A Protein Kinase C Site Highly Conserved in P2X Subunits Controls the Desensitization Kinetics of P2X2 ATP-gated Channels

Boué‐Grabot, Éric; Archambault, Vincent; Séguéla, Philippe

doi:10.1074/jbc.275.14.10190

Cited by 159 publications

(170 citation statements)

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“…4A). Activation of P2X 2 TR with 100 M ATP evoked inward responses with slow to fast desensitizing kinetics, depending on oocytes as shown previously (27). As for wild-type P2X 2 , P2X 2 TR receptors expressed alone in oocytes were not gated or modulated by the application of GABA (Fig.…”

Section: Current Occlusion Was Observed At Low Receptor Densities Andmentioning

confidence: 69%

“…Plasmid Constructions-Wild-type rat P2X 2 , a C-terminal truncated form of P2X 2 (P2X 2 TR), and wild-type rat 1 clones were available from previous work (18,27). Carboxyl-terminal epitope-tagged P2X 2 subunits with either hexahistidine (His 6 )-tagged or enhanced YFP-tagged sequences and a C-terminal His 6 -tagged 1 subunit ( 1-HIS) were obtained as described previously (28).…”

Section: Methodsmentioning

confidence: 99%

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Cross-talk and Co-trafficking between ρ1/GABA Receptors and ATP-gated Channels

Boué‐Grabot

Émerit

Toulmé

et al. 2004

Journal of Biological Chemistry

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␥-Aminobutyric-acid (GABA) and ATP ionotropic receptors represent two structurally and functionally different classes of neurotransmitter-gated channels involved in fast synaptic transmission. We demonstrate here that, when the inhibitory 1/GABA and the excitatory P2X 2 receptor channels are co-expressed in Xenopus oocytes, activation of one channel reduces the currents mediated by the other one. This reciprocal inhibitory cross-talk is a receptor-mediated phenomenon independent of agonist cross-modulation, membrane potential, direction of ionic flux, or channel densities. Functional interaction is disrupted when the cytoplasmic C-terminal domain of P2X 2 is deleted or in competition experiments with minigenes coding for the Cterminal domain of P2X 2 or the main intracellular loop of 1 subunits. We also show a physical interaction between P2X 2 and 1 receptors expressed in oocytes and the co-clustering of these receptors in transfected hippocampal neurons. Co-expression with P2X 2 induces retargeting and recruitment of mainly intracellular 1/ GABA receptors to surface clusters. Therefore, molecular and functional cross-talk between inhibitory and excitatory ligand-gated channels may regulate synaptic strength both by activity-dependent current occlusion and synaptic receptors co-trafficking.Neuronal activity is regulated by a number of transmitters acting on different receptor types (1). Fast neurotransmission is achieved through different classes of transmitter-gated channels, including the P2X and nicotinic receptor superfamilies (1, 2). The family of P2X ATP-gated cation channels is composed of seven genes coding for subunits with two transmembrane domains, intracellular N and C termini, and a large extracellular loop (2). The nicotinic superfamily includes the GABA-gated 1 channels along with the acetylcholine, 5-HT 3 , and glycine receptors that share several structural features, including a large extracellular N-terminal domain, four hydrophobic transmembrane domains (M1-M4), and a long cytoplasmic loop connecting M3 and M4 (1). GABA receptor channels have been classified into two subtypes based on their pharmacological properties. GABA A receptors are inhibited by bicuculline, whereas GABA C receptors are insensitive to this antagonist (1). Diversity of GABA A receptors is achieved by pentameric assembly of multiple subunits, including ␣1-6, ␤1-3, ␥1-3, ␦, , and ⑀. GABA C receptors are composed of 1-3 subunits that can assemble into homo-oligomers (3, 4). Recent data suggest that a 1 subunit could co-assemble with a GABA A subunit (5, 6). Neuronal ATP and GABA C ionotropic receptors are involved in fast excitatory and inhibitory synaptic transmission, respectively, and display overlapping distribution in many regions of the nervous system, including DRG, dorsal horn of the spinal cord (7, 8), retina (9 -13), hippocampus (14 -15), cerebellum (14, 16), and anterior pituitary (17, 18).Recently, Jo and co-workers described ATP and GABA corelease from the same axon terminals into the dorsal horn of the spinal ...

