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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tips.2010.02.004 Byline: Liam E. Browne (1), Lin-Hua Jiang (2), R. Alan North (1) Abstract: P2X receptors are ATP-gated membrane ion channels with multifarious roles, including afferent sensation, autocrine feedback loops, and inflammation. Their molecular operation has been less well elucidated compared with other ligand-gated channels (nicotinic acetylcholine receptors, ionotropic glutamate receptors). This will change with the recent publication of the crystal structure of a closed P2X receptor. Here we re-interpret results from 15 years of experiments using site-directed mutagenesis with a model based on the new structure. Previous predictions of receptor stoichiometry, the extracellular ATP binding site, inter-subunit contacts, and many details of the permeation pathway fall into place in three dimensions. We can therefore quickly understand how the channel operates at the molecular level. This is important not only for ion- channel aficionados, but also those engaged in developing effective antagonists at P2X receptors for potential therapeutic use. Author Affiliation: (1) Faculty of Medical and Human Sciences, University of Manchester, UK (2) Faculty of Biological Sciences, University of Leeds, UK

Author(s): Samuel J. Fountain [1]; Katie Parkinson [1]; Mark T. Young [1]; Lishuang Cao [1]; Christopher R. L. Thompson (corresponding author) [1]; R. Alan North (corresponding author) [1] P2X receptors [...]

Transient currents occur at rest in cortical neurones that reflect the quantal release of transmitters such as glutamate and [gamma]-aminobutyric acid (GABA). We found a bimodal amplitude distribution for spontaneously occurring inward currents recorded from mouse pyramidal neurones in situ, in acutely isolated brain slices superfused with picrotoxin. Larger events were blocked by glutamate receptor (AMPA, kainate) antagonists; smaller events were partially inhibited by P2X receptor antagonists suramin and PPADS. The decay of the larger events was selectively prolonged by cyclothiazide. Stimulation of single intracortical axons elicited quantal glutamate-mediated currents and also quantal currents with amplitudes corresponding to the smaller spontaneous inward currents. It is likely that the lower amplitude spontaneous events reflect packaged ATP release. This occurs with a lower probability than that of glutamate, and evokes unitary currents about hall the amplitude of those mediated through AMPA receptors. Furthermore, the packets of ATP appear to be released from vesicle in a subset of glutamate-containing terminals.

Author(s): Baljit S. Khakh (corresponding author) [1, 2]; R. Alan North (corresponding author) [3] The nucleotide ATP is the source of free energy used to maintain order within most cells, [...]

Activation of membrane P2[X.sub.7] receptors by extracellular ATP [or its analog 2',3'-O-(4-benzoylbenzoyl)-ATP] results in the opening within several milliseconds of an integral ion channel that is permeable to small cations. If the ATP application is maintained for several seconds, two further sequelae occur: there is a gradual increase in permeability to the larger cation N-methyl-D-glucamine and the cationic propidium dye quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl] -1-[3-(triethylammonio)propyl]diiodide (YO-PRO-1) enters the cell. The similarity in the time course of these two events has led to the widespread view that N-methyl-D-glucamine and YO-PRO-1 enter through a common permeation pathway, the 'dilating' P2[X.sub.7] receptor pore. Here we provide two independent lines of evidence against this view. We studied single human embryonic kidney cells expressing rat P2[X.sub.7] receptors with patch-clamp recordings of membrane current and with fluorescence measurements of YO-PRO-1 uptake. First, we found that maintained application of the ATP analog did not cause any increase in N-methyl-D-glucamine permeability when the extracellular solution contained its normal sodium concentration, although YO-PRO-1 uptake was readily observed. Second, we deleted a cysteine-rich 18-amino acid segment in the intracellular juxtamembrane region of the P2[X.sub.7] receptor. This mutated receptor showed normal YO-PRO-1 uptake but had no permeability to N-methyl-D-glucamine. Together, the clear differential effects of extracellular sodium ions or of mutation of the receptor strongly suggest that N-methyl-D-glucamine and YO-PRO-1 do not enter the cell by the same permeation pathway. ATP; cation channel; permeability; quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl] -1-[3-(triethylammonio)propyl]diiodide

Human alveolar macrophages were obtained from macroscopically normal lung tissue obtained at surgical resections, isolated by adherence, and identified by morphology. Whole cell recordings were made from cells 1-3 h in culture, using electrodes containing potassium chloride. From a holding potential of -100 mV, depolarizing pulses to -40 mV or greater activated an outward current. Tail current reversals showed that this current was potassium selective. Margatoxin completely blocked the current; the concentration giving half-maximal block was 160 pM. In current clamp recordings, the resting membrane potential was -34 mV; margatoxin depolarized cells to close to 0 mV. A pure macrophage population was isolated by fluorescence-activated cell sorting, using the phagocytosis of BODIPY-labeled zymosan particles. Reverse transcription-polymerase chain reaction showed that, of 13 voltage-gated [K.sup.+] (Kv) potassium channels sought, only Kvl.3 mRNA was present. Margatoxin (1 nM) did not affect the percentage of cells showing phagocytosis sorted from the total population. Under these experimental conditions Ky1.3 sets the resting potential of the cells, but it is not required for Fc receptor-mediated phagocytosis. monocyte/macrophage; inflammation; phagocytosis

North, R. Alan. Molecular Physiology of P2X Receptors. Physiol Rev 82: 1013-1067, 2002; 10.1152/physrev.00015. 2002.-P2X receptors are membrane ion channels that open in response to the binding of extracellular ATP. [...]

