Extracellular ATP exerts its effects through P2 purinoceptors: these are ligand-gated ion channels (P2x) or G-protein-coupled receptors (P2Y, P2U). ATP at P2x receptors mediates synaptic transmission between neurons and from neurons to smooth muscle, being responsible, for example, for sympathetic vasoconstriction in small arteries and arterioles. We have now cloned a complementary DNA encoding the P2x receptor from rat vas deferens and expressed it in Xenopus oocytes and mammalian cells. ATP activates a cation-selective ion channel with relatively high calcium permeability. Structural predictions suggest that the protein (399 amino acids long) is mostly extracellular and contains only two transmembrane domains plus a pore-forming motif which resembles that of potassium channels. The P2x receptor thus defines a new family of ligand-gated ion channels.

cDNA and genomic clones encoding alpha 7, a novel neuronal nicotinic acetylcholine receptor (nAChR) alpha subunit, were isolated and sequenced. The mature alpha 7 protein (479 residues) has moderate homology with all other alpha and non-alpha nAChR subunits and probably assumes the same transmembrane topology. alpha 7 transcripts transiently accumulate in the developing optic tectum between E5 and E16. They are present in both the deep and the superficial layers of E12 tectum. In Xenopus oocytes, the alpha 7 protein assembles into a homo-oligomeric channel responding to acetylcholine and nicotine. The alpha 7 channel desensitizes very rapidly, rectifies strongly above -20 mV, and is blocked by alpha-bungarotoxin. A bacterial fusion protein encompassing residues 124-239 of alpha 7 binds labeled alpha-bungarotoxin. We conclude that alpha-bungarotoxin binding proteins in the vertebrate nervous system can function as nAChRs.

The major brain nicotinic acetylcholine receptor is assembled from two subunits termed a4 and nal. When expressed in Xenopus oocytes, these subunits reconstitute a functional acetylcholine receptor that is inhibited by progesterone levels similar to those found in serum. In this report, we show that the steroid interacts with a site located on the extracellular part of the protein, thus confirming that inhibition by progesterone is not due to a nonspecific perturbation of the membrane bilayer or to the activation of second messengers. Because inhibition by progesterone does not require the presence of agonist, is voltage-independent, and does not alter receptor desensitization, we conclude that the steroidis not an open channel blocker. In addition, we show that progesterone is not a competitive inhibitor but may interact with the acetylcholine binding site and that its effect is independent of the ionic permeability of the receptor.Steroid hormones are synthesized from cholesterol in the adrenal gland (the gluco-and mineralocorticoids) and in the gonads and placenta (androgens and estrogens). Their lipophilicity explains their passage of the blood-brain barrier. Steroids have also been demonstrated to reduce brain activity and alphaxalone is used clinically. The potency of anesthetics has been correlated with their liposolubility (2, 3), yet the molecular basis of these effects remains obscure.Some steroids, including 5a-pregnan-3a-hydroxy-20-one, enhance chloride fluxes at the y-aminobutyric acid (GABA) synapses of the type A GABA (GABAA) receptors (4, 5) and in transfected cells expressing GABAA receptors (6). In addition, progesterone attenuates cation fluxes evoked by excitatory amino acids in cerebellar Purkinje cells (7) and current induced by acetylcholine (AcCho) in chromaffin cells (8) and in reconstituted brain nicotinic AcCho receptor (nAcChoR) (9). In contrast, pregnenolone inhibits GABAA receptors in rat cortex neurons (10) and a progesteroneinduced reduction of glycine-evoked current has been reported for spinal cord neurons (11).The aim of this work is to investigate the mode of action of progesterone on the major brain neuronal nAcChoR a4/nal reconstituted in Xenopus oocytes. MATERIALS AND METHODSOocyte preparation and recording procedures were as described (12). Data acquisition, storage, and analysis were done on an IBM-PC/AT using the software DATAC (13).Inhibition curves were fitted to the empirical Hill equation. Inhibition by progesterone as a function of the AcCho concentration was fitted by using a Michaelis equation in the form:where y is the normalized current, a is a constant, x is the AcCho concentration, and IC50 is apparent inhibition constant. All steroids were purchased from Sigma. Stock solutions of steroids and progesterone-3-(O-carboxymethyl)oxime (P-3; Sigma P-3277) were at 0.01 M, in ethanol. Stock solutions were kept at -20TC and diluted in OR-2 (12) just before use. P-3-conjugated bovine serum albumin (P-3-BSA; Sigma P-4778, progesterone/BSA ratio = 38:1) and Hahyd...

In the immune system, apoptosis is involved in intrathymic elimination of self-reactive thymocytes and in peripheral T cell tolerance to exogenous antigens. Here, we describe the role in T cell apoptosis of P(2x1), a nonselective cation channel activated by ATP. P(2X1) molecules are up-regulated in thymocytes during dexamethasone-induced apoptosis, and antagonists to these receptors protect thymocytes from cell death. Moreover, P(2X1) mRNA and protein levels increase in thymocytes induced to die in vivo by the superantigen staphylococcal enterotoxin B. In contrast, T cells undergoing apoptosis in the periphery do not express P(2X1). The demonstration that P(2X1) ion channels play a role in the apoptosis of thymocytes but not peripheral T cells illustrates a novel mechanism contributing to thymocyte cell death and opens new possibilities for investigating clonal deletion in the thymus.

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