Endocytic uptake of SARS-CoV-2: the critical roles of pH, Ca2+, and NAADP

Endocytic acidification occurs at the same time as Ca2+ is lost from endosomes (Figure 1). The fluid taken into cells by endocytosis has, of course, the normal concentration of free Ca2+ found in the extracellular fluid, namely ∼1mM. However, as seen in Figure 1, at the earliest time points allowing assessment of endosomal [Ca2+], the concentration had already dropped to ∼30 μM, which is still markedly higher than the level in the cytosol (∼0.1 μM). The endosomal [Ca2+] then declined further to ∼4 μM in late endosomes (Figure 1). Endosomal acidification and loss of Ca2+ are interlinked. Blockade of the proton pump by bafilomycin prevented release of Ca2+ from the endosomes, with endosomal [Ca2+] remaining high.4 If [Ca2+] in the early endosomes was kept low, by reducing the extracellular [Ca2+] to less than 300 μM, acidification did not occur.4 The mechanism underlying the linkage between Ca2+ and H+ movements is not fully understood, but the fact that the uptake of H+ into endosomes occurs simultaneously with release of Ca2+, and that the two processes are interdependent, means that it is difficult to determine whether inhibition of virus entry by blockade of the proton pump or by TPC inhibition1,7 is primarily due to the high pH or to the relatively high [Ca2+] in the endosomes, or to a mixture of both.

It has recently been shown that SARS-CoV-2 enters cells via receptor-mediated endocytosis, as expected from previous studies of other coronaviruses. 1 ACE2 is the receptor for SARS-CoV-2, which unlocks access to the endocytic uptake of the virus. 2 It has also been demonstrated that a two-pore channel (TPC2) is critically important for SARS-CoV-2 entry into cells. 1 From earlier studies, we know that TPCs are channels in the endo-lysosomal system through which Ca 2þ is released when activated by the intracellular messenger nicotinic acid adenine dinucleotide phosphate (NAADP). 3 The new findings on SARS-CoV-2 entry 1 link two hitherto completely separate research fields, namely physiological Ca 2þ signaling and virology, opening up intriguing and important opportunities for future work on the cellular pathophysiology of COVID-19.
Endocytosis is a process whereby substances in the extracellular fluid can be taken into cells without endangering the integrity of the plasma membrane ( Figure 1). Endosomes have an acid interior due to the operation of a proton pump that can be very specifically inhibited by bafilomycin ( Figure 1). Ou et al. 1 have shown that SARS-CoV-2 entry is completely blocked when the proton pump is inhibited by 100 nM bafilomycin A, a concentration of the inhibitor that has previously been shown to prevent endosomal acidification in different cell types ( Figure 1). 4,6 It is particularly intriguing that pharmacological inhibition of TPC2, with tetrandrine, markedly reduced cellular entry of SARS-CoV-2. 1 This finding is similar to what has been shown in much more detail with regard to the entry of the Ebola virus. 7 Deletion of TPC1 or TPC2 resulted in marked inhibition of virus entry. 7 Furthermore, several known inhibitors of NAADPelicited Ca 2þ release from acid stores, for example, Ned-19 and tetrandrine, also severely reduced entry of this virus. 7 From earlier Ca 2þ signaling studies, it is known that deletion of TPC2 markedly reduced NAADP-elicited Ca 2þ release in permeabilized cells, as did an antibody to TPC2 (Figure 1). Ca 2þ release mediated by NAADP in intact cells was also markedly inhibited by Ned-19. 5 Endocytic acidification occurs at the same time as Ca 2þ is lost from endosomes ( Figure 1). The fluid taken into cells by endocytosis has, of course, the normal concentration of free Ca 2þ found in the extracellular fluid, namely 1mM. However, as seen in Figure 1, at the earliest time points allowing assessment of endosomal [Ca 2þ ], the concentration had already dropped to 30 lM, which is still markedly higher than the level in the cytosol (0.1 lM). The endosomal [Ca 2þ ] then declined further to 4 lM in late endosomes ( Figure 1). Endosomal acidification and loss of Ca 2þ are interlinked. Blockade of the proton pump by bafilomycin prevented release of Ca 2þ from the endosomes, with endosomal [Ca 2þ ] remaining high. 4 If [Ca 2þ ] in the early endosomes was kept low, by reducing the extracellular [Ca 2þ ] to less than 300 lM, acidification did not occur. 4 The mechanism underlying the linkage between Ca 2þ and H þ movements is not fully understood, but the fact that the uptake of H þ into endosomes occurs simultaneously with release of Ca 2þ , and that the two processes are interdependent, means that it is difficult to determine whether inhibition of virus entry by blockade of the proton pump or by TPC inhibition 1,7 is primarily due to the high pH or to the relatively high [Ca 2þ ] in the endosomes, or to a mixture of both.
