Neuronal activity regulates phosphorylation-dependent surface delivery of G protein-activated inwardly rectifying potassium channels

Abstract

G protein-activated inwardly rectifying K(+) (GIRK) channels regulate neuronal excitability by mediating inhibitory effects of G protein-coupled receptors for neurotransmitters and neuromodulators. Notwithstanding many studies reporting modulation of GIRK channel function, whether neuronal activity regulates GIRK channel trafficking remains an open question. Here we report that NMDA receptor activation in cultured dissociated hippocampal neurons elevates surface expression of the GIRK channel subunits GIRK1 and GIRK2 in the soma, dendrites, and dendritic spines within 15 min. This activity-induced increase in GIRK surface expression requires protein phosphatase-1-mediated dephosphorylation of a serine residue (Ser-9) preceding the GIRK2 Val-13/Leu-14 (VL) internalization motif, thereby promoting channel recycling. Because activation of GIRK channels hyperpolarizes neuronal membranes, the NMDA receptor-induced regulation of GIRK channel trafficking may represent a dynamic adjustment of neuronal excitability in response to inhibitory neurotransmitters and/or neuromodulators.

Fig. 1.

Neuronal activity increases surface expression of endogenous GIRK channels in cultured hippocampal neurons. (A) Immunostaining of hippocampal neurons (21 DIV) with anti-GIRK2 C-terminal (red) and anti-synaptophysin antibodies (green). Arrows point to excitatory synapses on dendritic spines, whereas arrowheads point to inhibitory synapses on dendritic shafts. (Scale bar, 2 μm.) (B) Surface biotinylation of hippocampal neurons (17 DIV) treated with 0.1% (vol/vol) H₂O (Control), 50 mM KCl for 20 min, 100 μM glutamate, or 100 μM NMDA and 1 μM glycine (NMDA) for 1 min, respectively, followed by incubation in ACSF for 20 min. Endogenous tubulin is shown as cytoplasmic control. (C) Surface biotinylation of hippocampal neurons (14–17 DIV, pretreated with 200 μM APV for 3 days) incubated in media with 200 μM APV (control) or without APV for 15 min (synaptic NMDAR activation). Endogenous tubulin is shown as cytoplasmic control. (D) Quantification of surface GIRK1, GIRK2, and NMDAR subunit NR1 proteins after NMDA/glycine bath application (n = 13, 14, and 5, respectively) or synaptic NMDAR activation (n = 18, 25, and 5, respectively). (E) NMDAR-induced GIRK2 surface expression was not affected by the voltage-gated Na⁺ channel blocker tetrodotoxin (TTX) (1 μM) (n = 7) but was abolished by omitting extracellular Ca²⁺ (n = 7). **P < 0.01; ***P < 0.001.

Fig. 2.

NMDAR-induced surface expression of GIRK channels requires activation of PP1 via PP2B-independent signaling pathway. (A) Surface biotinylation of hippocampal neurons (DIV 17) treated with 0.1% (vol/vol) DMSO, 10 μM cyclosporine A (CysA, n = 7), 20 nM or 1 μM okadaic acid (Oka-20, Oka-1, n = 6 and n = 8, respectively). (B) Surface biotinylation of hippocampal neurons (DIV 17) treated with 0.1% (vol/vol) DMSO or kinase inhibitors, including 5 μM chelerythrine (Chel), 10 μM RP-8-Br-cAMP (RPcAMP), 10 μM KN93, or 20 μM 4,5,6,7-tetrabromobenzotriazole (TBB). (C) Synaptic NMDAR activation induces dephosphorylation of PP1 at Thr-320 (n = 3), as determined by immunoblot analysis with phosphorylation site-specific anti-PP1-pThr320 and phosphorylation-independent anti-PP1 antibodies. (D) Synaptic NMDAR activation did not cause PP2B-dependent dephosphorylation of DARPP32/I1 at Thr-34/Thr-35 (n = 4), as determined by immunoblot analysis with phosphorylation site-specific anti-DARPP32-pThr34 and phosphorylation-independent anti-DARPP32 antibodies. *P < 0.05; **P < 0.01.

Fig. 3.

