to be well supported by the literature. Its activation in endolysosomal vesicles by PI(3,5)P₂ or NAADP has been confirmed by several research groups, although there is still an ongoing debate on the exact conditions under which TPCs are activated by NAADP and whether a mediator protein may be required for this activation. The identity of such a mediator needs to be determined. Interaction with SNARE proteins has been found by at least two independent groups, likewise data on the interaction with mTOR is very convincing. Unclear is the role in endolysosomal pH regulation, the role of TPCs in autophagy needs to be further elucidated and whether TPCs play a role in endolysosomal vesicle fusion of fission is lacking final proof. Whether PI(3,5)P₂ or NAADP are endogenous TPC ligands also remains uncertain. That TPCs are disease relevant has, however, been convincingly demonstrated by several studies. The fact that TPCs have been found to be relevant for a multitude of diseases will certainly encourage further research in this field. Both activation and inhibition of TPCs may be beneficial depending on the disease or condition to be treated, e.g., TPC inhibition may be beneficial in infectious diseases to prevent effective transport of virus (e.g., Ebola) or bacterial toxins (e.g., CT) in the endolysosomal system. On the other hand, TPC activation may promote, e.g., LDL cholesterol trafficking and may thus prevent subcellular accumulation of too much cholesterol. Likewise the accumulation of macromolecules and other material in lysosomes of neurons may be reverted or prevented by activating TPCs on time.

All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.