Ionic selectivity of native ATP‐activated (P2X) receptor channels in dissociated neurones from rat parasympathetic ganglia

Li, Dongmei; Adams, David J.

doi:10.1111/j.1469-7793.2001.00423.x

Cited by 39 publications

(22 citation statements)

References 53 publications

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“…The rat cells show a fast-onset, inwardly rectifying, cation-selective current that is desensitized by ␣␤meATP and blocked either by increasing the calcium concentration or by reactive blue 2 (IC 50 ϳ1 M). The relative permeabilities of the monovalent inorganic and organic cations were thoroughly measured in rat submandibular ganglion cells; these are similar to those of cloned rat P2X 2 receptors expressed in mammalian cells (285). The effects of protons were also similar to those seen for the cloned homomeric P2X 2 receptor.…”

Section: Parasympathetic Neuronsmentioning

confidence: 54%

Molecular Physiology of P2X Receptors

North

2002

Physiological Reviews

2,691

128

3,397

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P2X receptors are membrane ion channels that open in response to the binding of extracellular ATP. Seven genes in vertebrates encode P2X receptor subunits, which are 40–50% identical in amino acid sequence. Each subunit has two transmembrane domains, separated by an extracellular domain (∼280 amino acids). Channels form as multimers of several subunits. Homomeric P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7 channels and heteromeric P2X2/3 and P2X1/5 channels have been most fully characterized following heterologous expression. Some agonists (e.g., αβ-methylene ATP) and antagonists [e.g., 2′,3′- O-(2,4,6-trinitrophenyl)-ATP] are strongly selective for receptors containing P2X1 and P2X3subunits. All P2X receptors are permeable to small monovalent cations; some have significant calcium or anion permeability. In many cells, activation of homomeric P2X7 receptors induces a permeability increase to larger organic cations including some fluorescent dyes and also signals to the cytoskeleton; these changes probably involve additional interacting proteins. P2X receptors are abundantly distributed, and functional responses are seen in neurons, glia, epithelia, endothelia, bone, muscle, and hemopoietic tissues. The molecular composition of native receptors is becoming understood, and some cells express more than one type of P2X receptor. On smooth muscles, P2X receptors respond to ATP released from sympathetic motor nerves (e.g., in ejaculation). On sensory nerves, they are involved in the initiation of afferent signals in several viscera (e.g., bladder, intestine) and play a key role in sensing tissue-damaging and inflammatory stimuli. Paracrine roles for ATP signaling through P2X receptors are likely in neurohypophysis, ducted glands, airway epithelia, kidney, bone, and hemopoietic tissues. In the last case, P2X7 receptor activation stimulates cytokine release by engaging intracellular signaling pathways.

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Section: Parasympathetic Neuronsmentioning

confidence: 54%

Molecular Physiology of P2X Receptors

North

2002

Physiological Reviews

2,691

128

3,397

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“…The procedure for isolation of the neurones from rat submandibular ganglia have been described in detail previously (Liu and Adams, 2001). Electrical access to the cell interior was obtained using the whole-cell recording configuration of the patch clamp technique ( Hamill et al, 1981).…”

Section: Whole-cell Patch Clamp Recording Of Acutely Dissociated Neurmentioning

confidence: 99%

Pre- and postsynaptic actions of ATP on neurotransmission in rat submandibular ganglia

