P2X receptors within the CNS mediate excitatory synaptic transmission and also act presynaptically to modulate neurotransmitter release. We have studied the targeting and trafficking of P2X4 and P2X2 receptors heterologously expressed in cultured olfactory bulb neurons. Homomeric P2X4 receptors had a punctate distribution, and many of the puncta colocalized with early endosomes. In contrast, P2X2 receptors were primarily localized at the plasma membrane. By antibody-labeling of surface receptors in living neurons, we showed that P2X4 receptors undergo rapid constitutive internalization and subsequent reinsertion into the plasma membrane, whereas P2X2 receptors were not regulated in such a way. The internalization of P2X4 receptors was dynamin-dependent, and the binding of ATP enhanced the basal rate of retrieval in a Ca2+-independent manner. The presence of the P2X4 subunit in a P2X4/6 heteromer governed the trafficking properties of the receptor. P2X receptors acted presynaptically to enhance the release of glutamate, suggesting that the regulated cycling of P2X4-containing receptors might provide a mechanism for modulation of synaptic transmission.

Elementary Ca2+ release signals in nerve growth factor- (NGF-) differentiated PC12 cells and hippocampal neurons, functionally analogous to the "Ca2+ sparks" and "Ca2+ puffs" identified in other cell types, were characterized by confocal microscopy. They either occurred spontaneously or could be activated by caffeine and metabotropic agonists. The release events were dissimilar to the sparks and puffs described so far, as many arose from clusters of both ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (InsP3Rs). Increasing either the stimulus strength or loading of the intracellular stores enhanced the frequency of and coupling between elementary release sites and evoked global Ca2+ signals. In the PC12 cells, the elementary Ca2+ release preferentially occurred around the branch points. Spatio-temporal recruitment of such elementary release events may regulate neuronal activities.

Rapid modulation of the surface number of certain ionotropic receptors is achieved by altering the relative rates of insertion and internalization. These receptors are internalized by a clathrin-mediated pathway; however, a motif that is necessary for endocytosis of ionotropic receptors has not yet been identified. Here, we identified a motif that is required for constitutive and agonist-regulated internalization of the ionotropic P2X 4 receptor. Three amino acids in the C terminus of P2X 4 (Tyr 378 , Gly 381 , and Leu 382 ) compose a non-canonical tyrosine-based sorting signal of the form YXXGL. We found that P2X 4 protein was present in clathrin-coated vesicles isolated from rat brain and that a glutathione S-transferase fusion of the P2X 4 C terminus pulled down the adaptor protein-2 complex from brain extract. Mutation of either the tyrosine-binding pocket of the 2 subunit of adaptor protein-2 or the YXXGL motif in the receptor C terminus caused a decrease in receptor internalization and a dramatic increase in the surface expression of P2X 4 receptors. The YXXGL motif represents a non-canonical tyrosine-based sorting signal that is necessary for efficient endocytosis of the P2X 4 receptor. Similar motifs are present in other receptors and may be important for the control of their functional expression.

During clathrin-mediated endocytosis, proteins on the cell surface are selected for inclusion in clathrin-coated vesicles by clathrin adaptors, mainly the adaptor complex AP2. The P2X4 subtype of ATP-gated ion channel has in its C-terminus two putative endocytic motifs: a canonical YXXΦ motif and a non-canonical YXXGΦ motif (YEQGL). We demonstrate that endocytosis of P2X4 receptors is mediated preferentially by the YXXGΦ motif because the YXXΦ motif is inaccessible to AP2 owing to the structure of the channel. The crystal structure of a complex between residues 160-435 of the μ2 subunit of AP2 and a P2X4 C-terminal peptide showed that the YEQGL motif binds to μ2 at the same site as YXXΦ motifs. Y and Φ residues are accommodated in the same hydrophobic pockets in μ2 with the extra residue between them being accommodated by changes in the peptide's backbone configuration, when compared to YXXΦ motifs. These data demonstrate that the family of potential tyrosine-based endocytic signals must be expanded to include motifs with an additional glycine at Y+3 (YXXGΦ).

Members of the Rab3 (A-D) subfamily of small GTPases are believed to play a key role in regulated exocytosis. These proteins share approximately 80% identity at amino acid level. The question of whether isoforms of Rab3 are functionally redundant was the subject of this study. We used RT-PCR analysis, in situ hybridization histochemistry, and confocal microscope-based analysis of immunocytochemistry to show that rat melanotrophs contain about equal amounts of Rab3A and Rab3B transcripts as well as proteins. Therefore, these cells are a suitable model to study the subcellular distribution and the role of these paralogous isoforms in regulated exocytosis. Secretory activity of single cells was monitored with patch-clamp capacitance measurements, and the cytosol was dialyzed with a high-calcium-containing patch pipette solution. Preinjection of antisense oligodeoxyribonucleotides specific to Rab3A, but not to Rab3B, induced a specific blockage of calcium-dependent secretory responses, indicating an exclusive requirement for Rab3A in melanotroph cell-regulated secretion. Although the injection of purified Rab3B protein was ineffective, the injection of recombinant Rab3A proteins into rat melanotrophs revealed that regulated secretion was stimulated by a GTP-bound Rab3A with an intact COOH terminus and inhibited by Rab3AT36N, impaired in GTP binding. These results indicate that Rab3A, but not Rab3B, enhances secretory output from rat melanotrophs and that their function is not redundant.

We report the sequence, structure and distribution of a novel transient receptor potential (trp) homologue from Xenopus, Xtrp, determined by screening an oocyte cDNA library. On the basis of sequence similarity and predicted structure, Xtrp appears to be a homologue of mammalian trp1 proteins. Two polyclonal antibodies raised against distinct regions of the Xtrp sequence revealed Xtrp expression in various Xenopus tissues, and the localization of Xtrp at the plasma membrane of Xenopus oocytes and HeLa cells. Since capacitative calcium entry into Xenopus oocytes has been shown previously to be substantially inhibited by trp1 antisense oligonucleotides [Tomita, Kaneko, Funayama, Kondo, Satoh and Akaike (1998) Neurosci. Lett. 248, 195-198] we suggest that Xtrp may underlie capacitative calcium entry in Xenopus tissues.