AbstractTo establish comprehensive pharmacology of P2X7 receptors, membranecurrent recording, intracellular calcium transient recording and ethidium bromide uptake were carried out to examine several selective (A-740003, A- 438079) and non-selective (suramin) P2X7 antagonists across mammalian P2X7 receptors (human, mouse and rat). These P2X7 receptors demonstrated species-dependent sensitivities to antagonists. In each species, A-740003 revealed variant IC50 values with different assays, indicating the assay- dependent pharmacology of P2X7 receptors.Conventionally, pharmacology can be used to define a native current but not in the case of the human breast cancer cell line, Hs578T. It is found that P2X7 was expressed at both mRNA and protein level. The ATP-evoked currents recorded from Hs578T cells were P2X7-like with distinctive electrophysiological features. But the pharmacology profile of the currents did not fit with P2X7 receptor. Further experiments are needed to either include or exclude the existence of functional P2X7 receptors in Hs578T.Transmembrane domain 2 (TM2) is known as the pore-forming region for P2X receptors. TM2 of P2X7 receptor was investigated with cysteine substitution scanning. The predicted !-helix structure of the TM2 segment was in good agreement with the results from the substituted cysteine accessibility method (SCAM). Thr336, Ser339, Tyr343, Phe344 and Thr348 were found important for both channel dilation and aqueous pore formation.Ser339 was further studied. Various substitutions at Ser339 were explored. The results suggest that the polarity of the side chain at Ser339 is essential for the channel dilation. Furthermore, disulfide bond formation was identified between S339C in the trimeric receptor, implying that the side chains of Ser339 might turn very close to each other during the channel opening and dilation.
|Date of Award||31 Dec 2012|
|Supervisor||Alexej Verkhratsky (Supervisor)|
- amino acid substitution
- P2X7 receptor