β-blockers augment L-type Ca2+ channel activity by targeting spatially restricted β2AR signaling in neurons.

Ao Shen,Xi-Yong Yu,Dana Chen,Joseph M Martinez,Manpreet Kaur,Bing Xu,Yang K Xiang,Johannes W Hell,Qian Shi,Manuel F Navedo,Madeline Nieves-Cintron,Kwun-Nok Mimi Man,Peter Bartels

doi:10.7554/elife.49464

Ao Shen, Xi-Yong Yu + Show 11 more

Open Access

https://doi.org/10.7554/elife.49464

Copy DOI

Abstract

G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in mammalian cells. Here, we report neuronal excitability of β-blockers carvedilol and alprenolol at clinically relevant nanomolar concentrations. Carvedilol and alprenolol activate β2AR, which promote G protein signaling and cAMP/PKA activities without action of G protein receptor kinases (GRKs). The cAMP/PKA activities are restricted within the immediate vicinity of activated β2AR, leading to selectively enhance PKA-dependent phosphorylation and stimulation of endogenous L-type calcium channel (LTCC) but not AMPA receptor in rat hippocampal neurons. Moreover, we have engineered a mutant β2AR that lacks the catecholamine binding pocket. This mutant is preferentially activated by carvedilol but not the orthosteric agonist isoproterenol. Carvedilol activates the mutant β2AR in mouse hippocampal neurons augmenting LTCC activity through cAMP/PKA signaling. Together, our study identifies a mechanism by which β-blocker-dependent activation of GPCRs promotes spatially restricted cAMP/PKA signaling to selectively target membrane downstream effectors such as LTCC in neurons.

Highlights

G protein-coupled receptors (GPCRs) often signal through canonical G proteins and through noncanonical G proteinindependent signaling, frequently via G protein receptor kinases (GRKs) and b-arrestins (Lefkowitz, 2000; Xiang and Kobilka, 2003)
We found that various b-blockers including alprenolol (ALP), carvedilol (CAR), propranolol (PRO) and CGP12177 (177) were able to stimulate phosphorylation of b2 adrenergic receptor (b2AR) at protein kinase A (PKA) sites expressed in HEK293 cells, similar to the bAR agonist isoproterenol (ISO) (Figure 1A and Figure 1—figure supplement 1A)
In the central nervous system (CNS), b2AR emerges as a prevalent postsynaptic norepinephrine effector at glutamatergic synapses, where b2AR functionally interacts with AMPA receptor (AMPAR) and L-type Ca2+ channel (LTCC) CaV1.2, and regulates neuronal excitability and synaptic plasticity (Davare et al, 2001; Joiner et al, 2010; Wang et al, 2010; Qian et al, 2012)

Summary

Introduction

GPCRs often signal through canonical G proteins and through noncanonical G proteinindependent signaling, frequently via G protein receptor kinases (GRKs) and b-arrestins (Lefkowitz, 2000; Xiang and Kobilka, 2003). These b-blockers are emerging as partial agonists even at low concentrations rather than strict antagonists in mammalian cells This cAMP/PKA signaling is spatially restricted, selectively promoting phosphorylation of b2AR and CaV1.2 by PKA which augments LTCC activity in primary hippocampal neurons. We have engineered a mutant b2AR that can be selectively activated by carvedilol but not by the orthosteric agonist isoproterenol (ISO) to stimulate PKA but not GRK Together, these studies identify a unique mechanism by which b-blockers activate b2AR at low concentrations, which promotes Gs/cAMP/PKA signaling branch and selectively targets downstream LTCC channels in neurons. These studies identify a unique mechanism by which b-blockers activate b2AR at low concentrations, which promotes Gs/cAMP/PKA signaling branch and selectively targets downstream LTCC channels in neurons This observation may explain sympathomimetic effects of b-blockers in the CNS

Results

75 GRK-pβ2AR

Discussion

15 WT mutant

Materials and methods