An ancestral deuterostome family of two-pore channels mediates nicotinic acid adenine dinucleotide phosphate-dependent calcium release from acidic organelles

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent and widespread calcium-mobilizing messenger, the properties of which have been most extensively described in sea urchin eggs. The molecular basis for calcium release by NAADP, however, is not clear and subject to controversy. Recent studies have provided evidence that members of the two-pore channel (TPC) family in mammals are the long sought after target channels for NAADP. Here, we show that the TPC3 gene, which has yet to be functionally characterized, is present throughout the deuterostome lineage but is a pseudogene in humans and other primates. We report the molecular cloning of the complete ancestral TPC gene family from the sea urchin and demonstrate that all three isoforms localize to acidic organelles to mediate NAADP-dependent calcium release. Our data highlight the functional divergence of this novel gene family during deuterostome evolution and provide further evidence that NAADP mediates calcium release from acidic stores through activation of TPCs.

FIGURE 1.

TPC3 is a pseudogene in primates. A and B, deuterostome phylogeny of TPC genes (unscaled). Each isoform is represented by a numbered square. The TPC3 gene complement of mammals is shaded. B, chromosomal synteny between cow and human genomes in the vicinity of the TPC3 gene. Chr. 11, chromosome 11; Chr. 2, chromosome 2; B. taurus, Bos taurus; H. sapiens, Homo sapiens. C, multiple sequence alignment of translated amino acid sequences of cow TPC3 (BtaTPC3) and TPC3 pseudogenes from human (Hsa), monkey (Mmu), and chimpanzee (Ptr). Missing amino acids resulting from nucleotide deletion in primate genomes are marked with asterisks. The reading frame was manually adjusted at these positions to maximize subsequent sequence alignment with the cow sequence. In-frame stop codons (after adjustment) are indicated with filled circles.

FIGURE 2.

Sea urchin TPCs localize to acidic organelles. A–C, confocal fluorescence images of X. laevis oocytes (A), SKBR3 cells (B), and HEK cells (C) expressing the indicated sea urchin TPC fusion protein. A, images of the animal hemisphere taken in lateral (xy, scale = 20 μm) and axial (xz, scale = 10 μm) sections. The arrow indicates the position the of coverslip in z-scan (depth). In B, cells were loaded with LysoTracker Red, whereas in C, the cells were cotransfected with human TPCs.

FIGURE 3.

Sea urchin TPCs mediate NAADP-dependent calcium release from acidic calcium stores. A–D, cytosolic calcium responses of individual fura-2-loaded SKBR3 cells microinjected with either 10 nm NAADP (A–C) or 50 nm cADPR (D). Cells were from mock-transfected cultures (MOCK) or cultures expressing the indicated GFP-tagged TPC construct from sea urchin (SpTPC1–3) or human (HsTPC1–2). In some experiments, cells were pretreated with bafilomycin A1 (1 μm, 60 min) prior to injection. Data are expressed as mean fluorescence ratios (±S.E.) from 4–12 cells.