Gβ2 and Gβ4 participate in the opioid and adrenergic receptor‐mediated Ca²⁺ channel modulation in rat sympathetic neurons

Abstract

Key point• Sympathetic stellate ganglion (SG) neurons that innervate cardiac muscle play an important role in regulating heart rate and contractility.• In this study, we examined which Gβ protein subunit couples Ca 2+ channels to opioid and adrenergic receptors expressed in SG neurons.• We show that Gβ2 and Gβ4 are important signalling elements that maintain the transduction pathway of both receptor subtypes and Ca 2+ channels, and how their expression levels are differentially compensated.• Our results also indicate that Gβ1 subunits do not appear to play a role in either signalling pathway.• The elucidation of specific G proteins that couple Ca 2+ channels with opioid and adrenergic receptors will help us better understand how these receptors regulate synaptic transmission of SG neurons that innervate cardiac muscle.Abstract Cardiac function is regulated in part by the sympathetic branch of the autonomic nervous system via the stellate ganglion (SG) neurons. Neurotransmitters, such as noradrenaline (NA), and neuropeptides, including nociceptin (Noc), influence the excitability of SG neurons by modulating Ca 2+ channel function following activation of the adrenergic and nociceptin/orphanin FQ peptide (NOP) opioid receptors, respectively. The regulation of Ca 2+ channels is mediated by Gβγ, but the specific Gβ subunit that modulates the channels is not known. In the present study, small interference RNA (siRNA) was employed to silence the natively expressed Gβ proteins in rat SG tissue and to examine the coupling specificity of adrenergic and NOP opioid receptors to Ca 2+ channels employing the whole-cell variant of the patch-clamp technique. Western blotting analysis showed that Gβ1, Gβ2 and Gβ4 are natively expressed. The knockdown of Gβ2 or Gβ4 led to a significant decrease of the NA-and Noc-mediated Ca 2+ current inhibition, while Gβ1 silencing was without effect. However, sustaining low levels of Gβ2 resulted in an increased expression of Gβ4 and a concomitant compensation of both adrenergic and opioid signalling pathways modulating Ca 2+ channels. Conversely, Gβ4-directed siRNA was not accompanied with a compensation of the signalling pathway. Finally, the combined silencing of Gβ2 and Gβ4 prevented any additional compensatory mechanisms. Overall, our studies suggest that in SG neurons, Gβ2 and Gβ4 normally maintain the coupling of Ca 2+ channels with the receptors, with the latter subtype responsible for maintaining the integrity of both pathways.

The Expanding Roles of GβγSubunits in G Protein–Coupled Receptor Signaling and Drug Action

“…In a separate study aimed at identifying the subunit composition of the G protein coupled to M1-muscarinic acetylcholine receptors, it was shown using antisense oligonucleotides in rat basophilic leukemia RBL-2H3-hm1 (rat basophilic leukemia cell line) cells, that the loss of Gb 1 and Gb 4 reduced carbacholmediated Ca 2+ release (Dippel et al, 1996). Gb 2 and Gb 4 knockdown with siRNA demonstrated that these two Gb subunits mediate coupling of adrenergic and nociceptin receptors to voltage-gated calcium channels in stellate ganglion neurons (Mahmoud et al, 2012).…”

Alteration of the mu opioid receptor: Ca2+channel signaling pathway in a subset of rat sensory neurons following chronic femoral artery occlusion

“…The DRG tissue was transfected with small interference RNA (siRNA) using electroporation and lipofection, as described previously (Mahmoud et al 2012). Briefly, once L 4 -L 6 DRG tissue was removed, it was first electroporated with the Neon electroporator (Life Technologies, Carlsbad, CA) with three 1,000-V pulses for 20 ms duration in an electroporation solution, which consisted of R solution (Life Technologies), scrambled or G␣-targeted siRNA (3,000 nM; Life Technologies), and 2 mM 2,3-butanedione monoxime (BDM).…”

Gγ7 proteins contribute to coupling of nociceptin/orphanin FQ peptide (NOP) opioid receptors and voltage-gated Ca2+ channels in rat stellate ganglion neurons

“…SG neurons were transfected with siRNA employing both electroporation and lipofection as previously described [11]. Gγ7 subunit siRNA sequences designed in this study were chosen based on an eight point criteria with a scoring system (10 = best, 1 = worse) [17].…”

“…Following the transfection period, the SG tissue was enzymatically dissociated as previously described [11]. Briefly, the tissue was incubated in a shaking water bath (35°C) for 60 min in Earle's balanced salt solution containing 0.6 mg/ml collagenase Type D (Roche Diagnostics, Indianapolis, IN), 0.4 mg/ml trypsin (Worthington Biochemicals, Freehold, NJ) and 0.1 mg/ml DNase (Sigma-Aldrich).…”

“…Previously, we reported that the Noc-mediated modulation of Ca V currents occurred primarily via PTX-sensitive Gαi1 protein subunits in rat SG neurons [10]. Subsequently, employing small interference RNA (siRNA) approach, both Gβ2 and Gβ4 subunits were shown to mediate the coupling of NOP receptors with Ca V channels [11]. …”