Fast chemical communications in the nervous system are mediated by several classes of receptor channels believed to be independent functionally and physically. We show here that concurrent activation of P2X2 ATP-gated channels and 5-HT3 serotonin-gated channels leads to functional interaction and nonadditive currents (47-73% of the predicted sum) in mammalian myenteric neurons as well as in Xenopus oocytes or transfected human embryonic kidney (HEK) 293 cell heterologous systems. We also show that these two cation channels coimmunoprecipitate constitutively and are associated in clusters. In heterologous systems, the inhibitory cross talk between P2X2 and 5-HT3 receptors is disrupted when the intracellular C-terminal domain of the P2X2 receptor subunit is deleted and when minigenes coding for P2X2 or 5-HT3A receptor subunit cytoplasmic domains are overexpressed. Injection of fusion proteins containing the C-terminal domain of P2X2 receptors in myenteric neurons also disrupts the functional interaction between native P2X2 and 5-HT3 receptors. Therefore, activity-dependent intracellular coupling of distinct receptor channels underlies ionotropic cross talks that may significantly contribute to the regulation of neuronal excitability and synaptic plasticity.
ATP and ␥-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central synapses, where they co-activate excitatory P2X and inhibitory GABA A (GABA type A) receptors. We report here that co-activation of P2X 2 and various GABA A receptors, coexpressed in Xenopus oocytes, leads to a functional cross-inhibition dependent on GABA A subunit composition. Sequential applications of GABA and ATP revealed that ␣-or ␣␥-containing GABA A receptors inhibited P2X 2 channels, whereas P2X 2 channels failed to inhibit ␥-containing GABA A receptors. This functional crosstalk is independent of membrane potential, changes in current direction, and calcium. Non-additive responses observed between cation-selective GABA A and P2X 2 receptors further indicate the chloride independence of this process. Overexpression of minigenes encoding either the C-terminal fragment of P2X 2 or the intracellular loop of the 3 subunit disrupted the functional crossinhibition. We previously demonstrated functional and physical cross-talk between 1 and P2X 2 receptors, which induced a retargeting of 1 channels to surface clusters when co-expressed in hippocampal neurons (Boué -Grabot, E., Emerit, M. B., Toulme, E., Seguela, P., and Garret, M. (2004) J. Biol. Chem. 279, 6967-6975). Co-expression of P2X 2 and chimeric 1 receptors with the C-terminal sequences of ␣2, 3, or ␥2 subunits indicated that only 1-3 and P2X 2 channels exhibit both functional cross-inhibition in Xenopus oocytes and coclustering/retargeting in hippocampal neurons. Therefore, the C-terminal domain of P2X 2 and the intracellular loop of  GABA A subunits are required for the functional interaction between ATP-and GABA-gated channels. This ␥ subunit-dependent cross-talk may contribute to the regulation of synaptic activity.Synaptic transmission is achieved through the release of one or more neurotransmitters from the same presynaptic terminal, resulting in the activation of different classes of receptors co-localized at the same post-synaptic site. Recent reports have demonstrated that co-activation of distinct postsynaptic receptors by their respective transmitters induced cross-modulation of their functional properties (1). G-protein-coupled receptors and ligand-gated channels reciprocally affect their functions by direct interaction between intracellular domains, as illustrated for D5 and GABA A , 1 or D1 and N-methyl-D-aspartic acid (2, 3). Cross-talk between distinct ligand-gated channels has been described between ATP P2X receptors and either acetylcholine nicotinic receptors (4 -7), 5-hydroxytryptamine 3 receptors (8, 9), or GABA A receptors in dorsal root ganglia neurons (10), as well as between GABA and glycine receptors in spinal cord neurons (11). Cross-talk between ligand-gated channels is characterized by current occlusion during simultaneous agonist application, although the mechanisms remain unclear. Intracellular phosphorylation pathways underlie asymmetric cross-inhibition between GABA A and glycine receptors (11). Cross-talk between GABA A and ...
␥-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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