Neuronal autoregulation via nucleotide receptors

In non-neural tissues, such as liver, kidney, bones, and blood vessels, released nucleotides subserve autocrine/paracrine functions. This autocrine/paracrine regulation depends not only on the receptors involved, but also on the enzymatic activities of ectonucleotidases. Although feedback inhibition of neuronal transmitter release by co-released nucleotides has also been described, it remained unclear which receptors, nucleotidases, and signalling cascades were involved. One part of this project will identify the P2Y receptor subtype(s) and signal transduction mechanisms that mediate autoinhibition of transmitter release in PC12 cells and sympathetic neurons. Our preliminary data indicate that an inhibition of voltage-gated Ca2+ channels via P2Y 12 and/or 13 receptors and G protein beta/gamma subunits is involved. Future experiments will precisely delineate this signalling cascade. The initial results also show that a yet unknown second signalling component is involved and that one will also be elucidated. In our previous work (supported by the FWF project P14951), we found that endogenous nucleotides of PC12 cells activate P2Y receptors in an activity-dependent manner: activation of triphosphate-sensitive receptors required depolarization, whereas diphosphate-sensitive receptors were activated spontaneously. Since PC12 cells store and secrete 7 times more ATP than ADP, nucleotidases must be involved in the latter effect. In the present project, we will investigate how vesicle exocytosis, nucleotidases and P2Y receptors cooperate to guarantee this activity-dependent autoregulation. The experiments are designed (i) to reveal whether nucleotides are released from PC12 cells only by exocytosis or also by other mechanisms (as in non-neural cells), (ii) to clarify whether nucleotidases need to be colocalized with P2Y receptors to guarantee the supply of appropriate nucleotides, (iii) to resolve whether ectonucleotidases and P2Y receptors are in close contact with each other and whether there are subtype-specific interactions. In neurons, the modulation of N-type Ca2+ and M-type K+ channels via G protein- coupled receptors controls the cellular function. We and others found that these ion channels are also regulated via native and recombinant P2Y receptors. Preliminary data of this project show that another class of ion channels, Ca2+-activated K+ channels, are also regulated by P2Y receptors. Which P2Y receptor subtypes and associated sinalling cascades are able to modulate Ca2+-activated K+ channels will also be elucidated in this project. Taken together, the results of this project will pinpoint the network by which nucleotides contribute to neuronal autoregulation.

In non-neural tissues, such as liver, kidney, bones, and blood vessels, nucleotides have long been known to subserve autocrine/paracrine functions which depend not only on the receptors involved, but also on the enzymatic activities of ectonucleotidases. This project investigated autoregulation via nucleotides in neurons and revealed how vesicle exocytosis, nucleotide receptors and nucleotidases cooperate to regulate various types of effector systems. In experiments using botulinum toxin C1 to prevent vesicle exocytosis, spontaneous nucleotide release from neuroendocrine cells turned out to be independent of exocytosis and to rely on mechanisms similar to those in non- neural cells. In contrast, activity-dependent nucleotide release was lost when exocytosis was prevented. P2Y 12 receptors were found to mediate autoinhibition of transmitter release in sympathetic neurons and PC12 cells. This effect is based on an inhibition of voltage-gated Ca2+ channels via G protein beta/gamma subunits. In addition, a presynaptic P2Y 1 receptor was identified activation of which led to an inhibition of KV 7 channels and to an enhancement of transmitter release. This is the first report on a facilitatory presynaptic P2Y receptor. Considering that these two P2Y receptors are co-localized at presynaptic nerve terminals and are activated by ADP rather than ATP (which is the prime nucleotide released from neurons) prompted us to investigate whether P2Y receptors might directly interact with each other and with ectonucleotidases that degrade ATP. Indeed, via FRET (Fluorescence-Resonance-Energy-Transfer) microscopy and co-immunoprecipitation P2Y 1 , P2Y 2 , P2Y 12, and P2Y 13 as well as adenosine receptors were found to form heteromeric complexes with each other and to directly interact with the ectoenzyme NTPDase 1, but not with NTPDase 2. In electrophysiological experiments, a novel target for neuronal P2Y receptors was identified. Via P2Y 1 receptors heterologously expressed in PC12 cells, ADP triggered increases in intracellular Ca2+ through phospholipase C activation and thereby activated Ca2+-dependent K+ channels. This adds this latter family of ion channels to CaV 2, Kir 3, and KV 7 channels that had been found before to be controlled by P2Y receptors. Taken together, the results of this project delineate a signalling network of vesicles, receptors, enzymes and ion channels which are employed by nucleotides to contribute to neuronal autoregulation.