Neuronal P2Y receptors, i.e. nucleotide-sensitive G protein-coupled receptors (GPCRs), are known to control various voltage-gated ion channels, in particular KV7 K+ and CaV2.2 Ca2+ channels. The differential modulation of these ion channels via GPCRs was shown to rely on the presence or absence of scaffolding proteins such as AKAP79/150 and NHERF-2. Since scaffold proteins are believed to bring GPCRs and ion channels in close proximity to guarantee efficient G protein-mediated modulation, this project evaluates whether a tight contact between P2Y receptors and ion channels is a prerequisite for their functional interaction.
P2Y1 or P2Y12 receptors with fluorescent tags (CFP or YFP) were expressed together with fluorescently labeled KV7.2/7.3 or CaV2.2 channels in tsA 201 cells and the channel modulation by nucleotides was determined by measuring the according currents. To evaluate the behavior of the receptors and channels in the membrane, fluorescence recovery after photobleaching (FRAP) was determined by confocal laser microscopy.
Activation of P2Y1 but not of P2Y12 receptors by ADP inhibited the K+ currents in a concentration-dependent manner by up to 20.5 ± 1.9%. Conversely, activation of both, P2Y1 and P2Y12 receptors, reduced the Ca2+ currents by up to 60.1 ± 7.4% and 76.3 ± 4.2%, respectively. In initial FRAP experiments, the YFP-labeled receptors showed similar half-times of around 80 seconds. Upon coexpression of the P2Y1 receptor with the KV7 channel the half-time increased significantly (p < 0.009) to 116 seconds compared to single expression of receptor or channel only. In the case of P2Y12, coexpression with KV7 showed no significant change compared to P2Y12 or KV7 alone.
These findings suggest that distinct ion channels are modulated by different P2Y receptors. KV7 currents are inhibited by P2Y1 whereas CaV2.2 currents are reduced by both P2Y1 and P2Y12. Additionally, FRAP data show that the presence of KV7 slows down the movement of P2Y1 in the membrane, but not that of P2Y12. This suggests that there is a physical interaction between P2Y1 and KV7 which is not present between P2Y12 and KV7. The influence on movement of P2Y1 and P2Y12 receptors in the membrane by the presence CaV2.2 remains to be elucidated.
This study is supported by the FWF-funded doctoral program CCHD (W1205).