P2Y(1) , P2Y(2) , P2Y(4) , P2Y(12) and P2Y(13) receptors for nucleotides have been reported to mediate presynaptic inhibition, but unequivocal evidence for facilitatory presynaptic P2Y receptors is not available. The search for such receptors was the purpose of this study.

Molecularly defined P2Y receptor subtypes are known to regulate the functions of neurons through an inhibition of K(V)7 K(+) and Ca(V)2 Ca(2+) channels and via an activation or inhibition of Kir3 channels. Here, we searched for additional neuronal ion channels as targets for P2Y receptors. Rat P2Y(1) receptors were expressed in PC12 cells via an inducible expression system, and the effects of nucleotides on membrane currents and intracellular Ca(2+) were investigated. At a membrane potential of 30 mV, ADP induced transient outward currents in a concentration-dependent manner with half-maximal effects at 4 μm. These currents had reversal potentials close to the K(+) equilibrium potential and changed direction when extracellular Na(+) was largely replaced by K(+), but remained unaltered when extracellular Cl() was changed. Currents were abolished by P2Y(1) antagonists and by blockade of phospholipase C. ADP also caused rises in intracellular Ca(2+), and ADP-evoked currents were abolished when inositol trisphosphate-sensitive Ca(2+) stores were depleted. Blockers of K(Ca)2, but not those of K(Ca)1.1 or K(Ca)3.1, channels largely reduced ADP-evoked currents. In hippocampal neurons, ADP also triggered outward currents at 30 mV which were attenuated by P2Y(1) antagonists, depletion of Ca(2+) stores, or a blocker of K(Ca)2 channels. These results demonstrate that activation of neuronal P2Y(1) receptors may gate Ca(2+)-dependent K(+) (K(Ca)2) channels via phospholipase C-dependent increases in intracellular Ca(2+) and thereby define an additional class of neuronal ion channels as novel effectors for P2Y receptors. This mechanism may form the basis for the control of synaptic plasticity via P2Y(1) receptors.

Phosphatidylinositol-4,5-bisphosphate (PIP2 ) is a key regulator of many membrane proteins, including voltage-gated Kv7.2 channels. In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP2 -binding domains in Kv7.2. Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP2 . Dephosphorylation of Kv7.2 affected channel inhibition via M1 muscarinic receptors, but not via bradykinin receptors. Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP2 , thereby ensuring the tight regulation of the channel via G protein-coupled receptors.