Cloned G-protein-coupled nucleotide P2Y₁, P2Y₂, P2Y₄ and P2Y₆ receptors mediate inhibition of neuronal Ca²⁺ and M-type K⁺ currents when expressed in rat superior cervical sympathetic (SCG) neurones (Brown et al. 2000). The recently cloned P2Y₁₂ receptor also produces stable activation of G-protein-gated inward rectifier K⁺ (GIRK) current (Simon et al. 2002). We report here that P2Y₁, P2Y₄ and P2Y₆ receptors also modulate GIRK currents when expressed in SCG neurones.

cDNA for individual P2Y receptors was co-injected with cDNA for GIRK1 and GIRK2 channels into the nucleus of cultured SCG neurones (isolated from rats killed by approved Schedule 1 methods). After overnight incubation cells expressed both receptors and functional GIRK channels. Currents were recorded using either perforated-patch or whole-cell (disrupted patch) techniques, with similar results.

Stimulation of P2Y₁ receptors with 2-MeSADP (1 µM) induced activation of GIRK current followed by inhibition (n = 15). In contrast, stimulation of endogenous α₂-adrenoceptors by noradrenaline produced stable activation without inhibition. P2Y₁-mediated inhibition was also seen when 2-MeSADP was applied after GIRK current preactivation by noradrenaline or expression of Gβ₁λ₂ subunits (n = 10).

Stimulation of P2Y₄ receptors with UTP (100 µM) (n = 13) or P2Y₆ receptors with UDP (10 µM) (n = 15) only weakly activated GIRK current but significantly inhibited preactivated current.

Current activation by P2Y₁ receptors was prevented by overnight pretreatment with Pertussis toxin (PTX), whereas inhibition of GIRK current by all three nucleotide receptors was insensitive to PTX. Inhibition of the GIRK current was not affected after co-expression of RGS11 (n = 3) that interferes with G_qβλ pathway or after co-expression of PLCδ-PH (n = 5), a construct that sequesters the membrane phospholipid PI(4,5)P₂. In contrast, inhibition was significantly reduced after co-expression of RGS2 protein (n = 9), which is known to inhibit G_qα signalling. Buffering of intracellular Ca²⁺ after 1-4 h pre-incubation with BAPTA-AM (10 µM) (n = 2), or pre-treatment with protein kinase C inhibitor, GF 109203X (1 µM) (n = 5), did not change GIRK current inhibition.

We conclude that activation of GIRK current by cloned P2Y receptors can be related to G_i/oβλ while inhibition – to G_qα. These effects may provide a mechanism for the modulation of excitability in the brain by P2Y receptors.

This work was supported by The Wellcome Trust.