NMDAR activation increases GIRK surface expression in neuronal soma, dendrites, and some spines. (A and C) Surface expression of GIRK1 with extracellular HA and C-terminal GFP tags (HA-GIRK1-GFP) cotransfected with GIRK2A in hippocampal neurons (14 DIV) after synaptic NMDAR activation for 15 min. Surface HA-GIRK1-GFP was labeled using anti-HA antibody without permeabilization (Surface), whereas total proteins were visualized with GFP fluorescence (Total). (B and C) Surface staining of HA-GIRK2-GFP after synaptic NMDAR activation. (Scale bars, 10 μm in A and B, 2 μm in C.) (D) Quantification of surface HA-GIRK1-GFP (cotransfected with GIRK2A) in dendrites after synaptic NMDAR activation (n = 21) or APV control solution change (n = 16). (E) Quantification of surface expression of HA-GIRK2-GFP wild type (WT), deletion mutants (ΔN15, ΔN45, ΔC19), and a VL/AA mutant (in which the VL internalization motif was mutated to AA) after NMDA/glycine bath application, or synaptic NMDAR activation. **P < 0.01; ***P < 0.001.

Fig. 4.

NMDAR-induced GIRK surface expression requires Rme1-dependent trafficking of GIRK channels from recycling endosomes to plasma membrane. (A) NMDAR-induced surface expression of endogenous GIRK2 was abolished by pretreating neurons with primaquine (60 μM, n = 4). (B) Coexpression of dominant negative Rme1 G429R traps GIRK2 in recycling endosomes in dendrites. Arrows indicate GIRK2 proteins that colocalized with dominant negative Rme1 G429R (Bottom). (Scale bar, 2 μm.) (C and D) Quantification of dendritic surface expression of HA-GIRK2 after NMDA/glycine bath application (C) or synaptic NMDAR activation (D) in hippocampal neurons transfected with HA-GIRK2 and GFP, GFP-Rme1 or dominant negative GFP-Rme1 G429R, which blocks recycling. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.

NMDAR activation induces dephosphorylation of GIRK2 Ser-9. (A) NMDA/glycine treatment (n = 8) and synaptic NMDAR activation (n = 6) significantly decreased Ser-9 phosphorylation of GIRK2 in hippocampal neurons, as determined by immunoblot analysis with phosphorylation site-specific anti-GIRK2-pSer9 antibody (pSer9) and phosphorylation-independent anti-GIRK2 N terminus antibody (Total). (B) In vitro dephosphorylation reaction of immunoprecipitated GIRK2 proteins with purified PP1 (2.5 U), PP2A (0.1 U), PP2B (50 U), or λ-phosphatase (400 U). (C and D) Quantitative immunoblot analysis of GIRK2 Ser-9-phosphorylation in neurons treated with 0.1% (vol/vol) DMSO (n = 8), 10 μM cyclosporine A (CysA, n = 5), 20 nM or 1 μM okadaic acid (Oka-20, Oka-1, n = 3 and n = 5, respectively), or 5 μM FK520 (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6.

NMDAR-induced GIRK surface expression requires dephosphorylation of Ser-9 of GIRK2, which promotes channel delivery from recycling endosomes. (A) Comparison between a typical dileucine internalization motif ([D/E]xxxL[L/I]) and GIRK2 N-terminal sequence (–15) containing serine (Ser-9) instead of [D/E] at −4 position from VL. (B) Channel endocytosis assay in COS7 cells with or without 60 μM primaquine (PQ) treatment. COS7 cells were transfected with HA-GIRK2 wild-type (WT) or mutants (S9A, S9D, and VL/AA). Mutation of Ser-9 to alanine (S9A) and aspartate (S9D) mimics dephosphorylated and phosphorylated Ser-9, respectively. The mean fluorescence intensity of surface HA-GIRK2 proteins that have been internalized and remained inside the cells at 80 min was divided by the mean fluorescence intensity of the surface GIRK2 proteins at 0 min (Fig. S8 A, B, and D) to compute the relative level of internalization. (C) Surface staining of HA-GIRK2-GFP WT, S9A, or S9D in a dendritic process after APV control solution change (Control) or synaptic NMDAR activation. (Scale bar, 2 μm.) (D) Quantification of dendritic surface expression of HA-GIRK2-GFP WT, S9A, or S9D after NMDA/glycine bath application or synaptic NMDAR activation. (E) Quantification of dendritic surface expression of HA-GIRK1-GFP coexpressed with GIRK2 WT, S9A, or S9D after NMDA/glycine bath application or synaptic NMDAR activation. **P < 0.01; ***P < 0.001.