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.

A variety of ligand-gated ion channels undergo a fast activation process after the rapid application of agonist and also a slower transition towards desensitized or inactivated closed channel states when exposure to agonist is prolonged. Desensitization involves at least two distinct closed states in the acetylcholine receptor, each with an affinity for agonists higher than those of the resting or active conformations. Here we investigate how structural elements could be involved in the desensitization of the acetylcholine-gated ion channel from the chick brain alpha-bungarotoxin sensitive homo-oligomeric alpha 7 receptor, using site-directed mutagenesis and expression in Xenopus oocytes. Mutations of the highly conserved leucine 247 residue from the uncharged MII segment of alpha 7 suppress inhibition by the open-channel blocker QX-222, indicating that this residue, like others from MII, faces the lumen of the channel. But, unexpectedly, the same mutations decrease the rate of desensitization of the response, increase the apparent affinity for acetylcholine and abolish current rectification. Moreover, unlike wild-type alpha 7, which has channels with a single conductance level, the leucine-to-threonine mutant has an additional conducting state active at low acetylcholine concentrations. It is possible that mutation of Leu 247 renders conductive one of the high-affinity desensitized states of the receptor.

Introduction by site-directed mutagenesis of three amino acids from the MII segment of glycine or gamma-aminobutyric acid (GABAA) receptors into the MII segment of alpha 7 nicotinic receptor was sufficient to convert a cation-selective channel into an anion-selective channel gated by acetylcholine. A critical mutation was the insertion of an uncharged residue at the amino-terminal end of MII, stressing the importance of protein geometrical constraints on ion selectivity.

The putative channel-forming MU domains of the nicotinic, y-aminobutyric acid type A, and glycine receptors contain a highly conserved leucine residue. Mutation of this hydrophobic amino acid in the neuronal nicotinic receptor a7 (Leu-247), reconstituted in Xenopus oocytes, modifies the ionic response to acetylcholine and alters desensitization. Furthermore, the Leu -+ Thr (L247T) mutant has two conducting states (46 pS and 80 pS), in contrast with the wild-type (WT) receptor, which has only one (45 pS). We now show that this mutant possesses a rather paradoxical pharmacology: antagonists of the WT receptor such as dihydro-flerythroidin, hexamethonium, or (+)-tubocurarine elicit ionic currents when applied to the L247T a7 mutant and these responses are blocked by a-bungarotoxin. Furthermore, prolonged application of acetylcholine causes desensitization in the WT but leads to a potentiation of the responses to acetylcholine or dihydro-13-erythroidin in the mutant. These data are consistent with a scheme in which mutation of Leu-247 renders a desensitized state in the WT channel a conducting state. They also strengthen the proposal that, in the WT, some competitive antagonists may stabilize desensitized states. Finally, these observations may shed light on properties ofother ion channels, in particular the glutamate receptors, which display multiple conductance levels associated with various pharmacological agents.The structural principles by which the nicotinic acetylcholine receptor (AcChoR) mediates the effect of acetylcholine (AcCho) on ion-channel activation and desensitization have been explored by photolabeling methods and site-directed mutagenesis (for reviews, see refs. 1 and 2). In particular, photolabeling with the channel blockers chlorpromazine (3-6) or triphenylmethylphosphonium (7,8) suggested that MII, the second hydrophobic segment of each subunit, was a possible component of the ion channel. Chlorpromazine was found to label three rings of residues from MII, two polar rings and a leucine ring, spaced one a-helical turn apart (3-6). Sitedirected mutagenesis experiments of charged or polar amino acid residues bordering (9, 10) or located within MII (11-13) confirmed and further extended the photolabeling data, strongly supporting the hypothesis that amino acid side chains from MII line the ion channel. Among the three rings of amino acids labeled by chlorpromazine in Torpedo marmorata AcChoR, the hydrophobic leucine ring is conserved at the same position in almost all subunits of nicotinic, glycine, and y-aminobutyric acid type A receptors (14). By using, as a model system, the neuronal homooligomeric and a-bungarotoxin-sensitive a7 nicotinic receptor (15, 16) expressed in Xenopus oocyte, the function of this leucine ring was investigated by site-directed mutagenesis (17). Replacement of Leu-247 with a polar residue (serine or threonine) altered blocking by QX-222 indicating that this residue faces the lumen of the channel. In addition, these mutations abolished current rectification, reduc...

To evaluate the implication of taurine in the physiology of supraoptic neurones, we (i) investigated the agonist properties of taurine on glycine and GABAA receptors of supraoptic magnocellular neurones acutely dissociated from adult rats, using whole‐cell voltage clamp, (ii) studied the effects of taurine and strychnine in vivo by extracellular recordings of supraoptic vasopressin neurones in anaesthetized rats, and (iii) measured the osmolarity‐dependent release of endogenous taurine from isolated supraoptic nuclei by HPLC. GABA, glycine and taurine evoked rapidly activating currents that all reversed close to the equilibrium potential for Cl−, indicating activation of Cl− selective channels. Glycine‐activated currents were reversibly blocked by strychnine (IC50 of 35 nM with 100μm glycine), but were unaffected by the GABAA antagonist gabazine (1–3 μm). GABA‐activated currents were reversibly antagonized by 3 μm gabazine, but not by strychnine (up to 1 μm). Responses to 1 mm taurine were blocked by strychnine but not by gabazine and showed no additivity with glycine‐induced currents, indicating selective activation of glycine receptors. Responses to 10 mm taurine were partially antagonized by gabazine, the residual current being blocked by strychnine. Thus, taurine is also a weak agonist of GABAA receptors. In the presence of gabazine, taurine activated glycine receptors with an EC50 of 406 μm. Taurine activated at most 70% of maximal glycine currents, suggesting that it is a partial agonist of glycine receptors. In vivo, locally applied strychnine (300 mm) increased and taurine (1 mm) decreased the basal electrical activity of vasopressin neurones in normally hydrated rats. The effect of strychnine was markedly more pronounced in water‐loaded rats. Taurine, which is concentrated in supraoptic glial cells, could be released from isolated supraoptic nuclei upon hyposmotic stimulation. Decreases in osmolarity of 15 and 30% specifically enhanced basal release of taurine by 42 and 124%, respectively. We conclude that supraoptic neurones express high amounts of glycine receptors, of which taurine may be regarded as a major natural agonist. We postulate that taurine, which can be released in hyposmotic situations, acts on glycine receptors to exert an inhibitory control on magnocellular neurones during alterations of body fluid homeostasis, implicating an active participation of glial cells in this neuroendocrine regulatory loop.