The effect of overexpression of ABHD6 on the reduction of peak current in AMPARs.

(A) Plasmid abbreviations and variant combinations of AMPAR. (B-D) Representative traces (left) and summary graphs of the peak amplitudes (right) of 10 mM glutamate-induced currents in HEK 293T cells transfected with GluA1-3 (black), GluA1-3 + ABHD6 (orange), GluA1-3 + TARP γ-2 (blue), and GluA1-3 + TARP γ-2 + ABHD6 (red). The statistical method was one-way ANOVA followed by a two-way comparison (*P < 0.05; **P < 0.01; ***P < 0.001. Table. EV1.2).

Overexpression of ABHD6 accelerated the deactivation of AMPARs-TARP

γ-2 complexes in HEK 293T cells. The normalized traces and the summary bar graphs of the τ w, deactof glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch from HEK 293T cells transfected with GluA (black), GluA + ABHD6 (orange), GluA + TARP γ-2 (blue), and GluA + TARP γ-2 + ABHD6 (red). (A) GluA1i. (B) GluA1o. (C) GluA2(Q)i-R. (D) GluA2(Q)o-R. (E) GluA2(Q)i-G. (F) GluA2(Q)o-G. (G) GluA2(Q)i-R-TARP γ-2 tandem. (I) GluA1i-TARP γ-2 tandem. γ2-containing GluA receptors could be isolated when 50 μM spermine was in the internal solution and recorded at +50 mV, the average traces and the normalized traces (right), and the summary bar graphs of the τ w, deact of glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch recorded at +50 mV from HEK 293T cells transfected with GluA-TARP γ-2 tandem (blue) and GluA-TARP γ-2 tandem + ABHD6 (red). (H) GluA2(Q)i-R-TARP γ-2. (J) GluA1i-TARP γ-2. The statistical method was one-way ANOVA followed by a two-way comparison (*P < 0.05; **P < 0.01; ***P < 0.001. Table. EV2.2).

Overexpression of ABHD6 accelerated the desensitization of AMPARs-TARP

γ-2 complexes in HEK 293T cells. The normalized traces and the summary bar graphs of the τ w, des of glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch from HEK 293T cells transfected with GluA (black), GluA + ABHD6 (orange), GluA + TARP γ-2 (blue), and GluA + TARP γ-2 + ABHD6 (red). (A) GluA1i. (B) GluA1o. (C) GluA2(Q)i-R. (D) GluA2(Q)o-R. (E) GluA2(Q)i-G. (F) GluA2(Q)o-G. (G) GluA2(Q)i-R-TARP γ-2 tandem. (I) GluA1i-TARP γ-2 tandem. TARP γ-2-containing GluA receptors could be isolated when 50 μM spermine was in the internal solution and recorded at +50 mV, the average traces and the normalized traces (right), and the summary bar graphs of the τ w, des, and peak amplitude of glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch recorded at +50 mV from HEK 293T cells transfected with GluA-TARP γ-2 tandem (blue) and GluA-TARP γ-2 tandem + ABHD6 (red). (H) GluA2(Q)i-R-TARP γ-2. (J) GluA1i-TARP γ-2. The statistical method was one-way ANOVA followed by a two-way comparison (*P < 0.05; **P < 0.01; ***P < 0.001. Table. EV3.2).

Overexpression of ABHD6 slows the recovery from desensitization of GluA1i-TARP

γ-2 complexes in HEK 293T cells. (A-F) Glutamate (Glu, 10 mM) induced currents in an outside-out patch excised from an HEK 293T cell transfected with GluA (black), GluA + ABHD6 (orange), GluA + TARP γ-2 (blue), and GluA+ TARP γ-2 + ABHD6 (red). The recovery ratio curves from desensitization and the summary bar graphs of the τ w, rec. (A) GluA1i. (B) GluA1o. (C) GluA2(Q)i-R. (D) GluA2(Q)o-R. (E) GluA2(Q)i-G. (F) GluA2(Q)o-G. (G) GluA2(Q)i-R-TARP γ-2 tandem. (H) GluA1i-TARP γ-2 tandem. The statistical method was one-way ANOVA followed by a two-way comparison (*P < 0.05; **P < 0.01; ***P < 0.001. Table. EV4.2).

Overexpression of ABHD6 accelerated the deactivation and desensitization of GluA1i/GluA2(R)i-G (or GluA2(R)i-G/GluA3(R)i) receptors-TARP

γ-2 complexes in HEK 293T cells and slowed the recovery of GluA1i/GluA2(R)i-G receptors in the presence and absence of TARP γ-2. (A) The normalized traces and the summary bar graphs of the τ w, deact of glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch recorded at-60 mV from HEK 293T cells transfected with GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i (black), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + ABHD6 (orange), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 + ABHD6 (red). (B) The normalized traces and the summary bar graphs of the τ w, des, and peak amplitude of glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch recorded at-60 mV from HEK 293T cells transfected with GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i (black), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + ABHD6 (orange), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 + ABHD6 (red). (C-D) Glutamate (Glu, 10 mM) induced currents in an outside-out patch excised from an HEK 293T cell transfected with GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i (black), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + ABHD6 (orange), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 + ABHD6 (red). The first application of 100 ms glutamate was followed by a second glutamate application at increasing pulse intervals at-60 mV. The recovery ratio curves from desensitization (C) and the summary bar graphs of the τ w, rec (D) The statistical method was one-way ANOVA followed by a two-way comparison (*P < 0.05; **P < 0.01; ***P < 0.001. Table. EV5.2 and Table. EV6.2)

