2011bevansphd.pdf (3.67 MB)
P2x Receptor Function in Lymnaea Stagnalis and Other Lower Organisms
thesisposted on 2012-02-03, 13:35 authored by Selvan Bavan
The mollusc Lymnaea stagnalis has a relatively simple central nervous system (CNS) consisting of large and easily identifiable neurons. This feature together with well characterized neuronal circuitry for important physiological processes and an established adenosine 5’-triphosphate (ATP) release system puts Lymnaea forward as an attractive model to examine the CNS function of P2X receptors. Furthermore, lower organism P2X receptors may provide structure function insights by virtue of conservation of functionally important domains. Following the identification of a P2X receptor-like sequence (LymP2X) in the Lymnaea CNS, the cloned complementary deoxyribonucleic acid encoding LymP2X when heterologously expressed in Xenopus oocytes exhibited ATP-evoked inward currents (EC50: 6.2 µM) with slow desensitisation. 2’, 3’-O-(4-Benzoylbenzoyl) adenosine 5’-triphosphate (BzATP) was a partial agonist (EC50: 2.4 µM), whilst pyridoxalphosphate-6-azophenyl-2', 5'-disulphonic acid (PPADS) and suramin were antagonists (IC50: 8.1 and 27.4 µM respectively). A P2X receptor from another invertebrate Hypsibius Dujardini (HdP2X) was also characterised in a comparative study. This tardigrade receptor displayed ATP-evoked currents (EC50: 28.5 µM) with extremely rapid activation and desensitization kinetics, which were concentration-dependently inhibited by copper and zinc. Histidine 306 in HdP2X was found to have a minor role in copper inhibition. Quantitative reverse transcription-polymerase chain reaction and in situ hybridization detected LymP2X expression in all CNS ganglia, including buccal suggesting a possible role in the feeding network. Intracellular recording of motoneuron activity demonstrated that application of ATP (1 mM) to buccal ganglia increased the rate of fictive feeding whereas PPADS abolished this response. The application of BzATP, in an attempt to selectively activate P2X receptors, did not trigger fictive feeding, suggesting that LymP2X activation alone does not initiate the feeding rhythm. BzATP however did cause a significant hyperpolarisation of a key feeding motoneuron, suggesting that LymP2X activation may be required for the latter phase in the ATP-evoked feeding cycle.
Supervisor(s)Ennion, Steven; Straub, Volko
Date of award2011-01-01
Awarding institutionUniversity of Leicester