Endogenous Gαq-Coupled Neuromodulator Receptors Activate Protein Kinase A

Abstract

Protein kinase A (PKA) integrates inputs from G-protein-coupled neuromodulator receptors to modulate synaptic and cellular function. Gαs signaling stimulates PKA activity, whereas Gαi inhibits PKA activity. Gαq, on the other hand, signals through phospholipase C, and it remains unclear whether Gαq-coupled receptors signal to PKA in their native context. Here, using two independent optical reporters of PKA activity in acute mouse hippocampus slices, we show that endogenous Gαq-coupled muscarinic acetylcholine receptors activate PKA. Mechanistically, this effect is mediated by parallel signaling via either calcium or protein kinase C. Furthermore, multiple Gαq-coupled receptors modulate phosphorylation by PKA, a classical Gαs/Gαi effector. Thus, these results highlight PKA as a biochemical integrator of three major types of GPCRs and necessitate reconsideration of classic models used to predict neuronal signaling in response to the large family of Gαq-coupled receptors.

Keywords: G protein-coupled receptor; Gαq signaling; acetylcholine; designer receptors; fluorescence lifetime imaging microscopy; hippocampus; muscarinic receptors; neuromodulation; optical reporters; protein kinase A.

Figure 1. An Optical FRET-FLIM Reporter Reveals Net PKA Activation by Muscarinic Receptors in the Hippocampus

(A) Schematic of the PKA activity reporter FLIM-AKAR. Upon phosphorylation of the threonine residue (shown as T for before, and pT for after phosphorylation) by PKA, the substrate region binds the FHA phosphopeptide binding domain. This brings the donor and acceptor fluorophores together, resulting in FRET and decreased donor fluorescence lifetime. (B) Image of an acute brain slice expressing FLIM-AKAR in hippocampal CA1 and subiculum (top) with the boxed region illustrating soma and dendritic branches of CA1 neurons shown enlarged (bottom). (C) Heat map of FLIM-AKAR lifetime in a hippocampal CA1 neuron (top: soma; bottom: primary apical dendrite), in response to acetylcholine (ACh, 100 μM) and subsequent forskolin (FSK, 50 μM) application. On the right are fluorescence intensity images to illustrate morphology. (D) The time course of net PKA activity in 3 subcellular compartments of the neuron in (C) during application of ACh and FSK. (E) Summaries of lifetime changes in the somatic cytoplasm during baseline, and in response to ACh and FSK (*: p<0.05 vs. baseline; **: p<0.05 vs ACh). Each square represents a data point and black lines show median values with interquartile intervals. (F) Image with maximal projection of a z stack in the hippocampal CA1 region that shows the design of the ACh puffing experiment: ACh was puffed via a patching pipette (shown in red) onto an apical dendrite of a CA1 pyramidal neuron expressing FLIM-AKAR in acute hippocampal slices, and both apical and basal dendrites (imaged regions shown in the rectangles) were monitored for FLIM-AKAR lifetime change. (G) The time course of net PKA activity in apical and basal dendrites of the neuron imaged in (F) in response to 3 second (3s) and 10 second (10s) ACh (200 μM) puffs. (H) X–Y summary plots of lifetime changes in the apical versus basal dendrites in response to 3s or 10s ACh puffs. (I) As in (D) for a representative neuron during application of muscarine (mus, 10 μM) and FSK. (J) As in (E) in response to mus and subsequent FSK addition. (*: p<0.05 vs. baseline; **: p<0.05 vs. mus). The composite data include stand-alone data as well as control data sets presented in other figures of this paper. Figures 1B and 1F were stitched from multiple images. See also Figure S1, Table S1.

Figure 2. Specificity Controls for PKA and an Independent Reporter Validate Activation of PKA by Muscarinic Receptor Activation

(A) Schematic of the non-phosphorylatable reporter FLIM-AKAR^T391A in which the PKA phosphorylation site was mutated to alanine. (B) Example plot showing FLIM-AKAR^T391A (mutant, abbreviated as mut) lifetime in CA1 neurons in response to 10 μM mus followed by 50 μM FSK in the somatic cytoplasm, nucleus and dendrite. (C) Summary plots showing the amplitudes of FLIM-AKAR or FLIM-AKAR^T391A lifetime changes in response to mus and FSK in the somatic cytoplasm and dendrite. *: p<0.05 vs. wt. (D) Schematic illustrating PKI inhibition of PKA. Binding of cAMP to the regulatory subunits (R) of PKA dissociates the regulatory and catalytic subunits (C), resulting in activation of PKA. PKI binds to the free catalytic subunits of PKA, thus inhibiting their activity. (E) Example plots showing FLIM-AKAR response in the presence of PKI in response to mus followed by FSK in the cytoplasm and dendrite. Nucleus data were not shown because PKI is a nuclear excluded protein. (F) Summary plots showing that PKI reduces the amplitudes of FLIM-AKAR lifetime changes in response to mus and FSK in the cytoplasm and dendrite. *: p<0.05 vs. no PKI. (G) Schematic of the PKAc-Substrate-Interaction (PSI) reporter. Binding of cAMP to the regulatory subunits (R) of PKA liberates the catalytic subunits (C), which then bind to a consensus PKA substrate that has been mutated so that it is not phosphorylatable. The physical interaction between PKAc-mEGFP and substrate-mCherry brings the donor and acceptor fluorophores together, resulting in FRET and decreased donor fluorescence lifetime. (H) Example plot showing PSI lifetime in CA1 neurons in response to 10 μM mus followed by 50 μM FSK in the somatic cytoplasm. Nucleus data were not shown because PKAc-mEGFP is excluded from the nucleus. (I) Summary plot showing the amplitudes of PSI lifetime changes in response to mus and FSK in the somatic cytoplasm. *: p<0.05 vs. baseline; **: p<0.05 vs. mus. See also Figures S2, S3, Table S1.