ATP-gated ion channels (P2X receptors) are abundantly expressed in both neuronal and nonneuronal tissues, where they can serve as postsynaptic receptors. The response to ATP shows marked desensitization in some tissues but not others. Currents induced by ATP in Xenopus oocytes expressing cloned P2[X.sub.1] (or P2[X.sub.3]) receptors had strong desensitization, whereas currents in cells expressing P2[X.sub.2] receptors desensitized relatively little (90% vs. 14% decline of current in a 10-s application). In chimetic receptors, substitution into the P2[X.sub.1] receptor of either one of two 34-residue segments from the P2[X.sub.2] receptor removed the desensitization; these segments included the first or the second hydrophobic domain. In contrast, desensitization was introduced into the P2[X.sub.2] receptor only by providing both these segments of the P2[X.sub.1] (or P2[X.sub.3]) receptor. This suggests that desensitization requires interaction between the two hydrophobic domains of the receptor, and supports the view that these are membrane-spanning segments.

[P.sub.2x] receptors are cation-selective ion channels that open on binding to extracellular ATP; they play a role in fast synaptic transmission between neurones, and from autonomic nerves to smooth muscles. Isolation of cDNAs that encode [P.sub.2x] receptors in the smooth muscle of vas deferens and in phaeochromocytoma cells indicates that the receptors are not related to other ligand-gated ion channels. Their overall structure resembles more closely that of epithelial [Na.sup.+] channels and the proteins that are thought to form mechanosensitive channels in Caenorhabditis elegans. The type of [P.sub.2x] RNA that is found in vas deferens is expressed preferentially by apoptotic thymocytes, and the type of [P.sub.2x] RNA that is found in PCI2 cells is abundant in the pituitary gland, suggesting hitherto unsuspected roles for ATP-gated channels in endocrine and immune function.

Analysis of a complementary DNA that encodes the ligand-gated ion channel P2x receptor cloned from rat vas deferens and expressed in Xenopus oocytes and mammalian cells reveals that the P2x receptor for extracellular ATP defines a new family of ligand-gated ion channels. ATP induces an ion channel that is partial to cations with high calcium permeability. The protein is extracellular and has two transmembrane units and a pore-forming pattern that is similar to that of potassium channels.

The effect of opiates on the central nervous system is dependent on the type and distribution of receptors present. Morphine has an affinity for mu receptors which determines the extent of its pharmacological actions. It promotes a rewarding effect on animals whose tendency is to seek and prolong its effects. This dependence to the drug promotes biological changes which lead to physical dependence and full-blown withdrawal manifestations. Maintenance of opiate addiction was attributed to motivational changes different from physical dependence and withdrawal.

Investigations of calcium-dependent potassium (KCa) channel regulation reveal that protein phosphorylation regulates the Drosophila Slo Kca channel activity, exhibited in xenopus oocytes. Mutation of a single serine residue in the channel protein inhibits regulation through ATP-gammaS and suggests that phosphorylation of Slo channel protein regulates channel activity. An endogenous PKA protein kinase also regulates the KCa channels in oocyte membrane patches.

Dopamine-containing neurons of the mammalian midbrain are required for normal behavior and movements. In vivo they fire action potentials in bursts, but in vitro they discharge regularly spaced action potentials. Burst firing in vitro has now been shown to be robustly induced by the glutamate agonist N-methyl-D-aspartate (NMDA) although not by the non-NMDA agonists kainate or quisqualate. The hyperpolarization between bursts of action potentials results from electrogenic sodium ion extrusion by a ouabain-sensitive pump. This mechanism of burst generation in mammalian neurons may be important in the pathophysiology of schizophrenia and Parkinson's disease., The dopamine-containing neurons of the ventral midbrain project predominantly to the prefrontal cortex, nucleus accumbens, and striatum and are involved in the control of affect, movement, and drug-seeking behavior (1). [...]

Dopamine-containing neurons of the mammalian midbrain are required for normal behavior and movements. In vivo they fire action potentials in bursts, but in vitro they discharge regularly spaced action potentials. Burst firing in vitro has now been shown to be robustly induced by the glutamate agonist N-methyl-D-aspartate (NMDA) although not by the non-NMDA agonists kainate or quisqualate. The hyperpolarization between bursts of action potentials results from electrogenic sodium ion extrusion by a ouabain-sensitive pump. This mechanism of burst generation in mammalian neurons may be important in the pathophysiology of schizophrenia and Parkinson's disease., The dopamine-containing neurons of the ventral midbrain project predominantly to the prefrontal cortex, nucleus accumbens, and striatum and are involved in the control of affect, movement, and drug-seeking behavior [1]. [...]

A slowly activating, voltage-dependent potassium channel protein cloned from rat kidney was expressed in Xenopus oocytes. Two activators of protein kinase C, 1-oleoyl-2-acetyl-rac-glycerol and phorbol 12,13-didecanoate, inhibited the current. This [...]

A member of the 2P potassium channel family called TASK may be involved in transmitter-inactivated K+ channels in motoneurons and cerebellar granule cells. Research to support this conclusion is described.

Expression of a Cloned Rat Brain Potassium Channel in Xenopus Oocytes POTASSIUM CHANNELS ARE MEMbrane proteins that are selectively permeable to K.sup.+ ions. Some kinds of K.sup.+ channels are opened [...]