Cloning and characterization of a P2X receptor expressed in the central nervous system of Lymnaea stagnalis

PLoS One. 2012;7(11):e50487. doi: 10.1371/journal.pone.0050487. Epub 2012 Nov 29.

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 (EC(50) 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 EC(50) 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 IC(50) values of 8.1 and 27.4 µM respectively. LymP2X is inhibited by acidic pH whereas Zn(2+) and Cu(2+) 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Animals
  • Central Nervous System / metabolism*
  • Copper / metabolism
  • Hydrogen-Ion Concentration
  • In Situ Hybridization
  • Lymnaea / metabolism*
  • Neurons / drug effects
  • Neurons / metabolism
  • Purinergic P2 Receptor Agonists / pharmacology
  • Purinergic P2 Receptor Antagonists / pharmacology
  • Receptors, Purinergic P2X2 / chemistry*
  • Receptors, Purinergic P2X2 / drug effects
  • Receptors, Purinergic P2X2 / genetics
  • Receptors, Purinergic P2X2 / metabolism*
  • Uridine Triphosphate / metabolism
  • Zinc / metabolism

Substances

  • Purinergic P2 Receptor Agonists
  • Purinergic P2 Receptor Antagonists
  • Receptors, Purinergic P2X2
  • Copper
  • Adenosine Triphosphate
  • Zinc
  • Uridine Triphosphate