Regulatory mechanisms underlying the modulation of GIRK1/GIRK4 heteromeric channels by P2Y receptors

Abstract

The muscarinic K(+) channel (I (K,ACh)) is a heterotetramer composed of GIRK1 (Kir3.1) and GIRK4 (Kir3.4) subunits of a G protein-coupled inwardly rectifying channel, and plays an important role in mediating electrical responses to the vagal stimulation in the heart. I (K,ACh) displays biphasic changes (activation followed by inhibition) through the stimulation of the purinergic P2Y receptors, but the regulatory mechanism involved in these modulation of I (K,ACh) by P2Y receptors remains to be fully elucidated. Various P2Y receptor subtypes and GIRK1/GIRK4 (I (GIRK)) were co-expressed in Chinese hamster ovary cells, and the effect of stimulation of P2Y receptor subtypes on I (GIRK) were examined using the whole-cell patch-clamp method. Extracellular application of 10 μM ATP induced a transient activation of I (GIRK) through the P2Y(1) receptor, which was completely abolished by pretreatment with pertussis toxin. ATP initially caused an additive transient increase in ACh-activated I (GIRK) (via M(2) receptor), which was followed by subsequent inhibition. This inhibition of I (GIRK) by ATP was attenuated by co-expression of regulator of G-protein signaling 2, or phosphatidylinositol-4-phosphate-5-kinase, or intracellular phosphatidylinositol 4,5-bisphosphate loading, but not by the exposure to protein kinase C inhibitors. P2Y(4) stimulation also persistently suppressed the ACh-activated I (GIRK). In addition, I (GIRK) evoked by the stimulation of the P2Y(4) receptor exhibited a transient activation, but that evoked by the stimulation of P2Y(2) or P2Y(12) receptor showed a rather persistent activation. These results reveal (1) that P2Y(1) and P2Y(4) are primarily coupled to the G(q)-phospholipase C-pathway, while being weakly linked to G(i/o), and (2) that P2Y(2) and P2Y(12) involve G(i/o) activation.

Coupling of P2Y receptors to G proteins and other signaling pathways

“…In general, opening of GIRK channels by P2Y receptors and other GPCRs is mediated by the βγ subunit of G i/o proteins, whereas closing of GIRK channels is mediated by the α subunit of G q proteins 103,121. The P2Y 1 receptor, which couples primarily to G q proteins, has been shown to both open and close GIRK channels when expressed and activated in rat sympathetic neurons or CHO cells 121,122. Furthermore, closing of GIRK channels mediated by the P2Y 1 receptor in these studies was PTX‐sensitive, indicating that the P2Y 1 receptor in this instance is capable of coupling to G i/o proteins.…”