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Section: Current Occlusion Was Observed At Low Receptor Densities Andmentioning

confidence: 69%

Section: Methodsmentioning

confidence: 99%

Cross-talk and Co-trafficking between ρ1/GABA Receptors and ATP-gated Channels

Boué‐Grabot

Émerit

Toulmé

et al. 2004

Journal of Biological Chemistry

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“…3 I and J) and minor increases in NMDG ϩ permeability (the shift in the NMDG ϩ E rev value was 4 Ϯ 0.5 mV; n ϭ 8). We also studied P2X 2 -NT-4C and P2X 2 -CT-4C receptors carrying T18A mutations (30), which results in channels that do not enter the I 2 state (3, 30). We detected ATP-evoked currents but no changes in ⌬F (Fig.…”

Section: Resultsmentioning

confidence: 99%

Patch–clamp coordinated spectroscopy shows P2X ₂ receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

Chaumont

Khakh

2008

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

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ATP-gated P2X receptors display ion permeability increases within seconds of receptor activation as the channels enter the I 2 state, which is permeable to organic cations and dye molecules. The mechanisms underlying this important behavior are not completely understood. In one model, the I 2 state is thought to be due to opening of Pannexin-1 (Panx-1) channels, and, in the second, it is thought to be an intrinsic P2X property. We tested both models by measuring ion and dye permeability and used a patch-clamp coordinated spectroscopy approach to measure conformational changes. Our data show that Panx-1 channels make no detectable contribution to the P2X 2 receptor I2 state. However, P2X 2 receptors display permeability dynamics, which are correlated with conformational changes in the cytosolic domain remote from the selectivity filter itself. Finally, the data illustrate the utility of a new approach, using tetracysteine tags and biarsenical fluorophores to measure site-specific conformational changes in membrane proteins. (Fig. 1A), which is permeable to cations, such as Na ϩ and Ca 2ϩ (7,8). In P2X 2 , P2X 4 , and P2X 7 receptors after seconds of activation by ATP, the channel undergoes additional seconds time-scale changes that increase permeability to organic cations, such as N-methyl-D-glucamine (NMDG) ϩ , and dyes, such as YOPRO1 (2,(4)(5)(6)9). This second open state is referred to as I 2 (Fig. 1 A). The first reports of the I 2 state permeable to large molecules go back 30 years, when ATP was shown to permeabilize cells (10). The mechanisms that underlie opening to the I 2 state remain unclear, even though it is a trigger for a variety of pathophysiological processes, including blebbing, microvesicle shedding, release of signaling molecules, permeablization, and cell death (7,11). In addition to the obvious relevance to ATP signaling, a better understanding of the I 2 state would also advance our understanding of the diversity of mechanisms used by ion channels. This is because ion permeability and/or selectivity filter dynamics are not unique to P2X but also occur for other channels (12, 13). In particular, the organic cation and dye permeable I 2 state of TRP channels (14, 15) shares similarity to P2X 2 receptors. A key open question is how the organic cation and dye permeable I 2 state arises.Two models exist to explain the I 2 state for P2X receptors. The first model proposes that P2X 7 -mediated dye uptake (16, 17) occurs via Pannexin-1 (Panx-1) channels, leading to the suggestion that the I 2 state is due to Panx-1 channels rather than P2X (11,18). In this study, we call this the ''Panx-1 model'' (Fig. 1 A). The second model posits that the I 2 state is an intrinsic property of P2X receptors involving slow conformational changes that allow organic cations to flow (2-6, 19). We call this the ''gating model'' (Fig. 1 A). We tested both models under a common set of recording conditions. We also used a patch-clamp coordinated imaging approach to directly measure protein conformational changes. Results...

