Abstract
P2X receptors are membrane ion channels gated by extracellular ATP. Mammals possess seven distinct P2X subtypes (P2X1-7) that have important functions in a wide array of physiological processes including roles in the central nervous system (CNS) where they have been linked to modulation of neurotransmitter release. We report here the cloning and functional characterization of a P2X receptor from the mollusc Lymnaea stagnalis. This model organism has a relatively simple CNS consisting of large readily identifiable neurones, a feature which together with a well characterized neuronal circuitry for important physiological processes such as feeding and respiration makes it an attractive potential model to examine P2X function. Using CODEHOP PCR we identified a single P2X receptor (LymP2X) in Lymnaea CNS which was subsequently cloned by RT-PCR. When heterologously expressed in Xenopus oocytes, LymP2X exhibited ATP evoked inward currents (EC50 6.2 µM) which decayed during the continued presence of agonist. UTP and ADP did not activate the receptor whereas αβmeATP was a weak agonist. BzATP was a partial agonist with an EC50 of 2.4 µM and a maximal response 33% smaller than that of ATP. The general P2 receptor antagonists PPADS and suramin both inhibited LymP2X currents with IC50 values of 8.1 and 27.4 µM respectively. LymP2X is inhibited by acidic pH whereas Zn2+ and Cu2+ ions exhibited a biphasic effect, potentiating currents up to 100 µM and inhibiting at higher concentrations. Quantitative RT-PCR and in situ hybridization detected expression of LymP2X mRNA in neurones of all CNS ganglia suggesting this ion channel may have widespread roles in Lymnaea CNS function.
Highlights
In addition to its role as the primary energy source within cells, adenosine 59-triphosphate (ATP) acts as a signalling molecule and is thought to be one of the earliest transmitters to have appeared during the evolution of eukaryotes [1]
CODEHOP PCR to Identify a P2X Receptor Expressed in Lymnaea central nervous system (CNS)
In this study we describe the cloning and functional characterization of a P2X receptor expressed in the CNS of the model organism Lymnaea stagnalis
Summary
In addition to its role as the primary energy source within cells, adenosine 59-triphosphate (ATP) acts as a signalling molecule and is thought to be one of the earliest transmitters to have appeared during the evolution of eukaryotes [1]. ATP initiates intracellular signalling pathways by activating either fast acting P2X ion channels or slower acting P2Y G-protein coupled receptors. Mammalian species possess seven P2X receptor subtypes (P2X17), each encoded by a separate gene, which form non-selective cation channels in the plasma membrane upon gating by extracellular ATP [6,7]. The first P2X receptor identified in an invertebrate organism was from the blood fluke Schistosoma mansoni [10] and subsequently P2X receptors from more primitive organisms such as the amoeba Dictyostelium discoideum [11,12], the green algae Ostreococcus tauri [13], the choanoflagellate Monosiga brevicollis [13] and three species of basal fungi [14,15] were described as well as additional invertebrate P2X receptors from the tick Boophilus microplus [16] and the tardigrade Hypsibius dujardini [17]. The five P2X receptors present in Dictyostelium discoideum are localized to the contractile vacuole, an intracellular organelle involved in osmoregulation and Ca2+ release [11,12,18]
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