Coupling of the nucleotide P2Y4 receptor to neuronal ion channels

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Abstract
1. G protein-linked P2Y nucleotide receptors are known commonly to stimulate the phosphoinositide signalling pathway. However, we have previously demonstrated that the cloned P2Y(2), P2Y(6) and P2Y(1) receptors couple to neuronal N-type Ca(2+) channels and to M-type K(+) channels. Here we investigate the coupling of recombinant, neuronally expressed rat- and human P2Y(4) receptors (rP2Y(4), hP2Y(4)) to those channels. 2. Rat sympathetic neurones were nuclear-injected with a P2Y(4) cDNA plasmid. A subsequent activation of rP2Y(4) or hP2Y(4) by UTP (100 micro M) in whole-cell (ruptured-patch) mode produced only about 12% inhibition of the N-type Ca(2+) current (I(Ca(N))). Surprisingly, in perforated patch mode, UTP produced much more inhibition of I(Ca(N)) (maximally 51%), with an IC(50) value of 273 nM. This inhibition was voltage-dependent and was blocked by co-expression of the betagamma-binding transducin Galpha-subunit. Pertussis toxin (PTX) pretreatment also suppressed I(Ca(N)) inhibition. 3. UTP inhibited the M-current, recorded in perforated patch mode, by (maximally) 52%, with IC(50) values of 21 nM for rP2Y(4) and 28 nM for hP2Y(4). This inhibition was not affected by PTX pretreatment. 4. With rP2Y(4), ATP inhibited the M-current (IC(50) 524 nM, 26 times weaker than UTP), whereas ATP had no agonist activity at hP2Y(4). This suggests a difference in agonist binding site between rP2Y(4) and hP2Y(4). 5. We conclude that, in contrast to other nucleotide receptors studied, the P2Y(4) receptor couples much more effectively to M-type K(+) channels than to Ca(2+) channels. Coupling to the Ca(2+) channels involves the betagamma-subunits of G(i/o)-proteins and requires a diffusible intracellular component that is lost in ruptured-patch recording.