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“…The term P2X 2A distinguishes the nondesensitizing full-length P2X 2A subunit from the desensitizing C-terminal splice variant termed P2X 2B , which lacks a stretch of 69 amino acids C-terminal to M2 (8). The different rates of desensitization have been attributed by mutational analysis to various structural motifs, including N-and C-terminal domains (9, 10) and a highly conserved protein kinase C site (11).…”

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confidence: 99%

Desensitization Masks Nanomolar Potency of ATP for the P2X1 Receptor

Rettinger

Schmalzing²

2004

Journal of Biological Chemistry

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ATP-gated P2X 1 receptors feature fast activation and fast desensitization combined with slow recovery from desensitized states. Here, we exploited a non-desensitizing P2X 2 /P2X 1 chimera that includes the entire P2X 1 ectodomain (Werner, P., Seward, E. P., Buell, G. N., and North, R. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 15485-15490) to obtain a macroscopic representation of intrinsic receptor kinetics without bias arising from the overlap of channel activation and desensitization. From the stationary currents made amenable to analysis by this chimera, an EC 50 for ATP of 3.3 nM was derived, representing a >200-and >7000-fold higher ATP potency than observed for the parental P2X 1 and P2X 2A receptors, respectively. Also, other agonists activated the P2X 2 /P2X 1 chimera with nanomolar EC 50 values ranging from 3.5 to 73 nM in the following rank order: 2-methylthio-ATP, 2 ,3 -O-(4-benzoylbenzoyl)-ATP, ␣,␤-methylene-ATP, adenosine 5 -O-(3-thiotriphosphate). Upon washout, the P2X 2 /P2X 1 chimera deactivated slowly with a time constant (ranging from 63 to 2.5 s) that is inversely related to the EC 50 value for the corresponding agonist. This suggests that deactivation time courses reflect unbinding rates, which by themselves define agonist potencies. The P2X 2 /P2X 1 chimera and the P2X 1 receptor possess virtually identical sensitivity to inhibition by the P2X 1 receptor-selective antagonist NF279, a suramin analog. These results suggest that the P2X 1 ectodomain confers nanomolar ATP sensitivity, which, within the wild-type P2X 1 receptor, is obscured by desensitization such that only a micromolar ATP potency can be deduced from peak current measurements, representing an amalgam of activation and desensitization.Extracellular ATP is a ubiquitous signaling molecule that exerts fast effects by directly gating cation-conducting channels designated P2X receptors, which exist on a large variety of cells, including many excitable cells and different leukocytes (1). Seven genes encoding P2X subunits (P2X 1-7 ) are known in mammals. All P2X subunits share a common membrane topology, with two hydrophobic membrane-spanning segments (M1 and M2) separated by a large extracellular loop of ϳ300 amino acid residues, which comprise the ATP-binding domain. At least part of the pore of the P2X receptor is lined by M2 (2), which includes a conserved glycine residue that is likely to constitute the channel gate (3), but also residues of the Cterminal cytoplasmic tail appear to play a role in controlling channel permeability (4). The cytoplasmic N-terminal tails of the various subunit isoforms are approximately the same length (ϳ30 amino acid residues), whereas the cytoplasmic C-terminal tails are highly variable in length. Both chemical cross-linking studies and blue native polyacrylamide gel electrophoresis analysis of wild-type and concatenated P2X 1 and P2X 3 receptors indicate that P2X receptors feature a trimeric architecture, which is unique among ligand-gated ion channels (5-7). Further details of the structure of ...

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A Protein Kinase C Site Highly Conserved in P2X Subunits Controls the Desensitization Kinetics of P2X2 ATP-gated Channels

Cited by 159 publications

References 29 publications

Cross-talk and Co-trafficking between ρ1/GABA Receptors and ATP-gated Channels

Cross-talk and Co-trafficking between ρ1/GABA Receptors and ATP-gated Channels

Patch–clamp coordinated spectroscopy shows P2X ₂ receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

Desensitization Masks Nanomolar Potency of ATP for the P2X1 Receptor

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A Protein Kinase C Site Highly Conserved in P2X Subunits Controls the Desensitization Kinetics of P2X2 ATP-gated Channels

Cited by 159 publications

References 29 publications

Cross-talk and Co-trafficking between ρ1/GABA Receptors and ATP-gated Channels

Cross-talk and Co-trafficking between ρ1/GABA Receptors and ATP-gated Channels

Patch–clamp coordinated spectroscopy shows P2X 2 receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

Desensitization Masks Nanomolar Potency of ATP for the P2X1 Receptor

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Patch–clamp coordinated spectroscopy shows P2X ₂ receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels