ATP-gated ionotropic receptors (P2X receptors) are distributed widely in the nervous system. For example, a hetero-oligomeric receptor containing both P2X2 and P2X3 subunits is involved in primary afferent sensation. Each subunit has two membrane-spanning domains. We have used disulfide bond formation between engineered cysteines to demonstrate close proximity between the outer ends of the first transmembrane domain of one subunit and the second transmembrane domain of another. After expression in HEK 293 cells of such modified P2X2 or P2X4 subunits, the disulfide bond formation is evident because an ATP-evoked channel opening requires previous reduction with dithiothreitol. In the hetero-oligomeric P2X2/3 receptor the coexpression of doubly substituted subunits with wild-type partners allows us to deduce that the hetero-oligomeric channel contains adjacent P2X3 subunits but does not contain adjacent P2X2 subunits. The results suggest a "head-to-tail" subunit arrangement in the quaternary structure of P2X receptors and show that a trimeric P2X2/3 receptor would have the composition P2X2(P2X3)2.

The first hydrophobic segment of the rat P2X 2 receptor extends from residue Leu 29 to Val 51 . In the rat P2X 2 receptor, we mutated amino acids in this segment and adjoining flanking regions (Asp 15 through Thr 60 ) individually to cysteine and expressed the constructs in human embryonic kidney cells. Whole-cell recordings were used to measure membrane currents evoked by brief (2-s) applications of ATP (0.3-100 M). Currents were normal except for Y16C, R34C, Y43C, Y55C, and Q56C (no currents but normal membrane expression by immunohistochemistry), Q37C (small currents), and F44C (normal current but increased sensitivity to ATP, as well as ␣␤-methylene-ATP). We used methanethiosulfonates of positive, negative, or no charge to test the accessibility of the substituted cysteines. D15C, P19C, V23C, V24C, G30C, Q37C, F44C, and V48C were strongly inhibited by neutral, membrane-permeant methanethiosulfonates. Only V48C was also inhibited by positively and negatively charged methanethiosulfonates, consistent with an extracellular position; however, accessibility of V48C was increased by channel opening. V48C could disulfide with I328C, as shown by the large increase in ATPevoked current caused by reducing agents. The results suggest that Val 48 at the outer end of the first hydrophobic segment takes part in the gating movement of channel opening.P2X receptors are a family of multimeric membrane proteins that function as ion channels gated by extracellular ATP. Hydrophobicity plots for P2X receptors suggest that two parts of the protein are sufficiently long and hydrophobic to cross the plasma membrane (1). and Ile 331 faces the extracellular aspect. First, antibodies against N-and C-terminal epitopes work only in permeabilized cells (2). Second, the proteins can be glycosylated at both natural and artificially introduced consensus sequences (NX(S/T)) at several positions in the extracellular domain from Pro 62 to Lys 324 (in the P2X 2 receptor), though not at such positions in the N terminus (positions 9, 16, or 26) (2-4). Third, concatenated cDNAs in which the C terminus of one construct is joined to the N terminus of a second form functional channels (5).There is now biochemical evidence that the P2X receptors form channels as trimers (6, 7). However, the parts of the individual subunits that contribute to different functions of the receptor are little understood. Mutations of several positively charged residues have been shown to decrease the effectiveness of ATP as an agonist at the P2X 1 (8) and P2X 2 (9) receptors, and these residues occupy corresponding positions (e. 67 is mutated to cysteine (I67C). However, the attachment of a negatively charged methanethiosulfonate ((2-sulfonatoethyl) methanethiosulfonate; MTSES 1 ) led to a parallel rightward shift in the ATP concentration-response curve that was not seen with neutral (methyl methanethiosulfonate; MTSM) or positively charged methanethiosulfonate ([2-(trimethylammonium)ethyl] methanethiosulfonate; MTSET). Point mutations that introduced a negative charge (I6...

SUMMARY Ischemic pain – examples include the chest pain of a heart attack and the leg pain of a 30 second sprint – occurs when muscle gets too little oxygen for its metabolic need. Lactic acid cannot act alone to trigger ischemic pain because the pH change is so small. Here we show that another compound released from ischemic muscle, ATP (adenosine tri-phosphate), works together with acid by increasing the pH sensitivity of ASIC3 (acid sensing ion channel #3), the molecule used by sensory neurons to detect lactic acidosis. Our data argue that ATP acts by binding to P2X receptors that form a molecular complex with ASICs; the receptor on sensory neurons appears to be P2X5, an electrically quiet ion channel. Coincident detection of acid and ATP should confer sensory selectivity for ischemia over other conditions of acidosis.

