Calcium signaling via two-pore channels: local or global, that is the question

Abstract

Recently, we identified, for the first time, two-pore channels (TPCs, TPCN for gene name) as a novel family of nicotinic acid adenine dinucleotide phosphate (NAADP)-gated, endolysosome-targeted calcium release channels. Significantly, three subtypes of TPCs have been characterized, TPC1-3, with each being targeted to discrete acidic calcium stores, namely lysosomes (TPC2) and endosomes (TPC1 and TPC3). That TPCs act as NAADP-gated calcium release channels is clear, given that NAADP binds to high- and low-affinity sites associated with TPC2 and thereby induces calcium release and homologous desensitization, as observed in the case of endogenous NAADP receptors. Moreover, NAADP-evoked calcium signals via TPC2 are ablated by short hairpin RNA knockdown of TPC2 and by depletion of acidic calcium stores with bafilomycin. Importantly, however, NAADP-evoked calcium signals were biphasic in nature, with an initial phase of calcium release from lysosomes via TPC2, being subsequently amplified by calcium-induced calcium release (CICR) from the endoplasmic reticulum (ER). In marked contrast, calcium release via endosome-targeted TPC1 induced only spatially restricted calcium signals that were not amplified by CICR from the ER. These findings provide new insights into the mechanisms that cells may utilize to "filter" calcium signals via junctional complexes to determine whether a given signal remains local or is converted into a propagating global signal. Essentially, endosomes and lysosomes represent vesicular calcium stores, quite unlike the ER network, and TPCs do not themselves support CICR or, therefore, propagating regenerative calcium waves. Thus "quantal" vesicular calcium release via TPCs must subsequently recruit inositol 1,4,5-trisphoshpate receptors and/or ryanodine receptors on the ER by CICR to evoke a propagating calcium wave. This may call for a revision of current views on the mechanisms of intracellular calcium signaling. The purpose of this review is, therefore, to provide an appropriate framework for future studies in this area.

Fig. 1.

Intracellular location, membrane topology, and sequence homology of two-pore channels (TPCs). A: diagram of acidic organelle depicting the function of vacuolar (V) H⁺-ATPase, putative Ca²⁺/H⁺ exchanger, and TPC2. TPC2 is enlarged to show predicted transmembrane organization of TPCs based on hydrophobicity analysis and membrane orientation of voltage-gated Ca²⁺ channels and transient receptor potential (TRP) channels. P loop, pore loop; NAADP, nicotinic acid adenine dinucleotide phosphate. B: pairwise comparison of amino acid identities at different domains of TPCs. NH₂-termini (N-ter), COOH-termini (C ter), and transmembrane (TM) repeats are indicated. Note: higher homology is found at TM segments 4–6, but, overall, the three TPCs are quite distant from each other.

Fig. 2.

Schematic representation depicts the targeting, in human embryonic kidney-293 (HEK-293) cells, of human TPC1 and chicken TPC3 to different endosome populations, and of human TPC2 to lysosomes, and the functional role of these acidic organelles. SOC and ROC, store- and receptor-operated channel, respectively; VGCC, voltage-gated Ca²⁺ channel; TGN, trans-Golgi network. [Adapted from Luzio et al. (62)].

Fig. 3.

Schematic diagram depicts partial family trees from the animal kingdom and identifies key examples of species in which the genes encoding TPC1, TPC2, and TPC3 are absent or present. “>” denotes possible presence of additional TPCs.

Fig. 4.

Record depicts the relative contribution to NAADP-evoked global Ca²⁺ waves of quantal Ca²⁺ release from lysosomal stores via TPC2 and that provided by subsequent amplification by Ca²⁺-induced Ca²⁺ release from sarco(endo)plasmic reticulum (S/ER). ER, endoplasmic reticulum.

Fig. 5.

Schematic diagram illustrates the possible relationship between a mobile lysosome and the junctional complex formed between “docked” lysosome clusters and the S/ER. Also depicted is the generation of local, monoquantal Ca²⁺ release (A) and the development of a propagating Ca²⁺ wave by quantal recruitment of inositol 1,4,5-trisphosphate receptor (IP₃R) or ryanodine receptor (RyR) clusters on the S/ER (B). SERCA, S/ER Ca²⁺-ATPase.

Fig. 6.

Cell showing the colocalization between Alexa 488 labeled human TPC2 (green; excitation 488 nm, emission 520 nm) and Texas Red labeled LAMP2 (red; excitation 568 nm, emission 617 nm). Left: confocal Z-section taken through the cell with the distribution of hemagglutinin (HA)-tagged hTPC2 (green). Middle: same as in left, but shows the distribution of LAMP2 labeling (red). Right: a merged image of other two panels with areas of colocalization shown in yellow. In addition to the regular lysosomes, both elements of labeling highlight a very large lysosome-related vesicular structure.