ATP signaling in the integrative neural center of Aplysia californica

ATP and its ionotropic P2X receptors are components of the most ancient signaling system. However, little is known about the distribution and function of purinergic transmission in invertebrates. Here, we cloned, expressed, and pharmacologically characterized the P2X receptors in the sea slug Aplysia californica—a prominent neuroscience model. AcP2X receptors were successfully expressed in Xenopus oocytes and displayed activation by ATP with two-phased kinetics and Na+-dependence. Pharmacologically, they were different from other P2X receptors. The ATP analog, Bz-ATP, was a less effective agonist than ATP, and PPADS was a more potent inhibitor of the AcP2X receptors than the suramin. AcP2X were uniquely expressed within the cerebral F-cluster, the multifunctional integrative neurosecretory center. AcP2X receptors were also detected in the chemosensory structures and the early cleavage stages. Therefore, in molluscs, rapid ATP-dependent signaling can be implicated both in development and diverse homeostatic functions. Furthermore, this study illuminates novel cellular and systemic features of P2X-type ligand-gated ion channels for deciphering the evolution of neurotransmitters.

ATP Signaling in the Integrative Neural Center of Aplysia Californica

ATP and its ionotropic P2X receptors are components of the most ancient signaling systems. However, little is known about the distribution and function of purinergic transmission in invertebrates. Here, we cloned, expressed, and pharmacologically characterized P2X receptors in the sea slug Aplysia californica – the prominent neuroscience model. acP2X receptors were successfully expressed in Xenopus oocytes and were displayed activation by ATP with two-phased kinetics and Na+-dependence. Pharmacologically, they were quite different from other P2X receptors. The ATP analog, Bz-ATP, was a less effective agonist than ATP, and PPADS was a more potent inhibitor of the acP2X receptors than the suramin. acP2X were uniquely expressed within the cerebral F-cluster, which contains multiple secretory peptides (e.g., insulins, interleukins, and potential toxins), ecdysone-type receptors, and a district subset of ion channels. We view F-cluster as the multifunctional integrative center, remarkably different from other neurosecretory cells. acP2X receptors were also found in the chemosensory structures and the early cleavage stages. Therefore, in molluscs, rapid ATP-dependent signaling can be implicated both in development and diverse homeostatic functions. Furthermore, this study illuminates novel cellular and systemic features of P2X-type ligand-gated ion channels for deciphering evolution of neurotransmitters.

ATP signaling in the integrative neural center of Aplysia californica

ATP and its ionotropic P2X receptors are components of one of the most ancient signaling systems. However, little is known about the distribution and function of purinergic transmission in invertebrates. Here, we cloned, expressed, and pharmacologically characterized P2X receptors in the sea slug Aplysia californica – the prominent model in cellular and system neuroscience. These functional P2X receptors were successfully expressed in Xenopus oocytes and displayed activation by ATP (EC50=306 μM) with two-phased kinetics as well as Na+-dependence. The ATP analog, Bz-ATP, was a less effective agonist (~20%) than ATP, and PPADS was a more potent inhibitor of the P2X receptors than the suramin. We showed that P2X receptors are uniquely expressed within Aplysia’s cerebral bioenergetic center (also known as F-cluster). Using RNA-seq, we found that the F-cluster contains more than a dozen unique secretory peptides, including three insulins, interleukins, and potential toxins, as well as ecdysone-type receptors and a district subset of ion channels. This structure is one of the most prominent integrative centers in the entire CNS and remarkably different from the morphologically similar neurosecretory center (bag cluster) involved in egg-laying behavior. Using RNA-seq, we also characterized the expression of P2X receptors across more than a dozen Aplysia peripheral tissues and developmental stages. We showed that P2X receptors are predominantly expressed in chemosensory structures and during early cleavage stages. The localization and pharmacology of P2X receptors in Aplysia highlight the evolutionary conservation of bioenergetic sensors and chemosensory purinergic transmission across animals. This study also provides a foundation to decipher homeostatic mechanisms in development and neuroendocrine systems.Graphical AbstractWe show that ATP and its ligand-gated P2X receptors are essential signaling components within both the chemosensory systems and the unique integrative neurosecretory center, present in the CNS of the sea slug Aplysia – a prominent model in neuroscience. Expression and pharmacology of P2X receptors in Aplysia confirms the preservation of evolutionary conserved bioenergetic sensors across animals and provide new tools to decipher homeostatic mechanisms in neuro-endocrine systems in general.