ATP-gated P2X 7 purinoceptors are found in most immune cells of the periphery and the brain where their activation leads to multiple downstream events such as cell permeabilization, apoptosis, and/or cytokine release. P2X 7 receptors do not form heteromeric receptors with any of the other six P2X subunits, and it is not known what type of homomeric assemblies the P2X 7 subunit makes. We constructed and purified an ectodomain protein of the rat P2X 7 receptor (amino acids 60 -323) and used this to generate a monoclonal antibody (Ab) with which to probe P2X 7 receptors in central and peripheral immune cells. In HEK cells expressing rat P2X 7 receptors, the Ab increased the maximum current evoked by BzATP by 3-8-fold with a 5-fold leftward shift in EC 50 concentration. This Ab recognized only a nondenatured, multimeric form of the receptor on blue native-PAGE but did not recognize the denatured form on SDS-PAGE. A C-terminal polyclonal P2X 7 Ab recognized both monomeric subunits on SDS-PAGE and a multimeric complex on blue native-PAGE in this heterologous expression system. With Western blotting using these two Abs, native P2X 7 receptors in peritoneal macrophage and bone marrow cells are shown to exist as a strongly bound multimeric complex, whereas P2X 7 receptors in brain glia and/or astrocytes appear to form only as monomeric subunits.Like most neurotransmitters, extracellular ATP activates both metabotropic (G protein-coupled) receptors, the P2Y receptor family, and ionotropic (ligand-gated) receptors, the P2X receptor family (1). When compared with other ionotropic receptors (e.g. nicotinic), which are primarily or solely expressed in nerve and muscle, P2X receptors are unusual in their wide spread expression in both excitable and non-excitable cells (1-3). There are seven P2X receptors, six of which (P2X 1-6 ) are found in brain and peripheral neurons; additionally, mRNA for P2X 1 , P2X 4 , and the non-neuronal P2X 7 receptor are prominent in many immune cells, particularly monocytes, macrophage, bone marrow, and brain microglia (1-3).The P2X 7 receptor shows 40 -45% amino acid identity with any one of the other P2X receptors; it shares the overall membrane topology of two transmembrane domains, intracellular N and C termini, and the large ectodomain with conservation of the 10 extracellular cysteine residues, although its C-terminal is some 200 amino acids longer than other P2X receptors (1-3). However, the functional sequelae of P2X 7 receptor activation differ strikingly from other P2X receptors. Activation of P2X 7 receptors expressed in mammalian cells, such as HEK 1 or CHO cells, opens both a small cationic channel and a large (up to 900 Da) dye-permeable pore, followed within seconds by extensive membrane blebbing (4, 5); similar events occur upon activation of native P2X 7 receptors in some but not all immune cells expressing this receptor (6 -8). The mechanism(s) underlying formation of the permeabilizing pore and other downstream events such as membrane blebbing are not known but the intracellular ...

1 The morphological pattern and motor correlates of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) innervation in the human isolated gastric fundus was explored. 2 By using the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-diaphorase and speci®c rabbit polyclonal NO-synthase (NOS) and VIP antisera, NOS-and VIP-containing varicose nerve ®bres were identi®ed throughout the muscle layer or wrapping ganglion cell bodies of the myenteric plexus. NOS-immunoreactive (IR) neural cell bodies were more abundant than those positive for VIP-IR. The majority of myenteric neurones containing VIP coexpressed NADPHdiaphorase. 3 Electrical stimulation of fundus strips caused frequency-dependent NANC relaxations. N G -nitro-L-arginine (L-NOARG: 300 mM) enhanced the basal tone, abolished relaxations to 0.3 ± 3 Hz (5 s) and those to 1 Hz (5 min), markedly reduced (*50%) those elicited by 10 ± 50 Hz, and unmasked or potentiated excitatory cholinergic responses at frequencies 51 Hz. L-NOARG-resistant relaxations were virtually abolished by VIP (100 nM) desensitization at all frequencies. 4 Relaxations to graded low mechanical distension (41 g) were insensitive to tetrodotoxin (TTX: 1 mM) and L-NOARG (300 mM), while those to higher distensions (2 g) were slightly inhibited by both agents to the same extent (*25%). 5 In the human gastric fundus, NOS-and VIP immunoreactivities are colocalized in the majority of myenteric neurones. NO and VIP mediate electrically evoked relaxations: low frequency stimulation, irrespective of the duration, caused NO release only, whereas shortlasting stimulation at high frequencies induced NO and VIP release. Relaxations to graded mechanical distension were mostly due to passive viscoelastic properties, with a slight NO-mediated neurogenic component at 2 g distension. The di erence between NO and VIP release suggests that in human fundus accommodation is initiated by NO.