A membrane network of receptors and enzymes for adenine nucleotides and nucleosides

AbstractMost cells express more than one receptor plus degrading enzymes for adenine nucleotides or nucleosides, and cellular responses to purines are rarely compatible with the actions of single receptors. Therefore, these receptors are viewed as components of a combinatorial receptor web rather than self-dependent entities, but it remained unclear to what extent they can associate with each other to form signalling units. P2Y1, P2Y2, P2Y12, P2Y13, P2X2, A1, A2A receptors and NTPDase1 and -2 were expressed as fluorescent fusion proteins which were targeted to membranes and signalled like the unlabelled counterparts. When tested by FRET microscopy, all the G protein-coupled receptors proved able to form heterooligomers with each other, and P2Y1, P2Y12, P2Y13, A1, A2A, and P2X2 receptors also formed homooligomers. P2Y receptors did not associate with P2X, but G protein-coupled receptors formed heterooligomers with NTPDase1, but not NTPDase2. The specificity of prototypic interactions (P2Y1/P2Y1, A2A/P2Y1, A2A/P2Y12) was corroborated by FRET competition or co-immunoprecipitation. These results demonstrate that G protein-coupled purine receptors associate with each other and with NTPDase1 in a highly promiscuous manner. Thus, purinergic signalling is not only determined by the expression of receptors and enzymes but also by their direct interaction within a previously unrecognized multifarious membrane network

Membrane coordination of receptors and channels mediating the inhibition of neuronal ion currents by ADP

ADP and other nucleotides control ion currents in the nervous system via various P2Y receptors. In this respect, Cav2 and Kv7 channels have been investigated most frequently. The fine tuning of neuronal ion channel gating via G protein coupled receptors frequently relies on the formation of higher order protein complexes that are organized by scaffolding proteins and harbor receptors and channels together with interposed signaling components. However, ion channel complexes containing P2Y receptors have not been described. Therefore, the regulation of Cav2.2 and Kv7.2/7.3 channels via P2Y1 and P2Y12 receptors and the coordination of these ion channels and receptors in the plasma membranes of tsA 201 cells have been investigated here. ADP inhibited currents through Cav2.2 channels via both P2Y1 and P2Y12 receptors with phospholipase C and pertussis toxin-sensitive G proteins being involved, respectively. The nucleotide controlled the gating of Kv7 channels only via P2Y1 and phospholipase C. In fluorescence energy transfer assays using conventional as well as total internal reflection (TIRF) microscopy, both P2Y1 and P2Y12 receptors were found juxtaposed to Cav2.2 channels, but only P2Y1, and not P2Y12, was in close proximity to Kv7 channels. Using fluorescence recovery after photobleaching in TIRF microscopy, evidence for a physical interaction was obtained for the pair P2Y12/Cav2.2, but not for any other receptor/channel combination. These results reveal a membrane juxtaposition of P2Y receptors and ion channels in parallel with the control of neuronal ion currents by ADP. This juxtaposition may even result in apparent physical interactions between receptors and channels.W 1205-B09(VLID)309167

P2Y1 receptors mediate an activation of neuronal calcium-dependent K+ channels

Molecularly defined P2Y receptor subtypes are known to regulate the functions of neurons through an inhibition of KV7 K+ and CaV2 Ca2+ channels and via an activation or inhibition of Kir3 channels. Here, we searched for additional neuronal ion channels as targets for P2Y receptors. Rat P2Y1 receptors were expressed in PC12 cells via an inducible expression system, and the effects of nucleotides on membrane currents and intracellular Ca2+ were investigated. At a membrane potential of −30 mV, ADP induced transient outward currents in a concentration-dependent manner with half-maximal effects at 4 μm. These currents had reversal potentials close to the K+ equilibrium potential and changed direction when extracellular Na+ was largely replaced by K+, but remained unaltered when extracellular Cl− was changed. Currents were abolished by P2Y1 antagonists and by blockade of phospholipase C. ADP also caused rises in intracellular Ca2+, and ADP-evoked currents were abolished when inositol trisphosphate-sensitive Ca2+ stores were depleted. Blockers of KCa2, but not those of KCa1.1 or KCa3.1, channels largely reduced ADP-evoked currents. In hippocampal neurons, ADP also triggered outward currents at −30 mV which were attenuated by P2Y1 antagonists, depletion of Ca2+ stores, or a blocker of KCa2 channels. These results demonstrate that activation of neuronal P2Y1 receptors may gate Ca2+-dependent K+ (KCa2) channels via phospholipase C-dependent increases in intracellular Ca2+ and thereby define an additional class of neuronal ion channels as novel effectors for P2Y receptors. This mechanism may form the basis for the control of synaptic plasticity via P2Y1 receptors