Synaptic Activity Augments Muscarinic Acetylcholine Receptor-stimulated Inositol 1,4,5-Trisphosphate Production to Facilitate Ca2+ Release in Hippocampal Neurons

Abstract

Intracellular Ca2؉ store release contributes to activitydependent synaptic plasticity in the central nervous system by modulating the amplitude, propagation, and temporal dynamics of cytoplasmic Ca 2؉ changes. However, neuronal Ca 2؉ stores can be relatively insensitive to increases in the store-mobilizing messenger inositol 1,4,5-trisphosphate (IP 3 ). Using a fluorescent biosensor we have visualized M 1 muscarinic acetylcholine (mACh) receptor signaling in individual hippocampal neurons and observed increased IP 3 production in the absence of concurrent Ca 2؉ store release. However, coincident glutamate-mediated synaptic activity elicited enhanced and oscillatory IP 3 production that was dependent upon ongoing mACh receptor stimulation and S-␣-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid receptor activation of Ca 2؉ entry. Moreover, the enhanced levels of IP 3 now mobilized Ca 2؉ from intracellular stores that were refractory to the activation of mACh receptors alone. We conclude that convergent ionotropic and metabotropic receptor inputs can facilitate Ca 2؉ signaling by enhancing IP 3 production as well as augmenting release by Ca 2؉ -induced Ca 2؉ release.There is now substantial evidence that fundamental neuronal properties ranging from membrane excitability through gene expression to regulation of synaptic plasticity can be modulated by changes in intracellular free calcium (Ca 2ϩ i ) (1-4). In particular, it has been increasingly recognized that neuronal intracellular stores, far from just acting as a sink for Ca 2ϩ , can play a critical role in modulating the amplitude, localization, propagation, and temporal dynamics of neuronal Ca 2ϩ i transients (1-3, 5, 6). The key current questions relate to the mechanisms that might control local and global Ca 2ϩ signaling within the highly structured and functional domains of central neurons.A major player in Ca 2ϩ store release is the intracellular messenger IP 3 that can be generated by phospholipase C-mediated hydrolysis of the minor membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 1 (4). This signaling pathway is particularly prominent in the central nervous system, and a wide range of G protein-coupled receptors (GPCRs) can initiate the generation of IP 3 in neurons (1). Currently, models support a role for IP 3 receptors in the propagation of Ca 2ϩ waves observed in dendrites following activation of GPCRs such as muscarinic acetylcholine (mACh) and metabotropic glutamate receptors (2,7,8 (7, 9 -12). Alternatively or additionally, in view of the ability of Ca 2ϩ influx to activate PLC activity in neurons (13-16) enhanced production of IP 3 could further enhance regenerative Ca 2ϩ release (7,17). Indeed, the more favorable properties of IP 3 rather than Ca 2ϩ as a diffusible messenger (18) and the micro regional constraints imposed between the sites of generation and action of IP 3 in neurons such as hippocampal CA1 pyramidal cells (7) or sympathetic ganglia (19) would support such a model.In the present study we attempt to address these me...

Roles of Phosphorylation-dependent and -independent Mechanisms in the Regulation of M1 Muscarinic Acetylcholine Receptors by G Protein-coupled Receptor Kinase 2 in Hippocampal Neurons

“…Cell Culture and Transfections-Hippocampal neurons from 1-dayold Lister-hooded rat pups were isolated as described previously (22,23). Briefly, isolated hippocampi were dissociated with Pronase E (0.5 mg ml Ϫ1 ) and thermolysin (0.5 mg ml Ϫ1 ) in a HEPES-buffered salt solution (130 mM NaCl, 10 mM HEPES, 5.4 mM KCl, 1.0 mM MgSO 4 , 25 mM glucose, and 1.8 mM CaCl 2 , pH 7.2) for 30 min.…”

“…Measurement of PLC Activity in Neurons and Assessment of M 1 mACh Receptor Desensitization-PLC activity was assessed using the agonist-stimulated translocation of eGFP-tagged pleckstrin homology domain of PLC␦1 (eGFP-PH PLC␦ ) and was visualized using an Olympus Optical FV500 scanning laser confocal IX70 inverted microscope as described previously (18,22). All of the experiments were undertaken in the presence of tetrodotoxin (500 nM) to block action potential-dependent synaptic activity.…”

“…Phosphorylation-independent receptor regulation by GRK2 is mediated through a specific interaction of GTP-loaded G␣ q/11 with the RGS homology (RH) domain situated at the N terminus of GRK2 (14,15,19). Mutational and crystallographic analyses have identified specific amino acid residues within the RH domain that mediate G␣ q binding that map almost exclusively to the ␣5 helix of the RH domain of GRK2 (19 -21).We have previously used confocal imaging to detect the translocation of eGFP-PH PLC␦ as an IP 3 biosensor (18,22) to investigate M 1 mACh receptor regulation of PLC activity in hippocampal neurons. Using this approach, we showed that GRK2 inhibits M 1 mACh receptor signaling in hippocampal neurons via a phosphorylation-independent mechanism (18)…”

“…We have previously used confocal imaging to detect the translocation of eGFP-PH PLC␦ as an IP 3 biosensor (18,22) to investigate M 1 mACh receptor regulation of PLC activity in hippocampal neurons. Using this approach, we showed that GRK2 inhibits M 1 mACh receptor signaling in hippocampal neurons via a phosphorylation-independent mechanism (18)…”

“…We have previously used confocal imaging to detect the translocation of eGFP-PH PLC␦ as an IP 3 biosensor (18,22) to investigate M 1 mACh receptor regulation of PLC activity in hippocampal neurons. Using this approach, we showed that GRK2 inhibits M 1 mACh receptor signaling in hippocampal neurons via a phosphorylation-independent mechanism (18) 1 The abbreviations used are: mACh, muscarinic acetylcholine; PLC, phosphoinositide-specific phospholipase C; GPCR, G protein-coupled receptor; PKC, protein kinase C; CHO, Chinese hamster ovary; MCh, methacholine; IP 3 , inositol 1,4,5-trisphosphate; GRK, G protein-coupled receptor kinase; RH, RGS homology; eGFP, enhanced green fluorescent protein; HA, hemagglutinin; PDBu, phorbol 12,13-dibutyrate; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK kinase; JNK, c-Jun N-terminal kinase; AM, acetoxymethyl ester; BAPTA, 1,2-bis(O-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid; BAPTA-AM, 1,2-bis(O-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid tetra(acetoxymethyl) ester.…”