Since TPCs are NAADP-sensitive channels, it is important to understand the mechanism by which NAADP is formed. The CD38 enzyme responsible for NAADP generation is an ectoenzyme that is internalized by endocytosis, 8 and requires an acid environment to perform its task. 8 In pancreatic acinar cells, the process of NAADP formation and the mechanism of action of this messenger have been investigated in some detail. 5,8 In this cell type, there is evidence showing that it is in the acidic endosomes that NAADP generation takes place. 8 Furthermore, the initial Ca 2þ release triggered by NAADP may depend on actions of this messenger on both endosomes and lysosomes. 5 At least part of the effect of proton pump inhibition on virus entry 1,7 could therefore be explained by lack of NAADP formation 8 and action, thereby preventing TPC activation. 5 SARS-CoV-2 is not only entering cells in the respiratory tract, 2 but is also taken up in the central nervous system, where it can cause serious damage, 9 as well as in the gastro-intestinal tract, including the pancreas. 10 From a mechanistic perspective, it may be of particular interest to study virus uptake in pancreatic acinar cells, since in these cells there is clear evidence for a physiological role of NAADP. 5 In this context, it is interesting that COVID-19 can be associated with acute pancreatitis and that ACE2 is present in the pancreas. 10 In acute pancreatitis, a human disease in which the pancreas digests itself, there is a secretion defect and large endocytic vacuoles, which are different from endosomes and post-exocytic in nature, appear in the acinar cells. 6 Nevertheless, these vacuoles have functional features in common with endosomes. When NAADP is generated by hormonal stimulation, the vacuole membrane is very permeable to Ca 2þ , as demonstrated by the rapid loss of Ca 2þ from vacuoles following a sudden increase in [Ca 2þ ] elicited by uncaging of caged Ca 2þ by UV light ( Figure 1D). The endocytic vacuoles are also acidic, with a pH similar to that found in endosomes, and bafilomycin A1-the very specific proton pump blocker-rapidly increased pH inside the vacuoles ( Figure 1E).
The endocytic vacuoles may provide an additional and important entry pathway for viruses, including SARS-CoV-2. Furthermore, the post-exocytic vacuoles contain large amounts of many different proteases, 6 which could also play a role in SARS-CoV-2 infection.
Exocrine pancreatic secretion is principally controlled by the neurotransmitter acetylcholine (ACh) and the hormone cholecystokinin (CCK). Both agents elicit secretion by mobilizing intracellular Ca 2þ , which subsequently triggers Ca 2þ entry from the extracellular solution. CCK primarily evokes Ca 2þ release mediated by NAADP acting not only on TPCs-but also on ryanodine receptors-in a complex arrangement involving endosomes and lysosomes. 5 In contrast, ACh primarily causes intracellular Ca 2þ release mediated by inositol trisphosphate (IP 3 ), acting on IP 3 receptors principally located in the endoplasmic reticulum. 5 Given that endosomal acidification depends on endosomal Ca 2þ release, 4 and that the entry of SARS-CoV-2 and Ebola virus is abolished or severely reduced when the proton pump is blocked or when TPCs are inhibited, 1,7 it would be of considerable interest to know if and how NAADP-dependent Ca 2þ signaling influences SARS-CoV-2 entry. Taking advantage of the co-existence in pancreatic acinar cells of two different Ca 2þ signaling mechanisms, activated by separate receptor pathways, it would seem possible to provide well-defined test and control conditions in such studies.
We conclude that there is evidence for the involvement of acidic endosomes and TPC-mediated Ca 2þ release from the endo-lysosomal system in both SARS-CoV-2 entry and NAADP- mediated Ca 2þ signaling. The pancreatic acinar cells may prove to be a particularly useful system for exploring the relationship between these phenomena.