Figure 3. Gαq-coupled Hippocampal mAChR Activation Increases Net PKA Activity

(A&B) Example plot (A) and summaries (B) showing modulation of net PKA activity by 77-LH-28-1 (LH, 10 μM), primarily an agonist of the Gαq-coupled M1 receptor, followed by application of mus and FSK. *: P<0.05 vs. baseline. (C&D) Example plot (C) and summaries (D) showing the effect on mus- and FSK-induced change in net PKA activity in the cytoplasm by an inhibitor of the Gαq/G₁₁/G₁₄ family, YM-254890 (YM, 1 μM). *: p<0.05 vs. control See also Figures S2, S4, S5, S6, Table S1.

Figure 4. Calcium Transients Are Not Necessary for mAChR-induced Net PKA Activation

(A) Schematic illustrating model of Gαq signal transduction. Activation of Gαq-coupled receptors triggers PLCβ to cleave PIP2 into IP3 and DAG. IP3 activates IP3 receptors, leading to Ca²⁺ release from intracellular stores. DAG or Ca²⁺ binds and activates PKC. (B) Images of two GCaMP3-expressing hippocampal CA1 neurons showing fluorescence transients in response to bath application of mus in the reservoir at 0 sec. (C) Examples of GCaMP3 fluorescence changes in a CA1 pyramidal neuron in response to mus followed by 50 mM KCl application in control conditions (left) or in slices pre-incubated with 30 μM cyclopiazonic acid (CPA) and with nominal 0 Ca²⁺ ACSF (right). (D) Summary of amplitudes of peak changes in GCaMP3 fluorescence in response to mus under control and treatment conditions (labelled as øCa²⁺, corresponding to pre-incubation with CPA and using ACSF with nominal 0 Ca²⁺). *: p<0.05 vs. control. (E) Time course of FLIM-AKAR lifetime changes in response to mus and FSK, with pre-incubation with CPA, and with nominal 0 Ca²⁺ in the ACSF. (F) Summaries of FLIM-AKAR lifetime changes showing the effect of blocking Ca²⁺ transients on mus-induced PKA activity in the cytoplasm. See also Figure S7, Table S1.

Figure 5. PKC Activation Is Sufficient but Not Necessary for mAChR-induced Net PKA Activation

(A–C) Example time courses (left) and summaries (right) of FLIM-AKAR lifetime changes in response to the PKC activator phorbol 12, 13-dibutyrate (PDBu, 1 μM) and FSK, in HEK293 cells (A), acute hippocampal slice (B), and HEK 293 cells transfected with adenylyl cyclase 2 (AC2) (C). *: p<0.05 vs. baseline; **: p<0.05 vs. AC2 negative cells. (D) Example time course (left) and summaries (right) of FLIM-AKAR lifetime changes showing the effect of blocking PKC with the inhibitor GF109203X (GF, 2 μM). *: p<0.05 vs. no GF. See also Figures S2, S7, Table S1.

Figure 6. mAChR-mediated Phosphorylation by PKA Is Mediated by Signaling via Either Ca²⁺ or PKC

(A) Example time courses of PKA activity reporter lifetime changes in response to mus and FSK with inhibition of both Ca²⁺ transients and PKC (nominal 0 Ca²⁺ in the external solution, pre-incubation with CPA and GF109203X, including GF109203X in the perfusing solution). Additional application of the Gαs-coupled β-adrenergic receptor agonist isoproterenol (iso, 1 μM) was used to assess if net PKA modulation by Gαq- and Gαs-coupled receptors employ the same mechanism. (B) Summaries of FLIM-AKAR lifetime changes showing the effect of blocking both Ca²⁺ transients and PKC activity on mus-induced PKA activity in the cytoplasm. *: p<0.05 vs. control. (C) Summary plots showing the amplitudes of FLIM-AKAR lifetime change in response to mus and subsequent addition of iso with inhibition of both Ca²⁺ transients and PKC. *: p<0.05 vs. baseline. (D) Model based on our data illustrating that Gαq-coupled mAChR activation increases net PKA activity via PKC or Ca²⁺ transient dependent pathways. See also Figure S7, Table S1.

Figure 7. Gαq-coupled Receptor Modulation of PKA Activity Occurs with Multiple Receptors

(A) Activation of Group I mGluRs by their agonist (S)-3,5-DHPG (DHPG) or activation of the designer receptor hM3Dqs by their agonist Clozapine-N-oxide (CNO) leads to Gαq signaling. (B) Example (left) and summary plot (right) showing response of FLIM-AKAR to DHPG (50 μM) and FSK in the cytoplasm of hippocampal CA1 pyramidal neurons. *: p<0.05 vs. baseline. (C) CNO (10 μM), an agonist of the Gαq-coupled designer receptor hM3Dq, increased net PKA activity in a CA1 pyramidal neurons expressing FLIM-AKAR and hM3Dq. (D) Example plot (left) from the neuron in (C) and summaries (right) showing lifetime changes in the cytoplasm in response to CNO and subsequent application of mus and FSK. *: p<0.05 vs. baseline. See also Figure S8, Table S1.

Figure 8. Revised Model of GPCR Modulation of Net PKA Activity

Classically, Gαs- and Gαi-coupled GPCRs stimulate and inhibit PKA activity respectively. Our data reveal an additional pathway wherein Gαq-coupled neuromodulator GPCR activation increases net PKA activity via PKC or Ca²⁺ transient dependent pathways.