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At the majority of mutants in the region Glu181-Val200 incorporating a conserved AsnPheThrΦΦxLys motif cysteine substitution had no effect on sensitivity to ATP, partial agonists, or methanethiosulfonate (MTS) compounds. For the F185C mutant the efficacy of partial agonists was reduced by ∼ 90% but there was no effect on ATP potency or the actions of MTS reagents. At T186C, F188C and K190C mutants ATP potency and partial agonists responses were reduced. The ATP sensitivity of the K190C mutant was rescued towards WT levels by positively charged (2-aminoethyl)methanethiosulfonate hydrobromide and reduced by negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate. Both MTS reagents decreased ATP potency at the T186C mutant, and abolished responses at the F195C mutant. 32P-2-azido ATP binding to the mutants T186C and K190C was sensitive to MTS reagents consistent with an effect on binding, however binding at F195C was unaffected indicating an effect on gating. The accessibility of the introduced cysteines was probed with (2-aminoethyl)methanethiosulfonate hydrobromide-biotin, this showed that the region Thr186-Ser192 is likely to form a beta sheet and that accessibility is blocked by ATP. Taken together these results suggest that Thr186, Phe188 and Lys190 are involved in ATP binding to the receptor and Phe185 and Phe195 contribute to agonist evoked conformational changes. [ABSTRACT FROM AUTHOR]

The potentiation of P2X1 receptor currents by phorbol ester (PMA) treatment and stimulation of mGluR1α receptors was sensitive to inhibition of novel forms of protein kinase C. Potentiation was also reduced by co-expression of an amino terminal P2X1 receptor minigene. Cysteine point mutants of residues Tyr16-Gly30 were expressed in Xenopus oocytes. Peak current amplitudes to ATP for Y16C, T18C and R20C mutants were reduced, however this did not result from a decrease in surface expression of the channels. The majority of the mutants showed changes in the time-course of desensitization of ATP evoked currents indicating the important role of this region in regulation of channel properties. PMA and mGluR1α potentiation was abolished for the mutants Y16C, T18C, R20C, K27C and G30C. Minigenes incorporating either Y16C, K27C, V29C or G30C still inhibited PMA responses. However D17C, T18C or R20C mutant minigenes were no longer effective suggesting that these residues are important for interaction with regulatory factors. These results demonstrate that the conserved YXTXK/R sequence and a region with a conserved glycine residue close to the first transmembrane segment contribute to PMA and GPCR regulation of P2X1 receptors. [ABSTRACT FROM AUTHOR]

The role of conserved polar glutamine, asparagine and threonine residues in the large extracellular loop, and glycosylation, to agonist action at human P2X1 receptors was tested by generating alanine substitution mutants. For the majority of mutants (Q56A, Q95A, T104A, T109A, Q112A, Q114A, T146A, N153A, T158A, N184A, N191A, N242A, N300A) alanine substitution had no effect on ATP potency. The mutants Q95A, Q112A, Q114A and T158A showed changes in efficacy for the partial agonists BzATP and Ap5A, suggesting that these polar residues may contribute to the gating of the channel. The mutants T186A, N204A and N290A had six-, three- and 60-fold decreases in ATP potency, respectively. For T186A and N290A, the partial agonists BzATP and Ap5A were no longer agonists but still bind to the receptor as shown by the ability to modulate the response to co-applied ATP. N153, N184 and N242 are glycosylated in the endoplasmic reticulum and N300 acquires complex glycosylation in the golgi. These results aid in refining a model for ATP binding at the P2X1 receptor where the residues F185T186, and the conserved triplet N290F291R292, are likely to play a role in ATP action at the receptor. [ABSTRACT FROM AUTHOR]

Glycine residues can introduce flexibility in proteins, give rise to turns and breaks in secondary structure and are key components of some nucleotide binding motifs. In the P2X receptor extracellular ATP binding domain, 11 glycine residues are completely conserved and an additional five are conserved in at least five of the seven family members. We have mutated individual conserved glycine residues and determined their effect on the ATP sensitivity and time-course of P2X1 receptors expressed in Xenopus oocytes. In the majority of cases, replacement by alanine had no or a less than 3-fold effect on ATP sensitivity and time-course of responses. G71A resulted in a 6-fold decrease in ATP potency and ATP (10 m m) failed to evoke functional responses from G96A, G250A and G301A mutant receptors. However, proline or cysteine could substitute for glycine at positions 96 and 301, giving receptors that were essentially normal. At glycine 250 substitution by serine gave functional responses to ATP with no effect on ATP sensitivity but a reduction in peak amplitude; in contrast, functional responses were not recorded when glycine 250 was replaced by the amino acids alanine, cysteine, aspartate, phenylalanine, isoleucine, lysine, proline or asparagine. These results suggest that glycine 250 plays an important role in determining the function of P2X receptors. [ABSTRACT FROM AUTHOR]

Proline residues can play a major role in the secondary structure of proteins. In the extracellular ATP binding loop of P2X receptors there are four totally conserved proline residues (P2X1 receptor numbering; P93, P166, P228 and P272) and three less conserved residues P196 (six of seven isoforms), P174 and P225 (five of seven isoforms). We have mutated individual conserved proline residues in the human P2X1 receptor and determined their properties. Mutants were expressed inXenopusoocytes and characterized using a two-electrode voltage clamp. Mutants P166A, P174A, P196A, P225A and P228A had no effect on ATP potency compared with wild-typeand P93A had a fourfold decrease in ATP potency. The P272A, P272D and P272K receptor mutants were expressed at the cell surface; however, these mutants were non-functional. In contrast, P272I, P272G and P272F produced functional channels, with either no effect or a 2.5- or 6.5-fold increase in ATP potency, respectively. At P272F receptors the apparent affinity of the ATP analogue antagonist 2′,3′-O-(2,4,6-trinitrophenyl)-ATP was increased by 12.5-fold. These results suggest that individual proline residues are not essential for normal P2X receptor function and that the receptor conformation around P272 contributes to ATP binding at the receptor. [ABSTRACT FROM AUTHOR]

At the majority of mutants in the region Glu181-Val200 incorporating a conserved AsnPheThrΦΦxLys motif cysteine substitution had no effect on sensitivity to ATP, partial agonists, or methanethiosulfonate (MTS) compounds. For the F185C mutant the efficacy of partial agonists was reduced by ∼ 90% but there was no effect on ATP potency or the actions of MTS reagents. At T186C, F188C and K190C mutants ATP potency and partial agonists responses were reduced. The ATP sensitivity of the K190C mutant was rescued towards WT levels by positively charged (2-aminoethyl)methanethiosulfonate hydrobromide and reduced by negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate. Both MTS reagents decreased ATP potency at the T186C mutant, and abolished responses at the F195C mutant. 32P-2-azido ATP binding to the mutants T186C and K190C was sensitive to MTS reagents consistent with an effect on binding, however binding at F195C was unaffected indicating an effect on gating. The accessibility of the introduced cysteines was probed with (2-aminoethyl)methanethiosulfonate hydrobromide-biotin, this showed that the region Thr186-Ser192 is likely to form a beta sheet and that accessibility is blocked by ATP. Taken together these results suggest that Thr186, Phe188 and Lys190 are involved in ATP binding to the receptor and Phe185 and Phe195 contribute to agonist evoked conformational changes. [ABSTRACT FROM AUTHOR]