Purified TPC Isoforms Form NAADP Receptors with Distinct Roles for Ca 2+ Signaling and Endolysosomal Trafficking

Margarida Ruas,Katja Rietdorf,Abdelilah Arredouani,Lianne C Davis,Emyr Lloyd-Evans,Heidi Koegel,Timothy M Funnell,Anthony J Morgan,John A Ward,Keiko Watanabe,Xiaotong Cheng,Grant C Churchill,Michael X Zhu,Frances M Platt,Gary M Wessel,John Parrington,Antony Galione

doi:10.1016/j.cub.2010.02.049

Abstract

SummaryIntracellular Ca2+ signals constitute key elements in signal transduction. Of the three major Ca2+ mobilizing messengers described, the most potent, nicotinic acid adenine dinucleotide phosphate (NAADP) is the least well understood in terms of its molecular targets [1]. Recently, we showed that heterologous expression of two-pore channel (TPC) proteins enhances NAADP-induced Ca2+ release, whereas the NAADP response was abolished in pancreatic beta cells from Tpcn2 gene knockout mice [2]. However, whether TPCs constitute native NAADP receptors is unclear. Here we show that immunopurified endogenous TPC complexes possess the hallmark properties ascribed to NAADP receptors, including nanomolar ligand affinity [3–5]. Our study also reveals important functional differences between the three TPC isoforms. Thus, TPC1 and TPC2 both mediate NAADP-induced Ca2+ release, but the subsequent amplification of this trigger Ca2+ by IP3Rs is more tightly coupled for TPC2. In contrast, TPC3 expression suppressed NAADP-induced Ca2+ release. Finally, increased TPC expression has dramatic and contrasting effects on endolysosomal structures and dynamics, implicating a role for NAADP in the regulation of vesicular trafficking. We propose that NAADP regulates endolysosomal Ca2+ storage and release via TPCs and coordinates endoplasmic reticulum Ca2+ release in a role that impacts on Ca2+ signaling in health and disease [6].

Highlights

We demonstrated a central role for two-pore channels (TPCs) in the nicotinic acid adenine dinucleotide phosphate (NAADP) response [2], but whether they constitute the actual NAADP receptor remains unresolved
Sea Urchins Express Three TPC Isoforms We screened the Strongylocentrotus purpuratus (Sp) genome [9] and identified three distinct isoforms, SpTPC1, SpTPC2, and SpTPC3, which we cloned from urchin ovary
The antibodies recognized endogenous SpTPC2 in urchin egg membrane preparations, mainly in heavy fractions (P100), containing lysosome-related reserve granules [10], and recognized SpTPC3 in lower-density fractions (S10P100) (Figures 1A and 1B; Figure S1C), which correlates with the highest specific [32P]NAADP binding (Figure 1B)

Summary

Intracellular Ca2+ signals constitute key elements in signal transduction. Of the three major Ca2+ mobilizing messengers described, the most potent, nicotinic acid adenine dinucleotide phosphate (NAADP) is the least well understood in terms of its molecular targets [1]. We showed that heterologous expression of two-pore channel (TPC) proteins enhances NAADP-induced Ca2+ release, whereas the NAADP response was abolished in pancreatic beta cells from Tpcn gene knockout mice [2]. Whether TPCs constitute native NAADP receptors is unclear. We show that immunopurified endogenous TPC complexes possess the hallmark properties ascribed to NAADP receptors, including nanomolar ligand affinity [3,4,5]. Increased TPC expression has dramatic and contrasting effects on endolysosomal structures and dynamics, implicating a role for NAADP in the regulation of vesicular trafficking. We propose that NAADP regulates endolysosomal Ca2+ storage and release via TPCs and coordinates endoplasmic reticulum Ca2+ release in a role that impacts on Ca2+ signaling in health and disease [6]

Results and Discussion

H P10 S10 P100 P100

Conclusions