P2X receptors in Aplysia californica: Chemosensory systems, bio-energetic and development

ATP and its ionotropic P2X receptors are components of one of the most ancient signaling systems. However, little is known about the distribution and function of purinergic transmission in invertebrates. Here, we cloned, expressed, and pharmacologically characterized P2X receptors in the sea slug Aplysia californica – a prominent model in cellular and system neuroscience. We showed that ATP and P2X receptors are essential signaling components within the unique bioenergetic center located in the CNS of Aplysia, also known as the cerebral F-cluster of insulin-containing neurons. Functional P2X receptors were successfully expressed in Xenopus oocytes to characterize their ATP-dependence (EC50=306μM), two-phased kinetics, ion selectivity (Na+-dependence), sensitivity to the ATP analog Bz-ATP (~20% compare to ATP) and antagonists (with PPADS as a more potent inhibitor compared to suramin). Next, using RNA-seq, we characterized the expression of P2X receptors across more than a dozen Aplysia peripheral tissues and developmental stages. We showed that P2X receptors are predominantly expressed in chemosensory structures and during early cleavage stages. The localization and pharmacology of P2X receptors in Aplysia highlight the evolutionary conservation of bioenergetic sensors and chemosensory purinergic transmission across animals. This study also provides a foundation to decipher homeostatic mechanisms in development and neuroendocrine systems.Graphical AbstractWe show that ATP and its ligand-gated P2X receptors are essential signaling components within both the chemosensory systems and the unique bioenergetic center, present in the CNS of the sea slug Aplysia californica – a prominent model in neuroscience. Expression and pharmacology of P2X receptors in Aplysia confirms the preservation of evolutionary conserved bioenergetic sensors across animals and provide new tools to decipher homeostatic mechanisms in neuro-endocrine systems in general.

In Memoriam Geoffrey Burnstock: Creator of Purinergic Signaling

Geoff Burnstock (1929–2020) discovered purinergic signaling in a fastidious research that started in early 1960 and culminated in a concept of purinergic nerves in 1972. Subsequently, Geoff developed the concept of purinergic transmission and demonstrated ATP storage, release, and degradation in the context of cotransmission, which was another fundamental concept developed by him. Purinergic transmission contributes to the most fundamental physiological functions such as sensory transduction, regulation of heart rate, smooth muscle contraction, bile secretion, endocrine regulation, immune responses, as well as to various pathophysiological conditions, including inflammation, cancer, neuropathic pain, diabetes, and kidney failure.

ATP-Gated P2X Receptor Channels: Molecular Insights into Functional Roles

In the nervous system, ATP is co-stored in vesicles with classical transmitters and released in a regulated manner. ATP from the intracellular compartment can also exit the cell through hemichannels and following shear stress or membrane damage. In the past 30 years, the action of ATP as an extracellular transmitter at cell-surface receptors has evolved from somewhat of a novelty that was treated with skepticism to purinergic transmission being accepted as having widespread important functional roles mediated by ATP-gated ionotropic P2X receptors (P2XRs). This review focuses on work published in the last five years and provides an overview of ( a) structural studies, ( b) the molecular basis of channel properties and regulation of P2XRs, and ( c) the physiological and pathophysiological roles of ATP acting at defined P2XR subtypes.

Purinergic nerves

It was in 1972 that Burnstock laid the foundation of a new nerve type that he called ‘purinergic nerves’. In this article, he presented experimental data using five criteria to establish that adenosine triphosphate can be considered to be a neurotransmitter, including (1) the release of a purinergic molecule from terminal axons, (2) the structures of purinergic nerves, (3) the electrophysiological properties of purinergic transmission, (4) the pharmacology of adenyl compounds and purinergic transmission, and (5) the distribution and evolution of the purinergic nerves. However, in spite of convincing data, it took more than 20 years for the scientific community to accept this hypothesis. Since then, it has been recognized that the purinergic system is involved in multiple short-term actions such as cell proliferation and pain.