Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes.

Recombinant FlagHis(6) tagged Human P2X1 receptors expressed in HEK293 cells were purified, digested with trypsin and analysed by mass spectroscopy (96% coverage following de-glycosylation and reduction). The receptor was basally phosphorylated at residues S387, S388 and T389 in the carboxyl terminus, a triple alanine mutant of these residues had a modest ~ 25% increase in current amplitude and recovery from desensitization. Chemical modification showed that intracellular lysine residues close to the transmembrane domains and the membrane stabilization motif are accessible to the aqueous environment.

Heat shock protein 90 inhibitors reduce trafficking of ATP-gated P2X1 receptors and human platelet responsiveness.

We have used selective inhibitors to determine whether the molecular chaperone heat shock protein 90 (HSP90) has an effect on both recombinant and native human P2X1 receptors. P2X1 receptor currents in HEK293 cells were reduced by ∼70-85% by the selective HSP90 inhibitor geldanamycin (2 μM, 20 min). This was associated with a speeding in the time course of desensitization as well as a reduction in cell surface expression.

Agonist binding evokes extensive conformational changes in the extracellular domain of the ATP-gated human P2X1 receptor ion channel.

P2X receptors for ATP have a wide range of physiological roles and comprise a structurally distinct family of ligand-gated trimeric ion channels. The crystal structure of a P2X4 receptor, in combination with mutagenesis studies, has provided a model of the intersubunit ATP-binding sites and identified an extracellular lateral portal, adjacent to the membrane, that funnels ions to the channel pore. However, little is known about the extent of ATP-induced conformational changes in the extracellular domain of the receptor.

Identification of human P2X1 receptor-interacting proteins reveals a role of the cytoskeleton in receptor regulation.

P2X1 receptors are ATP-gated ion channels expressed by smooth muscle and blood cells. Carboxyl-terminally His-FLAG-tagged human P2X1 receptors were stably expressed in HEK293 cells and co-purified with cytoskeletal proteins including actin. Disruption of the actin cytoskeleton with cytochalasin D inhibited P2X1 receptor currents with no effect on the time course of the response or surface expression of the receptor.

Contribution of the region Glu181 to Val200 of the extracellular loop of the human P2X1 receptor to agonist binding and gating revealed using cysteine scanning mutagenesis.

At the majority of mutants in the region Glu181-Val200 incorporating a conserved AsnPheThrPhiPhixLys 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 approximately 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.

Cysteine substitution mutagenesis and the effects of methanethiosulfonate reagents at P2X2 and P2X4 receptors support a core common mode of ATP action at P2X receptors.

The agonist binding site of ATP-gated P2X receptors is distinct from other ATP-binding proteins. Mutagenesis on P2X(1) receptors of conserved residues in mammalian P2X receptors has established the paradigm that three lysine residues, as well as FT and NFR motifs, play an important role in mediating ATP action. In this study we have determined whether cysteine substitution mutations of equivalent residues in P2X(2) and P2X(4) receptors have similar effects and if these mutant receptors can be regulated by charged methanethiosulfonate (MTS) compounds.

Cysteine substitution mutants give structural insight and identify ATP binding and activation sites at P2X receptors.

P2X receptors for extracellular ATP are a distinct family of ligand-gated cation channels involved in physiological processes ranging from synaptic transmission to muscle contraction. Common ATP binding motifs are absent from P2X receptors, and the extent of the agonist binding site is unclear. We used cysteine-scanning mutagenesis, radiolabeled 2-azido ATP binding, and methanethiosulfonate (MTS) compounds to identify amino acid residues involved in ATP binding and gating of the human P2X1 receptor.

Molecular properties of P2X receptors.

P2X receptors for adenosine tri-phosphate (ATP) are a distinct family of ligand-gated cation channels with two transmembrane domains, intracellular amino and carboxy termini and a large extracellular ligand binding loop. Seven genes (P2X(1-7)) have been cloned and the channels form as either homo or heterotrimeric channels giving rise to a wide range of phenotypes. This review aims to give an account of recent work on the molecular basis of the properties of P2X receptors.

Contribution of conserved polar glutamine, asparagine and threonine residues and glycosylation to agonist action at human P2X1 receptors for ATP.

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.

Contribution of conserved glycine residues to ATP action at human P2X1 receptors: mutagenesis indicates that the glycine at position 250 is important for channel function.

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.

Mutagenesis studies of conserved proline residues of human P2X receptors for ATP indicate that proline 272 contributes to channel function.

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.

Molecular properties of ATP-gated P2X receptor ion channels.

P2X receptors for ATP are expressed throughout the body and mediate a multitude of functions, including muscle contraction, neuronal excitability and bone formation. In the mid-1990s seven genes encoding P2X receptors (P2X(1-7)) were identified. These receptors comprised a novel family of ligand-gated ion channels with subunits that possessed intracellular N- and C-termini, two transmembrane domains and an extracellular ligand-binding loop.

Analysis of the membrane topology of the acid-sensing ion channel 2a.

Acid-sensing ion channels, or ASICs, are members of the amiloride-sensitive cationic channel superfamily that are predicted to have intracellular amino and carboxyl termini and two transmembrane domains connected by a large extracellular loop. This prediction comes from biochemical studies of the mammalian epithelial sodium channels where glycosylation mutants identified the extracellular regions of the channel and a combination of antibody sensitivity and protease action substantiated the intracellular nature of the amino and carboxyl termini. However, although there are highly conserved regions within the different cation channel family members, membrane topology prediction programs provide several alternative structures for the ASICs.

ATP binding at human P2X1 receptors. Contribution of aromatic and basic amino acids revealed using mutagenesis and partial agonists.

P2X receptors comprise a family of ATP-gated ion channels with the basic amino acids Lys-68, Arg-292, and Lys-309 (P2X(1) receptor numbering) contributing to agonist potency. In many ATP-binding proteins aromatic amino acids coordinate the binding of the adenine group. There are 20 conserved aromatic amino acids in the extracellular ligand binding loop of at least 6 of the 7 P2X receptors.

Involvement of T-type calcium channels in excitatory junction potentials in rat resistance mesenteric arteries.

1. We investigated the role of voltage-operated calcium channels in sympathetic transmission and depolarization-induced contractions in the rat mesenteric artery. In particular, we investigated the role of the T-type voltage-operated calcium channels (T-channels) in mediating excitatory junction potentials (EJPs).