Differential trafficking of adenosine receptors in hippocampal neurons monitored using GFP- and super-ecliptic pHluorin-tagged receptors

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 A1Rs, A2ARs and A3Rs in primary hippocampal neurons. Furthermore, we developed a novel super-ecliptic pHluorin (SEP)-tagged A1R which, via the N-terminal SEP tag, reports the cell-surface expression and trafficking of A1Rs in real-time. We demonstrate the differential trafficking of ARs in neurons: A3Rs internalise more rapidly than A1Rs, with little evidence of appreciable A2AR trafficking over the time-course of the experiments. Furthermore, the novel SEP-A1R 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 A1R and A3Rs when expressed at the neuronal plasma membrane. Given the high levels of adenosine in the brain during ischaemia and seizures, internalisation of the inhibitory A1R may result in hyperexcitability, increased brain damage and the development of chronic epileptic states.

► Differential trafficking of GFP-tagged adenosine receptors in neurons is presented.
► The rate of agonist-induced internalisation is: A3R > A1R > A2AR.
► A novel N-terminal A1R construct with a pH-sensitive variant of GFP is described.
► SEP-A1R allows real-time measurement of A1R trafficking, which occurs rapidly.
► The labile AR pool may be relevant in conditions such as epilepsy and stroke.