An intracellular P2X receptor required for osmoregulation in Dictyostelium discoideum

Abstract

P2X receptors are membrane ion channels gated by extracellular ATP that are found widely in vertebrates, but not previously in microbes. Here we identify a weakly related gene in the genome of the social amoeba Dictyostelium discoideum, and show, with the use of heterologous expression in human embryonic kidney cells, that it encodes a membrane ion channel activated by ATP (30-100 muM). Site-directed mutagenesis revealed essential conservation of structure-function relations with P2X receptors of higher organisms. The receptor was insensitive to the usual P2X antagonists but was blocked by nanomolar concentrations of Cu2+ ions. In D. discoideum, the receptor was found on intracellular membranes, with prominent localization to an osmoregulatory organelle, the contractile vacuole. Targeted disruption of the gene in D. discoideum resulted in cells that were unable to regulate cell volume in hypotonic conditions. Cell swelling in these mutant cells was accompanied by a marked inhibition of contractile vacuole emptying. These findings demonstrate a new functional role for P2X receptors on intracellular organelles, in this case in osmoregulation.

Figure 1. DdP2X receptor is an ATP-gated ion channel

a, b, Single-channel openings in response to 100 μM ATP recorded from outside-out patches taken from transfected HEK 293 cells. c, Whole-cell current–voltage relationship shows modest inward rectification (red trace). Replacement of extracellular sodium by N-methyl-d-glucamine (each 147 mM) shifts reversal potential from 0 mV to −50 mV (blue trace). The current reverses close to zero with isotonic extracellular Ca²⁺ (green trace). Filled circles indicate amplitudes of unitary currents recorded in outside-out patches. Error bars indicate s.e.m. (n = 6). d, Concentration-dependent responses to ATP. Typical whole-cell recordings of inward currents evoked by different concentrations of ATP applied for 2 s, repeated at 2-min intervals. Cells were held at −60 mV. e, SDS–PAGE western blot analysis of whole-cell lysates from HEK −93 cells expressing His-tagged DdP2X shows bands at 48 and 55 kDa. The mass of both bands was decreased by treatment with PNGaseF (not shown), indicating that both were N-glycosylated, and both bands were present at the cell surface as measured by cell-surface biotinylation. His-tagged DdP2X migrates on perfluoro-octanoic acid (PFO)–PAGE predominantly as a trimer (although dimeric and monomeric species are also detectable, as indicated by the middle and lower arrows). Numbers at the left of the gels are molecular masses in kDa. f, Immunocytochemistry showing predominant localization of the His-tagged DdP2X receptor (red; secondary antibody was Cy3) at the membrane in HEK 293 cells. Scale bar, 10 μm.

Figure 2. Properties of DdP2X receptors

a, Inward currents evoked by ATP and analogues (each 300 μM, for 2 s) in HEK 293 cells expressing DdP2X receptors (holding potential −60 mV). b, Concentration–response curves for experiments such as those shown in a. Responses are normalized to each maximum response and show a rank order of potency βγimidoATP (filled squares) > αβmeATP (open circles) = ATP (filled circles) > βγmeATP (filled diamonds) > benzoyl-benzyl-ATP (BzATP; open squares); each point is the mean ± s.e.m. for five to ten cells. c, Responses to ATP (100 μM) before and after application of Cu²⁺, Ni²⁺ and P2X antagonists. The duration of preapplication of antagonists was 2 min (Cu²⁺ and Ni²⁺) or 4 min (2′,3′-O-(2,4,6-trinitrophenyl)-adenosine 5′-triphosphate (TNP-ATP), pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonate (PPADS) and suramin). d, Representative whole-cell currents evoked by ATP (3 mM) in HEK 293 cells expressing mutant receptors. Lower right: diagram illustrating the topology of the P2X receptor and the relative positions of the residues mutated. e, Comparison of ATP sensitivity between wild-type DdP2X receptors (filled circles) and the K67A (open circles), K289A (filled squares) and F156A (open squares) mutations. Each point is the mean ± s.e.m. for five to ten cells. The immunohistochemical appearance of cells expressing mutated receptors (including D330A) with the anti-His antibody was the same as that of wild-type cells. Biotinylation and SDS–PAGE followed by gel densitometry showed a highly significant expression of wild-type, K67A and D330A receptors at the cell surface.

Figure 3. DdP2X receptors are localized to the contractile vacuole and are required for cell volume regulation and contractile vacuole voiding

a, Co-localization of DdP2X–GFP (left, green) and calmodulin (middle, red), particularly on the membrane of the contractile vacuole (arrowheads indicate one such vacuole). Right, merged image. Scale bar, 10 μm. b, Structure of D. discoideum p2xA gene to illustrate position of cassette insertion. Disruption of the p2xA gene caused no observable differences in cell morphology or developmental cycle (data not shown). c, Bright-field micrographs of D. discoideum in HL5 growth medium, and 60 min after change from medium to distilled water. DdP2X mutant cells are swollen, whereas wild-type cells have recovered their normal volume. Scale bar, 10 μm. d, Time course of cell swelling and recovery. Wild-type cells (red circles) swell for 10–20 min and then regain their normal volume after about 30 min. DdP2X mutant cells (blue circles) continue to swell for 60 min. Wild-type cells treated with Cu²⁺ ions (10 μM; green squares) continue to swell. Mutant cells expressing DdP2X–GFP (black squares) are also similar to wild-type cells except that they show less swelling in the first 10 min. Error bars indicate s.e.m. e, DdP2X receptors are required for normal contractile vacuole voiding. In wild-type amoebae (top row) the vacuoles (arrows) within the cell move to the membrane and void within 20–30 s. In DdP2X-disrupted amoebae (bottom row) this cycle is much prolonged: the vacuole identified takes almost 3 min to discharge. Numbers indicate time in seconds. Scale bar, 5 μm.