Functional and physical interactions between P2Y receptors and ion channels

Background

Neuronal P2Y receptors, i.e. nucleotide-sensitive G protein-coupled receptors (GPCRs), are known to control various voltage-gated ion channels, in particular K_V7 K⁺ and Ca_V2.2 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.

Methods

P2Y₁ or P2Y₁₂ receptors with fluorescent tags (CFP or YFP) were expressed together with fluorescently labeled K_V7.2/7.3 or Ca_V2.2 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.

Results

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 K_V7 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 K_V7 showed no significant change compared to P2Y₁₂ or K_V7 alone.

Conclusions

These findings suggest that distinct ion channels are modulated by different P2Y receptors. K_V7 currents are inhibited by P2Y₁ whereas Ca_V2.2 currents are reduced by both P2Y₁ and P2Y₁₂. Additionally, FRAP data show that the presence of K_V7 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 K_V7 which is not present between P2Y₁₂ and K_V7. The influence on movement of P2Y₁ and P2Y₁₂ receptors in the membrane by the presence Ca_V2.2 remains to be elucidated.