PORCN Negatively Regulates AMPAR Function Independently of Subunit Composition and the Amino-Terminal and Carboxy-Terminal Domains of AMPARs

Abstract

Most fast excitatory synaptic transmissions in the mammalian brain are mediated by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs), which are ligand-gated cation channels. The membrane expression level of AMPARs is largely determined by auxiliary subunits in AMPAR macromolecules, including porcupine O-acyltransferase (PORCN), which negatively regulates AMPAR trafficking to the plasma membrane. However, whether PORCN-mediated regulation depends on AMPAR subunit composition or particular regions of a subunit has not been determined. We systematically examined the effects of PORCN on the ligand-gated current and surface expression level of GluA1, GluA2, and GluA3 AMPAR subunits, alone and in combination, as well as the PORCN-GluA interaction in heterologous HEK293T cells. PORCN inhibited glutamate-induced currents and the surface expression of investigated GluA AMPAR subunits in a subunit-independent manner. These inhibitory effects required neither the amino-terminal domain (ATD) nor the carboxy-terminal domain (CTD) of GluA subunits. In addition, PORCN interacted with AMPARs independently of their ATD or CTD. Thus, the functional inhibition of AMPARs by PORCN in transfected heterologous cells was independent of the ATD, CTD, and subunit composition of AMPARs.

Keywords: AMPA receptor; PORCN; glutamate-induced currents; protein-protein interactions; receptor trafficking.

FIGURE 1

The overexpression of PORCN suppressedglutamate-induced currents mediated by GluA2 and GluA3 in HEK293T cells with or without stargazin coexpression. (A) Representative traces (left) and summary graphs (right) of the peak amplitudes and plateaus of 10 mM glutamate-induced currents in HEK293T cells transfected with GluA1 and either PORCN or a control plasmid. (B) Representative traces (left) and summary graphs (right) of the peak amplitudes and plateaus of 10 mM glutamate-induced currents in HEK293T cells transfected with GluA2 and either PORCN or a control plasmid. (C) Representative traces (left) and summary graphs (right) of the peak amplitudes and plateaus of 10 mM glutamate-induced currents in HEK293T cells transfected with GluA3 and either PORCN or a control plasmid. (D) Representative traces and summary graphs of the peak amplitudes and plateaus of 10 mM glutamate-induced currents in HEK293T cells transfected with GluA1, stargazin, and either PORCN or a control plasmid. (E) Representative traces and summary graphs of the peak amplitudes and plateaus of 10 mM glutamate-induced currents in HEK293T cells transfected with GluA2, stargazin, and either PORCN or a control plasmid. (F) Representative traces and summary graphs of the peak amplitudes and plateaus of 10 mM glutamate-induced currents in HEK293T cells transfected with GluA3, stargazin, and either PORCN or a control plasmid. (G) Representative traces and summary graphs of the peak amplitudes and plateaus of 10 mM glutamate-induced currents in HEK293T cells transfected with GluA1 and GluA2 and either PORCN or a control plasmid. (H) Representative traces and summary graphs of the peak amplitudes and plateaus of 10 mM glutamate-induced currents in HEK293T cells transfected with GluA2 and GluA3 and either PORCN or a control plasmid. (I) Representative traces and summary graphs of the peak amplitudes and plateaus of 10 mM glutamate-induced currents in HEK293T cells transfected with GluA1, GluA2, and stargazin and either PORCN or a control plasmid. (J) Representative traces and summary graphs of the peak amplitudes and plateaus of 10 mM glutamate-induced currents in HEK293T cells transfected with GluA2, GluA3, and stargazin and either PORCN or a control plasmid. In all panels, the black traces and bars represent the control condition (no PORCN expression), while the red traces and bars represent PORCN overexpression. All summary graphs show means ± SEMs; statistical comparisons were performed with a student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 2

The overexpression of PORCN suppressed the surface expression of GluA1, GluA2, and GluA3 in transfected HEK293T cells. (A) Representative images (left) and quantification of the puncta intensity (right) of the surface expression of GluAs in HEK293T cells expressing GluA1 and stargazin (A1), GluA2 and stargazin (A2), or GluA3 and stargazin (A3) and transfected with either PORCN or a control plasmid. (B) Representative images (left) and quantification of the puncta intensity (right) of the total expression of GluAs in HEK293T cells expressing GluA1 and stargazin (B1), GluA2 and stargazin (B2), or GluA3 and stargazin (B3) and transfected with either PORCN or a control plasmid. The white lines in the images represent scale bars (scale bars = 10 μm). All summary graphs show means ± SEMs; statistical comparisons were performed with a student’s t-test (*p < 0.05; ***p < 0.001).

