Dual modulation of inward rectifier potassium currents in olfactory neuronal cells by promiscuous G protein coupling of the oxytocin receptor

Abstract

Oxytocin (OT) is a small nonapeptide that regulates multiple peripheral (i.e. parturition and lactation) as well as central biological functions. Within the brain, OT is especially involved in the regulation of social behavior hence promoting maternal care and aggression, pair bonding, sexual activity, social memory processes and recognition of conspecific in various animal species (Donaldson and Young 2008). The role of OT in processing social stimuli and modulating pro-social responses has also been confirmed in humans, where the peptide has been shown to reduce stress and anxiety, to increase trust and generosity, and to improve the ability of interpreting mental states (Heinrichs et al. 2009). Accordingly, a link between autism spectrum disorders and the OT system has been proposed.Oxytocin is mainly synthesized in the magnocellular neurons of the supraoptic nuclei (SON) and paraventricular nuclei of the hypothalamus, projecting to the posterior pituitary gland, where OT is released into the bloodstream to exert its peripheral effects. Moreover, the dendrites of magnocellular neurons are densely packed with OT-containing neurosecretory granules that, upon exocytosis, regulate Address correspondence and reprint requests to Bice Chini, CNR, Institute of Neuroscience, Milan, Italy. E-mail: b.chini@in.cnr.itAbbreviations used: AVP, arginine-vasopressin; GnRH, gonadotropin releasing hormone; IR, inward rectifier; IRK, inwardly-rectifying potassium channel; GIRK, G protein-coupled inwardly-rectifying potassium channel; GPCR, G protein-coupled receptor; GTPcS, guanosine 5¢-O-[gamma-thio]triphosphate; OTR, oxytocin receptor; PLCb, phospholipase C b; PTX, pertussis toxin; SON, supraoptic nucleus; Thr-OT, Thr 4 Gly 7 -oxytocin; V 1a R, V 1b R, V 2 R, AVP receptors of the V 1a , V 1b and V 2 subtype. AbstractOxytocin receptor is a seven transmembrane receptor widely expressed in the CNS that triggers G i or G q protein-mediated signaling cascades leading to the regulation of a variety of neuroendocrine and cognitive functions. We decided to investigate whether and how the promiscuous receptor/G protein coupling affects neuronal excitability. As an experimental model, we used the immortalized gonadotropinreleasing hormone-positive GN11 cell line displaying the features of immature, migrating olfactory neurons. Using RT-PCR analysis, we detected the presence of oxytocin receptors whose stimulation by oxytocin led to the accumulation of inositol phosphates and to the inhibition of cell proliferation, and the expression of several inward rectifier (IR) K + channel subtypes. Moreover, electrophysiological and pharmacological inspections using whole-cell patchclamp recordings evidenced that in GN11 cells, IR channel subtypes are responsive to oxytocin. In particular, we found that: (i) peptide activation of receptor either inhibited or stimulated IR conductances, and (ii) IR current inhibition was mediated by a pertussis toxin-resistant G protein presumably of the G q/11 subtype, and by phospholipase C, whereas IR current ac...

Functional Selective Oxytocin-derived Agonists Discriminate between Individual G Protein Family Subtypes

“…The expression vector for G proteins fused to Renilla luciferase G␣ q -97-Rluc, G␣ i1 -91-Rluc, G␣ i2 -91-Rluc, G␣ i3 -91-Rluc, G␣ oA -91-Rluc8, G␣ oB -91-Rluc8, and G␣ s -113-Rluc8 cDNAs are described elsewhere. 4 The G␣ q , G␣ i1 , G␣ i2 , G␣ i3 , G␣ s , G␣ oA , G␣ oB , and G␥ 2 cDNAs came from the Missouri S&T cDNA Research Center (Rolla, MO). The plasmids encoding GFP 10 -G␥ 2 and G␤ 1 have been described previously (13); the plasmids encoding for the human OTR and V 2 R are described in Refs.…”

“…Previously engineered to measure G␣ i1 activation (13), BRET probes have now been built for all of the G␣ protein isoforms. 4 An G␣ s -113-Rluc8 was first used to confirm that OT is unable to induce OTR-G s activation. No change in BRET was detected with increasing OT concentrations up to 10…”

“…The oxytocin receptor (OTR) is a GPCR whose promiscuous coupling to G q and G i heterotrimeric complexes has been described in several cell types (2)(3)(4)(5). In different cell systems, the multiple signaling pathways activated by OTRs may act synergistically (as in the case of the contraction induced in myometrial cells by OTR coupling to G␣ q-11 and to the small G proteins of the Rho family) (6).…”

“…In different cell systems, the multiple signaling pathways activated by OTRs may act synergistically (as in the case of the contraction induced in myometrial cells by OTR coupling to G␣ q-11 and to the small G proteins of the Rho family) (6). However, they may also have opposite effects on the same cell function, as in the case of neuronal cells in which OT can inhibit (via a PTX-resistant G protein pathway) or stimulate (via a PTX-sensitive G protein pathway) K ϩ conductances belonging to the inward rectifier family of K ϩ channels (4). Similarly, in human embryonic kidney HEK293 cells stably transfected with human OTRs, receptor coupling to G i is responsible for inhibiting cell growth, whereas receptor coupling to a pertussis toxin (PTX)-insensitive complex (possibly G q ) stimulates cell growth (5,7).…”

“…What are the structural features characterizing these analogues? To start answering these questions, we used a bioluminescent resonance energy transfer (BRET)-based biosensor 4 to screen a number of OT/AVP-derived peptides for their ability to activate G q -, G i1 -, G i2 -, G i3 -, G oA -, G oB -, and G s -transducing complexes.…”