Permeation properties of a P2X receptor in the green algae Ostreococcus tauri

Abstract

We have cloned a P2X receptor (OtP2X) from the green algae Ostreococcus tauri. The 42-kDa receptor shares approximately 28% identity with human P2X receptors and 23% with the Dictyostelium P2X receptor. ATP application evoked flickery single channel openings in outside-out membrane patches from human embryonic kidney 293 cells expressing OtP2X. Whole-cell recordings showed concentration-dependent cation currents reversing close to zero mV; ATP gave a half-maximal current at 250 mum. alphabeta-Methylene-ATP evoked only small currents in comparison to ATP (EC(50) > 5 mm). 2',3'-O-(4-Benzoylbenzoyl)-ATP, betagamma-imido-ATP, ADP, and several other nucleotide triphosphates did not activate any current. The currents evoked by 300 mum ATP were not inhibited by 100 microm suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, 2',3'-O-(2,4,6-trinitrophenol)-ATP, or copper. Ion substitution experiments indicated permeabilities relative to sodium with the rank order calcium >choline >Tris >tetraethylammonium >N-methyl-D-glucosamine. However, OtP2X had a low relative calcium permeability (P(Ca)/P(Na) = 0.4) in comparison with other P2X receptors. This was due at least in part to the presence of an asparagine residue (Asn(353)) at a position in the second transmembrane domain in place of the aspartate that is completely conserved in all other P2X receptor subunits, because replacement of Asn(353) with aspartate increased calcium permeability by approximately 50%. The results indicate that the ability of ATP to gate cation permeation across membranes exists in cells that diverged in evolutionary terms from animals about 1 billion years ago.

FIGURE 1.

Functional expression of algae P2X receptor. A, single channel recording from outside-out patch from HEK293 cells expressing OtP2X. Currents were evoked by 100 μm ATP (bar); holding potential -100 mV. B, whole-cell currents evoked by ATP (100 and 300 μm, 1, 3, and 5 mm). C, currents were evoked by αβ-methylene-ATP (αβmeATP) (3 mm), but not by UTP or ADP (1 mm). Holding potential -60 mV. D, concentration-response curves for whole-cell currents evoked by ATP (n = 10) and αβ-methylene-ATP (αβmeATP) (n = 8). E, ATP-evoked currents in the presence and absence of suramin, PPADS, or 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) (each at 100 μm). Superimposed records show effects of ATP applied in control conditions and 5 min later in the presence of antagonist. F, anti-Myc Western blot of whole-cell lysates from untransfected and HEK293 cells (-) and cells transfected with Myc-tagged OtP2X (+).

FIGURE 2.

Permeation properties of algae P2X receptor. A, whole-cell currents evoked by ATP (1 mm); holding potential -60 mV. Currents were evoked in external solution containing 147 mm sodium chloride, 98 mm calcium chloride, and 148 mm N-methyl-d-glucosamine; chloride (NMDG). B, current-voltage plots for ATP-evoked currents in external sodium, calcium, or NMDG. Note left shift in reversal potential in calcium (open arrowhead) or NMDG (filled arrowhead). C, permeability of five cations relative to that of sodium as a function of their mean geometric diameter. Points are mean ± S.E. of the mean; n = 8 cells.

FIGURE 3.

Asparagine residue in second transmembrane domain accounts for low calcium permeability. A, ATP (300 μm) evokes currents at OtP2X[N353D] but not at OtP2X[N353A] receptors. B, calcium permeability of OtP2X[N353D] receptors is significantly higher than that of wild-type receptors. Voltage ramps are shown for whole-cell currents evoked by 1 mm ATP for wild-type and OtP2X[N353D] mutant receptors, recorded in 98 mm external calcium. C, alignment of presumed second transmembrane domains of the OtP2X receptor (algae), with those of DdP2xA (dictyostelium, GenBank™ accession code EU047552), a flagellated protozoa (choanoflagellate),⁴ a nematode worm (schistome, GenBank accession code AJ783803), and a mammalian receptor (rat P2X₂, GenBank accession NM_053656). Open arrow indicates aspartate conserved in all P2Xreceptors except OtP2X.