FIGURE 3

PORCN suppressed the surface expression of GluA subunits of AMPARs in cultured neurons. (A) Representative images and quantification of the intensity and density of the surface expression of native GluA1. (B) Representative images and quantification of the intensity and density of the surface expression of native GluA2. (C) Representative images and quantification of the intensity and density of the surface expression of GluA3 in neurons transfected with N-flag tagged GluA3. The white lines in the images represent scale bars (scale bars = 10 μm). All summary graphs show means ± SEMs; statistical comparisons were performed with a student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 4

The ATD and CTD of GluAs are not required for the inhibitory effect of PORCN on glutamate-induced currents. (A) Representative traces (A1) and summary graphs (A2) of the normalized peak amplitudes of 10 mM glutamate-induced currents in HEK293T cells transfected with full-length GluA1 or GluA1 deletion constructs (GluA1-ΔATD, GluA1-Δ824, and GluA1-ΔC), stargazin, and either PORCN or a control plasmid. (B) Representative traces (B1) and summary graphs (B2) of the normalized peak amplitudes of 10 mM glutamate-induced currents in HEK293T cells transfected with full-length GluA2 or GluA2 deletion constructs (GluA2-ΔATD, GluA2-Δ824, or GluA2-ΔC), stargazin, and either PORCN or a control plasmid. (C) Representative traces (C1) and summary graphs (C2) of the normalized peak amplitudes of 10 mM glutamate-induced currents in HEK293T cells transfected with full-length GluA3 or GluA3 deletion constructs (GluA3-ΔATD, GluA3-Δ824, and GluA3-ΔMPR), stargazin, and either PORCN or a control plasmid. All summary graphs show means ± SEMs; statistical comparisons were performed with a student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 5

The ATD and CTD of GluAs are not required for the inhibitory effect of PORCN on the membrane expression of AMPARs in transfected HEK cells. (A) Representative images and quantification of the puncta intensity of the surface expression of GluA1 deletion constructs in HEK293T cells expressing GluA1 deletion constructs (A1: GluA1-ΔATD, A2: GluA1-Δ824, A3: GluA1-ΔC) and stargazin and transfected with either PORCN or a control plasmid. (B) Representative images and quantification of the puncta intensity of the surface expression of GluA2 deletion constructs in HEK293T cells expressing GluA2 deletion constructs (B1: GluA2-ΔATD, B2: GluA2-Δ824, B3: GluA2-ΔC) and stargazin and transfected with either PORCN or a control plasmid. (C) Representative images and quantification of the puncta intensity of the surface expression of GluA3 deletion constructs in HEK293T cells expressing GluA3 deletion constructs (C1: GluA3-ΔATD, C2: GluA3-Δ824, C3: GluA3-ΔMPR) and stargazin and transfected with either PORCN or a control plasmid. All summary graphs show means ± SEMs; statistical comparisons were performed with a student’s t-test (**p < 0.01; ***p < 0.001).

FIGURE 6

The ATD and CTD of GluAs are not required for the inhibitory effect of PORCN on the surface expression of AMPARs in cultured neurons. (A1,A2) Representative images and quantification of the intensity and density of the surface expression of GluA1 deletion constructs (A1: GluA1-ΔATD, A2: GluA1-ΔC) in cultured neurons expressing GluA1 deletion constructs. (B1,B2) Representative images and quantification of the intensity and density of the surface expression of GluA2 deletion constructs (B1: GluA2-ΔATD, B2: GluA2-ΔC) in cultured neurons expressing GluA2 deletion constructs. (C1–C3) Representative images and quantification of the intensity and density of the surface expression of GluA3 full length and deletion constructs (C1: GluA3-ΔATD, C1: GluA1-Δ824, C3: GluA3-ΔMPR) in cultured neurons expressing GluA3 deletion constructs. The white lines in the images represent scale bars (scale bars = 5 μm). All summary graphs show means ± SEMs; statistical comparisons were performed with a student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 7

The binding of PORCN to AMPARs is independent of the AMPAR ATD or CTD. (A) Pulldown of GluA1, GluA1-ΔATD, GluA1-Δ824, and GluA1-ΔC expressed in transfected HEK293T cells together with the pulldown of myc-tagged PORCN with an anti-myc antibody. (B) Pulldown of GluA2, GluA2-ΔATD, GluA2-Δ824, and GluA2-ΔC expressed in transfected HEK293T cells together with the pulldown of myc-tagged PORCN with an anti-myc antibody. (C) Pulldown of GluA3, GluA3-ΔATD, GluA3-Δ824, and GluA3-ΔMPR expressed in transfected HEK293T cells together with the pulldown of myc-tagged PORCN with an anti-myc antibody.