P2X receptors are nonselective cation channels gated by extracellular ATP. Recombinant mammalian P2X subunits assemble in homomeric ionotropic ATP receptors that differ by their agonist sensitivity and desensitization rate in heterologous expression systems. Using site-directed mutagenesis and voltage clamp recording in Xenopus oocytes, we identified the highly conserved protein kinase C site TX(K/R) located in the intracellular N terminus of P2X subunits as a critical determinant of kinetics in slowly desensitizing (time constant, >1 min) rat P2X 2 receptors. Mutant receptors P2X 2 T18A, T18N, and K20T devoid of this consensus site exhibited quickly desensitizing properties (time constant, <1 s). In contrast with wild-type receptors, mutant P2X 2 receptors with truncated C terminus exhibited variable cellspecific kinetics with quickly desensitizing currents converted to slowly desensitizing currents by phorbol ester-mediated stimulation of protein kinase C. Phosphorylation of Thr 18 was demonstrated directly by immunodetection using specific monoclonal antibodies directed against the phosphothreonine-proline motif. Our data indicate that both phosphorylation of the conserved threonine residue in the N-terminal domain by protein kinase C and interaction between the two cytoplasmic domains of P2X 2 subunits are necessary for the full expression of slowly desensitizing ATP-gated channels.Fast ionotropic responses to extracellular ATP are mediated by the activation of ATP-gated channels or P2X receptors present on the surface of various cell types. A family of genes coding for seven P2X channel subunits has been identified in human and rodents (1, 2). The electrophysiological characterization of recombinant homomeric and heteromeric P2X receptors expressed in heterologous systems led to their grouping in three functional categories based on their sensitivity to ATP and on their desensitization properties: 1) P2X 1 (3) and P2X 3 (4, 5) assemble in quickly desensitizing homomeric receptors highly sensitive to ATP and ␣␤-methylene ATP (EC 50 around 1 M); 2) P2X 2 (6), P2X 4 (7, 8), P2X 5 (9), P2X 2ϩ3 (5), P2X 1ϩ5 (10, 11), and P2X 4ϩ6 (12) receptors are less sensitive to ATP (EC 50 around 10 M) and desensitize at slow to moderate rate; 3) P2X 7 receptors (13) show low sensitivity to ATP (EC 50 around 500 M) and desensitize slowly. Modulation of the desensitization rate of neurotransmitter-gated channels is recognized as a potentially important mechanism for modulation of neuronal excitability (14). P2X receptors are nonselective cation channels with high permeability to calcium ions (15, 16), so the subtypespecific desensitization phenotype of ATP-mediated currents has a significant impact on the levels of intracellular calcium and subsequent activation of calcium-dependent effectors. Studies on the relationship between the slowly desensitization kinetics of P2X 2 receptor and its primary sequence have emphasized the requirement for several structural features including the transmembrane domains and their intracellula...

Ion channels are desirable therapeutic targets, yet ion channel-directed drugs with high selectivity and few side effects are still needed. Unlike small-molecule inhibitors, antibodies are highly selective for target antigens but mostly fail to antagonize ion channel functions. Nanobodies-small, single-domain antibody fragments-may overcome these problems. P2X7 is a ligand-gated ion channel that, upon sensing adenosine 5'-triphosphate released by damaged cells, initiates a proinflammatory signaling cascade, including release of cytokines, such as interleukin-1β (IL-1β). To further explore its function, we generated and characterized nanobodies against mouse P2X7 that effectively blocked (13A7) or potentiated (14D5) gating of the channel. Systemic injection of nanobody 13A7 in mice blocked P2X7 on T cells and macrophages in vivo and ameliorated experimental glomerulonephritis and allergic contact dermatitis. We also generated nanobody Dano1, which specifically inhibited human P2X7. In endotoxin-treated human blood, Dano1 was 1000 times more potent in preventing IL-1β release than small-molecule P2X7 antagonists currently in clinical development. Our results show that nanobody technology can generate potent, specific therapeutics against ion channels, confirm P2X7 as a therapeutic target for inflammatory disorders, and characterize a potent new drug candidate that targets P2X7.

l-DOPA-induced dyskinesia (LID), a detrimental consequence of dopamine replacement therapy for Parkinson's disease, is associated with an alteration in dopamine D1 receptor (D1R) and glutamate receptor interactions. We hypothesized that the synaptic scaffolding protein PSD-95 plays a pivotal role in this process, as it interacts with D1R, regulates its trafficking and function, and is overexpressed in LID. Here, we demonstrate in rat and macaque models that disrupting the interaction between D1R and PSD-95 in the striatum reduces LID development and severity. Single quantum dot imaging revealed that this benefit was achieved primarily by destabilizing D1R localization, via increased lateral diffusion followed by increased internalization and diminished surface expression. These findings indicate that altering D1R trafficking via synapse-associated scaffolding proteins may be useful in the treatment of dyskinesia in Parkinson's patients. IntroductionIn the striatum, dopamine (DA) terminals from the substantia nigra pars compacta (SNc) converge with glutamatergic signals from the cortex on dendritic spines of striatal medium spiny projecting GABAergic neurons (1, 2). The degeneration of the nigrostriatal pathway in Parkinson's disease (PD) induces complex modifications in both DA and glutamate signaling, leading to significant morphological and functional modifications in the striatal neuronal circuitry (3-5). Chronic DA replacement therapy with l-3,4-dihydroxyphenylalanine (l-DOPA) superimposes upon these DA depletion-induced changes, resulting in debilitating motor complications known as l-DOPAinduced dyskinesia (LID) (6-8). At the molecular level, the subcellular organization of and functional interactions between glutamate and DA receptors within the striatum are crucial both in the pathogenesis of PD (9) and in the development of LID (10, 11). LID has indeed been associated with plastic changes in postsynaptic neuronal targets in the striatum, including elevated extracellular levels of glutamate (12) and DA (13) and abnormal trafficking of DA D1 receptor (D1R) (14, 15) and of NMDA and AMPA glutamate receptor subunits (5,10,16,17). Such exaggerated DA and glutamate receptor expression at the plasma membrane results in abnormal activation of key signaling kinases (18)(19)(20)(21)(22). All these changes point to dysfunctional interactions between DA and glutamate neurotransmission in LID (5,23,24), although the molecular mechanisms remain elusive, despite recent progress (14, 25).The membrane-associated guanylate kinase (MAGUK) proteins, such as postsynaptic density 95 (PSD-95), organize ionotropic glutamate receptors and their associated signaling proteins, regulating the strength of synaptic activity. Interestingly, PSD-95 might also interact with DA D1R (26), thereby potentially regulating DA