School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK.
Neuropharmacology. 2011 Jul-Aug;61(1-2):1-11. doi: 10.1016/j.neuropharm.2011.02.005. Epub 2011 Feb 17.
Adenosine receptors (ARs) modulate many cellular and systems-level processes in the mammalian CNS. However, little is known about the trafficking of ARs in neurons, despite their importance in controlling seizure activity and in neuroprotection in cerebral ischaemia. To address this we examined the agonist-dependent internalisation of C-terminal GFP-tagged A(1)Rs, A(2A)Rs and A(3)Rs in primary hippocampal neurons. Furthermore, we developed a novel super-ecliptic pHluorin (SEP)-tagged A(1)R which, via the N-terminal SEP tag, reports the cell-surface expression and trafficking of A(1)Rs in real-time. We demonstrate the differential trafficking of ARs in neurons: A(3)Rs internalise more rapidly than A1Rs, with little evidence of appreciable A(2A)R trafficking over the time-course of the experiments. Furthermore, the novel SEP-A(1)R construct revealed the time-course of internalisation and recovery of cell-surface expression to occur within minutes of agonist exposure and removal, respectively. These observations highlight the labile nature of A(1)R and A(3)Rs when expressed at the neuronal plasma membrane. Given the high levels of adenosine in the brain during ischaemia and seizures, internalisation of the inhibitory A(1)R may result in hyperexcitability, increased brain damage and the development of chronic epileptic states.
腺苷受体（ARs）调节哺乳动物中枢神经系统中的许多细胞和系统水平过程。然而，尽管它们在控制癫痫发作活动和脑缺血中的神经保护中具有重要作用，但对于 ARs 在神经元中的转运知之甚少。为了解决这个问题，我们研究了原代海马神经元中 C 末端 GFP 标记的 A1R、A2AR 和 A3R 在激动剂依赖性内化的情况。此外，我们开发了一种新型超赤道 pHluorin（SEP）标记的 A1R，通过 N 末端 SEP 标记，实时报告 A1R 的细胞表面表达和转运。我们证明了 ARs 在神经元中的不同转运：A3R 的内化速度比 A1R 快，在实验过程中几乎没有明显的 A2AR 转运证据。此外，新型 SEP-A1R 构建体揭示了细胞表面表达的内化和恢复的时间过程分别在激动剂暴露和去除后的几分钟内发生。这些观察结果强调了在神经元质膜上表达时 A1R 和 A3R 的不稳定性质。鉴于在缺血和癫痫发作期间大脑中的腺苷水平较高，抑制性 A1R 的内化可能导致过度兴奋、增加脑损伤和慢性癫痫状态的发展。
