Caterina Virginio

The Permeabilizing ATP Receptor, P2X7 CLONING AND EXPRESSION OF A HUMAN cDNA

A cDNA was isolated from a human monocyte library that encodes the P2X7 receptor; the predicted protein is 80% identical to the rat receptor. Whole cell recordings were made from human embryonic kidney cells transfected with the human cDNA and from human macrophages. Brief applications (1-3 s) of ATP and 2′,3′-(4-benzoyl)-benzoyl-ATP elicited cation-selective currents. When compared with the rat P2X7 receptor, these effects required higher concentrations of agonists, were more potentiated by removal of extracellular magnesium ions, and reversed more rapidly on agonist removal. Longer applications of agonists permeabilized the cells, as evidenced by uptake of the propidium dye YO-PRO1, but this was less marked than for cells expressing the rat P2X7 receptor. Expression of chimeric molecules indicated that some of the differences between the rat and human receptor could be reversed by exchanging the intracellular C-terminal domain of the proteins.

Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X7 receptor

1 Extracellular ATP acting on P2X7 receptors opens a channel permeable to small cations, creates an access pathway for the entry of larger molecular weight dyes, and causes cell death. We used whole‐cell recording and fluorescence microscopy to measure the time courses of ionic currents, uptake of the propidium dye YO‐PRO‐1, and membrane disruption, in human embryonic kidney (HEK293) cells expressing the rat P2X7 receptor. 2 The ATP analogue 2′,3′‐O‐(benzoyl‐4‐benzoyl)‐ATP (30 μm) induced membrane blebbing within 30‐40 s of sustained application; this was 5‐10 times slower when extracellular sodium was replaced by larger cations. 3 Fluorescence of YO‐PRO‐1 was detectable within 3 s, and the uptake reached a steady rate within 10‐20 s; YO‐PRO‐1 uptake was greatly enhanced by removing extracellular sodium. 4 Electrophysiological measurements of current reversal potentials with intracellular sodium and extracellular cations of different sizes showed that the ionic channel progressively dilated during 10‐20 s to a diameter greater than 1 nm (10 Å). With short agonist applications (3‐5 s) the pore dilatation and YO‐PRO‐1 uptake were reversible and repeatable. 5 Polyethylene glycols having molecular weights ≥ 5000 blocked the increase in cation permeability, YO‐PRO‐1 uptake and membrane blebbing. 6 We conclude that maximum P2X7 receptor activation causes an exponential dilatation of the ion channel with a time constant of 25 s to a final diameter of 3‐5 nm from an initial minimum pore diameter of 0.8 nm.

Ionic permeability of, and divalent cation effects on, two ATP-gated cation channels (P2X receptors) expressed in mammalian cells.

1. Complementary DNAs for the ATP‐gated ion channel subunits P2X1 (from human bladder) and P2X2 (from rat phaeochromocytoma (PC12) cells) were used to express the receptors in human embryonic kidney cells by stable transfection, and in Chinese hamster ovary cells by viral infection. 2. Membrane currents evoked by ATP were recorded by the whole‐cell patch clamp method. The reversal potential of the current was measured with various intracellular and extracellular solutions and used to compute the relative permeability of the P2X receptor channels. 3. There was no difference between the two receptors with respect to their permeability to monovalent organic cations. The relative permeabilities (PX/PNa) were 2.3, 1.0, 1.0, 0.95, 0.72, 0.5, 0.29, 0.16, 0.04 and 0.03 for guanidinium, potassium, sodium, methylamine, caesium, dimethylamine, 2‐methylethanolamine, tris(hydroxymethyl)‐aminomethane, tetraethylammonium and N‐methyl‐D‐glucamine, respectively (values for P2X2 receptor). 4. The calcium permeability of P2X1 receptors was greater than that of P2X2 receptors. Under biionic conditions (112 mM calcium outside, 154 mM sodium inside), PCa/PNa values were 3.9 and 2.2, respectively (corrected for ionic activities). 5. ATP‐evoked currents in cells expressing the P2X2 receptor were strongly inhibited when the extracellular calcium concentration was increased (0.3‐30 mM); the action of ATP could be restored by increasing the ATP concentration. ATP‐evoked currents in cells expressing the P2X1 receptor were not inhibited by such increases in the extracellular calcium concentration.