Smith

Hansen

et al. 2001

Neuroscience

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The pre-and postsynaptic actions of exogenously applied ATP were investigated in intact and dissociated parasympathetic neurones of rat submandibular ganglia. Nerve-evoked excitatory postsynaptic potentials (EPSPs) were not inhibited by the purinergic receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), or the desensitising agonist, α,β-methylene ATP. In contrast, EPSPs were abolished by the nicotinic acetylcholine receptor antagonists, hexamethonium and mecamylamine. Focal application of ATP (100 μM) had no effect on membrane potential of the postsynaptic neurone or on the amplitude of spontaneous EPSPs. Taken together, these results suggest the absence of functional purinergic (P2) receptors on the postganglionic neurone in situ. In contrast, focally applied ATP (100 μM) reversibly inhibited nerve-evoked EPSPs. Similarly, bath application of the non-hydrolysable analogue of ATP, ATPγS, reversibly depressed EPSPs amplitude. The inhibitory effects of ATP and ATPγS on nerve-evoked transmitter release were antagonised by bath application of either PPADS or suramin, suggesting ATP activates a presynaptic P2 purinoceptor to inhibit acetylcholine release from preganglionic nerves in the submandibular ganglia. In acutely dissociated postganglionic neurones from rat submandibular ganglia, focal application of ATP (100 μM) evoked an inward current and subsequent excitatory response and action potential firing, which was reversibly inhibited by PPADS (10 μM).The expression of P2X purinoceptors in wholemount and dissociated submandibular ganglion neurones was examined using polyclonal antibodies raised against the extracellular domain of six P2X purinoceptor subtypes (P2X 1-6 ). In intact wholemount preparations, only the P2X 5 purinoceptor subtype was found to be expressed in the submandibular ganglion neurones and no P2X immunoreactivity was detected in the nerve fibres innervating the ganglion. Surprisingly, in dissociated submandibular ganglion neurones, high levels of P2X 2 and P2X 4 purinoceptors immunoreactivity were found on the cell surface. This increase in expression of P2X 2 and P2X 4 purinoceptors in dissociated submandibular neurones could explain the increased responsiveness of the neurones to exogenous ATP.We conclude that disruption of ganglionic transmission in vivo by either nerve damage or synaptic blockade may up-regulate P2X expression or availability and alter neuronal excitability.Author Keywords: acetylcholine; P2X receptors; synaptic transmission; electrophysiology; immunohistochemistry There is increasing evidence that ATP plays a role in neurotransmission in mammalian autonomic ganglia (for review see Ralevic and Burnstock, 1998). Extracellular ATP can activate a number of different receptors, termed P2 purinoceptors, which can be classified into two groups, P2X and P2Y purinoceptors. P2X purinoceptors are ligand-gated cation channels and P2Y purinoceptors are G protein-coupled receptors associated with intracellular second messengers. P2X re...

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“…The crystal structures of zebrafish P2X 4 receptor (zfP2X 4 ) (18,19), together with mutational analyses (20)(21)(22)(23)(24)(25)(26), provided the structural basis for the channel opening of P2X receptors upon ATP binding. In the crystal structures, zfP2X 4 forms a homotrimer (27,28), in which the transmembrane region of each subunit is composed of two helices (19).…”

mentioning

confidence: 99%

Conductance of P2X ₄ purinergic receptor is determined by conformational equilibrium in the transmembrane region

Minato

Suzuki

Hara

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cationselective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X 4 receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X 4 purinergic receptor in the apo, ATP-bound, and α,β-methylene ATP-bound states. Our NMR analyses revealed that, in the α,β-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s −1 ), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region.NMR | membrane proteins | ligand-gated ion channels | insect cell expression system | purinergic receptors I n chemical neurotransmission, various neurotransmitters bind to ligand-gated ion channels expressed in the plasma membrane of postsynaptic cells, such as the NMDA, AMPA, and P2X receptors, leading to changes in membrane potential and the concentration of intracellular ions. Each ligand for a ligand-gated ion channel has a distinct ability to evoke currents (1), and the ligands are classified according to the evoked current level: such as, full agonists, partial agonists, and antagonists. Partial agonists of ligand-gated ion channels reportedly offer clinical advantages over antagonists and full agonists in antidepressant and smoking-cessation treatment (2, 3).Two mechanisms have been proposed for the partial activation of the ligand-gated ion channels: the equilibrium between the open and closed conformations and the distinct conformation of the partial agonist-bound states from the closed and open conformations (4, 5). In the crystal structures of the extracellular region of the AMPA receptor, in which the distances between the two extracellular domains are changed upon agonist binding, the interdomain distances in the partial agonist-bound states correlated with the conductance level, suggesting that the AMPA receptor adopts specific intermediately permeable conformations (4, 6).The P2X receptors are a family of cation channe...

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Ionic selectivity of native ATP‐activated (P2X) receptor channels in dissociated neurones from rat parasympathetic ganglia

Cited by 39 publications

References 53 publications

Molecular Physiology of P2X Receptors

Molecular Physiology of P2X Receptors

Pre- and postsynaptic actions of ATP on neurotransmission in rat submandibular ganglia

Conductance of P2X ₄ purinergic receptor is determined by conformational equilibrium in the transmembrane region

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Ionic selectivity of native ATP‐activated (P2X) receptor channels in dissociated neurones from rat parasympathetic ganglia

Cited by 39 publications

References 53 publications

Molecular Physiology of P2X Receptors

Molecular Physiology of P2X Receptors

Pre- and postsynaptic actions of ATP on neurotransmission in rat submandibular ganglia

Conductance of P2X 4 purinergic receptor is determined by conformational equilibrium in the transmembrane region

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Conductance of P2X ₄ purinergic receptor is determined by conformational equilibrium in the transmembrane region