P2Y₁ receptors are linked to K_Ca2 channels in PC12 cells

Background

P2Y₁ receptors are widely expressed in the brain, but their signalling mechanisms in neurons remained largely unknown. In sympathetic neurons, recombinant P2Y₁ receptors inhibit voltage-gated Ca²⁺ currents (I_Ca) and M-type K⁺ currents.

Methods

Patch-clamp recordings were performed in PC12 cell cultures, P2Y receptor ligands and signaling interceptors were applied.

Results

In PC12 cells stably expressing rat P2Y₁ receptors (PC12-P2Y₁), but not in wild type PC12 cells (PC12-wt), ADP induced rises in intracellular Ca²⁺ with half-maximal effects at 15 ± 1.3 μM. In whole-cell patch-clamp recordings, ADP inhibited I_Ca of PC12-P2Y₁ cells (EC₅₀: 6.3 ± 1.7 μM) and of PC12-wt (EC₅₀: 3.8 ± 1.3 μM); this effect was not altered by the P2Y₁ antagonist MRS 2216 (1 μM), but abolished by P2Y₁₂ antagonists. In perforated-patch recordings, ADP inhibited I_M relaxation amplitudes of PC12-P2Y₁ cells with half-maximal effects at 2.0 ± 1.8 μM, but in PC12-wt no such effect was observed. In PC12-P2Y₁, but not in PC12-wt cells, ADP (1-100 μM) caused transient increases in outward currents determined at -30 mV in the perforated-patch, but not the whole-cell mode. ADP-induced currents had reversal potentials between -80 and -90 mV which was close to the calculated K⁺ equilibrium potential (-89 mV). Replacement of 100 mM extracellular Na⁺ by K⁺ shifted the reversal potential of ADP-induced currents to about -10 mV which was again close to the K⁺ equilibrium potential (-17 mV). ADP-induced currents were prevented by thapsigargin (1 μM) and by the phospholipase C inhibitor U73122 (3 μM), but not by an inactive analogue. Finally, the ADP-induced currents were significantly reduced by 100 nM apamin.

Conclusion

These results reveal channels of the K_Ca2 family as novel targets for P2Y₁ receptor signalling.