Cloning and Characterization of a P2X Receptor Expressed in the Central Nervous System of Lymnaea stagnalis

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.

Evolution of P2X receptors

“…Real‐time release of ATP from ganglia of the CNS of the freshwater snail, Lymnaea stagnalis , has been described 75. RT‐PCR with degenerate oligonucleotides was used to identify a P2X receptor fragment expressed in the CNS of L. stagnalis and the full‐length sequence was obtained by RACE‐PCR and the cloned receptor expressed in Xenopus oocytes to facilitate electrophysiological characterization 76. BzATP is a partial agonist at the Lymnea P2X receptor, but ADP and UTP are inactive.…”

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

“…The predicted structure of the Aplysia P2X reveals all major evolutionary conservative sites and posttranslational modifications (Supplementary Data, Fig. 1S), which are similar to its homolog in another gastropod, Lymnaea [30]. The genomic organization of the P2X receptors confirmed the overall evolutionary conservation of exons and intron-exon boundaries.…”

“…The release of ATP from the central ganglia of the pond snail, Lymnaea stagnalis was demonstrated [29], and, subsequently, P2X receptors were identified in this species with widespread expression across the CNS [30], but unknown function(s).…”

A set of endogenous control genes for use in quantitative real-time PCR experiments reveal that the wild-type formin Ldia2 is enriched in the early pond snail embryo

“…Specifically, as we set out to measure the expression of cytoskeletal genes in L. stagnalis , this precluded the use of actin and tubulin as appropriate endogenous controls, because they are themselves cytoskeletal genes. Unfortunately, many of the previously published qRT-PCR studies on L. stagnalis either used ribosomal RNA (rRNA), actin or tubulin genes as endogenous controls (Bavan et al , 2012; Bouetard et al , 2013; Carter et al , 2015; Hatakeyama et al , 2013; Lu & Feng, 2011; Ribeiro et al , 2010; van Kesteren et al , 2006; van Nierop et al , 2006). rRNA genes are potentially problematic because the over-abundance of rRNAs relative to the target mRNA sequence can lead to problems in accurate normalisation, and in any case, rRNA is transcribed through an independent pathway from mRNA and therefore not regulated in the same manner (Radonic et al , 2004).…”