Calcium permeability and block at homomeric and heteromeric P2X2 and P2X3 receptors, and P2X receptors in rat nodose neurones

1 Whole‐cell recordings were made from HEK 293 (human embryonic kidney) cells stably transfected with cDNAs encoding P2X2, P2X3 or both receptors (P2X2/3) and from cultured rat nodose neurones. Nodose neurones all showed immunoreactivity for both P2X2 and P2X3, but not P2X1, receptors. 2 Reversal potentials were measured in extracellular sodium, N‐methyl‐D‐glucamine (NMDG) and NMDG containing 5 mM Ca2+; the values were used to compute relative permeabilities (PNMDG/PNa and PCa/PNa). PNMDG/PNa was not different for P2X2, P2X2/3 and nodose neurones (0.03) but was significantly higher (0.07) for P2X3 receptors. PCa/PNa was not different among P2X3, P2X2/3 and nodose neurones (1.2‐1.5) but was significantly higher (2.5) for P2X2 receptors. 3 External Ca2+ inhibited purinoceptor currents with half‐maximal concentrations of 5 mM at the P2X2 receptor, 89 mM at the P2X3 receptor and 15 mM at both the P2X2/3 heteromeric receptor and nodose neurones. In each case, the inhibition was voltage independent and was overcome by increasing concentrations of agonist. 4 These results may indicate that Ca2+ permeability of the heteromeric (P2X2/3) channel is dominated by that of the P2X3 subunit, while Ca2+ block of the receptor involves both P2X2 and P2X3 subunits. The correspondence in properties between P2X2/3 receptors and nodose ganglion neurones further supports the conclusion that the native α,β‐methylene ATP‐sensitive receptor is a P2X2/3 heteromultimer.

Membrane Topology of an ATP-gated Ion Channel (P2X Receptor)

Western blots of Xenopus oocyte membrane preparations showed that the apparent molecular mass of the wild type P2X2 receptor (about 65 kDa) was reduced by pretreatment with endoglycosidase H. Mutagenesis of one or more of three potential asparagines (N182S, N239S, and N298S) followed by Western blots showed that each of the sites was glycosylated in the wild type receptor. Functional channels were formed by receptors lacking any single asparagine, but not by channels mutated in two or three positions. Artificial consensus sequences (N-X-S/T) introduced into the N-terminal region (asparagine at position 9, 16, or 26) were not glycosylated. Asparagines were glycosylated when introduced at the C-terminal end of the first hydrophobic domain (positions 62 and 66) and at the N-terminal end of the second hydrophobic domain (position 324). A protein in which the C terminus of one P2X2 subunit was joined to the N terminus of a second P2X2 subunit (from a concatenated cDNA) had twice the molecular mass of the P2X2 receptor subunit, and formed fully functional channels. The experiments provide direct evidence for the topology originally proposed for the P2X receptor, with intracellular N and C termini, two membrane-spanning domains, and a large extracellular loop.

The antagonist trinitrophenyl-ATP reveals co-existence of distinct P2X receptor channels in rat nodose neurones

1 Whole‐cell recordings were made from rat nodose ganglion neurones in culture and from human embryonic kidney (HEK293) cells stably transfected to express P2X2, P2X3 or both receptor subunits. We examined the blocking actions of 2′,3′‐O‐trinitrophenyl‐ATP (TNP‐ATP) on currents evoked by the agonists ATP and α,β‐methylene ATP. 2 In cells expressing only P2X2 or P2X3 receptor subunits, the inhibition by TNP‐ATP was fitted by a single binding site model with half‐maximal concentrations of about 3 μM and 3 nM, respectively. In cells expressing both P2X2 and P2X3 receptor subunits, currents showed little or no desensitization, thus excluding contributions from homomeric P2X3 receptors. When α,β‐methylene ATP was the agonist (activating heteromeric P2X2/3 receptors), the inhibition by TNP‐ATP conformed to a single binding site (half‐maximal concentration about 3 nM). When ATP (30 μM) was the agonist, activating both heteromeric P2X2/3 as well as homomeric P2X2 receptors, the inhibition curve was biphasic (half‐maximal concentrations about 3 nM and 3 μM); the proportion of high affinity sites in all six cells tested was about 40 %. 3 In nodose ganglion neurones, the inhibition by TNP‐ATP of currents evoked by ATP (30 μM) was also clearly biphasic. In this case, individual neurones showed more variability in the proportion of high and low affinity sites for TNP‐ATP. 4 We conclude that more than one form of multimeric P2X receptor channels are functionally expressed on the cell bodies of individual nodose ganglion neurones. On the basis of sensitivity to TNP‐ATP, and other properties, one of these may correspond to the homomeric P2X2 receptor and the other(s) to heteromeric P2X2/3 receptors.