Article date: January 2003
By: Alexander K Filippov, Joseph Simon, Eric A Barnard, David A Brown in Volume 138, Issue 2, pages 400-406
G protein‐linked P2Y nucleotide receptors are known commonly to stimulate the phosphoinositide signalling pathway. However, we have previously demonstrated that the cloned P2Y2, P2Y6 and P2Y1 receptors couple to neuronal N‐type Ca2+ channels and to M‐type K+ channels. Here we investigate the coupling of recombinant, neuronally expressed rat‐ and human P2Y4 receptors (rP2Y4, hP2Y4) to those channels.
Rat sympathetic neurones were nuclear‐injected with a P2Y4 cDNA plasmid. A subsequent activation of rP2Y4 or hP2Y4 by UTP (100 μM) in whole‐cell (ruptured‐patch) mode produced only about 12% inhibition of the N‐type Ca2+ current (ICa(N)). Surprisingly, in perforated patch mode, UTP produced much more inhibition of ICa(N) (maximally 51%), with an IC50 value of 273 nM. This inhibition was voltage‐dependent and was blocked by co‐expression of the βγ‐binding transducin Gα‐subunit. Pertussis toxin (PTX) pretreatment also suppressed ICa(N) inhibition.
UTP inhibited the M‐current, recorded in perforated patch mode, by (maximally) 52%, with IC50 values of 21 nM for rP2Y4 and 28 nM for hP2Y4. This inhibition was not affected by PTX pretreatment.
With rP2Y4, ATP inhibited the M‐current (IC50 524 nM, 26 times weaker than UTP), whereas ATP had no agonist activity at hP2Y4. This suggests a difference in agonist binding site between rP2Y4 and hP2Y4.
We conclude that, in contrast to other nucleotide receptors studied, the P2Y4 receptor couples much more effectively to M‐type K+ channels than to Ca2+ channels. Coupling to the Ca2+ channels involves the βγ‐subunits of Gi/o‐proteins and requires a diffusible intracellular component that is lost in ruptured‐patch recording.
G protein‐linked P2Y nucleotide receptors are known commonly to stimulate the phosphoinositide signalling pathway. However, we have previously demonstrated that the cloned P2Y2, P2Y6 and P2Y1 receptors couple to neuronal N‐type Ca2+ channels and to M‐type K+ channels. Here we investigate the coupling of recombinant, neuronally expressed rat‐ and human P2Y4 receptors (rP2Y4, hP2Y4) to those channels.
Rat sympathetic neurones were nuclear‐injected with a P2Y4 cDNA plasmid. A subsequent activation of rP2Y4 or hP2Y4 by UTP (100 μM) in whole‐cell (ruptured‐patch) mode produced only about 12% inhibition of the N‐type Ca2+ current (ICa(N)). Surprisingly, in perforated patch mode, UTP produced much more inhibition of ICa(N) (maximally 51%), with an IC50 value of 273 nM. This inhibition was voltage‐dependent and was blocked by co‐expression of the βγ‐binding transducin Gα‐subunit. Pertussis toxin (PTX) pretreatment also suppressed ICa(N) inhibition.
UTP inhibited the M‐current, recorded in perforated patch mode, by (maximally) 52%, with IC50 values of 21 nM for rP2Y4 and 28 nM for hP2Y4. This inhibition was not affected by PTX pretreatment.
With rP2Y4, ATP inhibited the M‐current (IC50 524 nM, 26 times weaker than UTP), whereas ATP had no agonist activity at hP2Y4. This suggests a difference in agonist binding site between rP2Y4 and hP2Y4.
We conclude that, in contrast to other nucleotide receptors studied, the P2Y4 receptor couples much more effectively to M‐type K+ channels than to Ca2+ channels. Coupling to the Ca2+ channels involves the βγ‐subunits of Gi/o‐proteins and requires a diffusible intracellular component that is lost in ruptured‐patch recording.
British Journal of Pharmacology (2003) 138, 400–406. doi:10.1038/sj.bjp.0705043
DOI: 10.1038/sj.bjp.0705043
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