Adenine nucleotides inhibit recombinant N‐type calcium channels via G protein‐coupled mechanisms in HEK 293 cells; involvement of the P2Y13 receptor‐type

N‐type Ca2+ channel modulation by an endogenous P2Y receptor was investigated by the whole‐cell patch‐clamp method in HEK 293 cells transfected with the functional rabbit N‐type calcium channel. The current responses (ICa(N)) to depolarizing voltage steps were depressed by ATP in a concentration‐dependent manner. Inclusion of either guanosine 5′‐O‐(3‐thiodiphosphate) or pertussis toxin into the pipette solution as well as a strongly depolarizing prepulse abolished the inhibitory action of ATP. In order to identify the P2Y receptor subtype responsible for this effect, several preferential agonists and antagonists were studied. Whereas the concentration–response curves of ADP and adenosine 5′‐O‐(2‐thiodiphosphate) indicated a higher potency of these agonists than that of ATP, α,β‐methylene ATP, UTP and UDP were considerably less active. The effect of ATP was abolished by the P2Y receptor antagonists suramin and N6‐(2‐methylthioethyl)‐2‐(3,3,3‐trifluoropropylthio)‐β,γ‐dichloromethylene‐ATP, but not by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid, 2′deoxy‐N6‐methyladenosine‐3′,5′‐diphosphate or 2‐methylthio AMP. Using reverse transcription and polymerase chain reaction, mRNA for the P2Y1, P2Y4, P2Y6, P2Y11 and P2Y13 receptor subtypes, but not the P2Y2, and P2Y12 subtypes, was detected in HEK 293 cells. Immunocytochemistry confirmed the presence of P2Y1, and to a minor extent that of P2Y4, but not of P2Y2 receptors. Hence, it is tempting to speculate that P2Y13 receptors may inhibit N‐type Ca2+ channels via the βγ subunits of the activated Gi protein. British Journal of Pharmacology (2004) 141, 141–151. doi:10.1038/sj.bjp.0705588