Deletion of ABHD6 decelerated the deactivation and desensitization in ABHD6 KO primary hippocampus neurons

(A) The normalized traces and the summary bar graphs of the τ w, deact of Glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch recorded at-70 mV from wild-type primary hippocampus neurons (black) and ABHD6 knockout (ABHD6 KO) primary hippocampus neurons (red); (A2) the average traces of the τ w, deact of Glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch recorded at - 70 mV from primary hippocampus neurons. (B) The normalized traces and the summary bar graphs of the τ w, des of Glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch recorded at-70 mV from wild-type primary hippocampus neurons (black) and ABHD6 knockout (ABHD6 KO) primary hippocampus neurons (red); (B2) the average traces of the τ w, des of Glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch recorded at - 70 mV from primary hippocampus neurons. (C) Glutamate (Glu, 10 mM) induced currents in an outside-out patch excised from wild-type primary hippocampus neurons (black) and ABHD6 knockout (ABHD6 KO) primary hippocampus neurons (red). The first application of 100 ms glutamate was followed by a second glutamate application at increasing pulse intervals at-70 mV. The recovery ratio curves from desensitization (C1, left), the summary bar graphs of the τ w, rec (C2, right), and the typical trace of recovery from desensitization (C2). (D) The typical trace and the summary bar graphs of glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch from WT primary hippocampus neurons (black) and ABHD6 knockout (ABHD6 KO) primary hippocampus neurons (red). The statistical method was one-way ANOVA followed by a two-way comparison (*P < 0.05; **P < 0.01; ***P < 0.001. Table. EV7.2)

ABHD6 accelerated the deactivation and desensitization of homomeric GluA4i-TARP

γ-2 complexes and negatively regulates the kinetics of GluA1i-TARP γ-8 complexes (A) The normalized traces and the summary bar graphs of the τ w, deact of glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch recorded at-60 mV from HEK 293T cells transfected with GluA4i or GluA1i (black), GluA4i or GluA1i (black) + ABHD6 (orange), GluA4i or GluA1i + TARP γ-2 (blue), or GluA4i or GluA1i + TARP γ-2 + ABHD6 (red). (B) The normalized traces and the summary bar graphs of the τ w, desof glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch recorded at-60 mV from HEK 293T cells transfected with GluA4i or GluA1i (black), GluA4i or GluA1i (black) + ABHD6 (orange), GluA4i or GluA1i + TARP γ-2 (blue), or GluA4i or GluA1i + TARP γ-2 + ABHD6 (red). (C-D) Glutamate (Glu, 10 mM) induced currents in an outside-out patch excised from an HEK 293T cell transfected with GluA4i or GluA1i (black), GluA4i or GluA1i (black) + ABHD6 (orange), GluA4i or GluA1i + TARP γ-2 (blue), or GluA4i or GluA1i + TARP γ-2 + ABHD6 (red). The statistical method was one-way ANOVA followed by a two-way comparison (*P < 0.05; **P < 0.01; ***P < 0.001. Table. EV8.2, EV 9.2)

Schematic illustration of the AMPAR subunit; the sequence alignment of RNA splice variants and editing of AMPAR; co-expression validation and Pearson’s correlation between natural logarithm peak amplitude (pA) and the

τ w, deact and τ w, des co-expression with various GluA subunits. (A) Topology of a single AMPAR subunit in the plasma membrane. Each subunit consists of an extracellular N-terminal domain (NTD), a ligand-binding domain (LBD), a transmembrane domain (TMD, M1–4), and an intracellular C-terminal domain (CTD); the flip/flop splice variants and the RNA editing sites (Q/R and R/G) are also shown. (B) Sequence alignment of RNA splice variants and editing. Q/R editing sites (red letters) (GluA2), R/G editing sites (purple letters) (GluA2, A3), flip/flop splice variants (grey box) (GluA1–A3). Complete sequences can be found in UniProt. Sequences are homologous and conserved in mouse, rat, and human. (C1) Representative confocal images of HEK 293T cells co-transfected with GluA1-flip, ABHD6, and TARP γ-2 plasmids. GluA1-flip: rabbit anti-GluR1 polyclonal antibody (Millipore, Cat. #AB1504, 1:1000) followed by Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 647 (Invitrogen, Cat. #A32733, 1:2000). ABHD6: detected via its Myc tag; primary antibody Myc-Tag (19C2) mAb (Abmart, Cat. #M20002, 1:1000) and secondary Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555 (Invitrogen, Cat. #A-21424, 1:2000). (C2) The summary bar graphs of GluA1, TARP γ-2, ABHD6, and co-expression. (D) Affinity chromatography for ABHD6 and TARP γ-2; ABHD6 and GluA1i (or TARP γ-2) were transfected into HEK 293T cells and anti-Myc Antibody (M20002, Abmart) or anti-Myc Antibody (AE070, ABclonal) with 24 μL of protein G beads was added to samples, and then the beads were washed. (E1-E9) Pearson’s correlation and Local Polynomial Regression (loess) with 95% confidence intervals between natural logarithm peak amplitude (pA) and the τ w, deact (ms) of Glutamate (10 mM Glu, 1 ms) induced currents and τ w, des (ms) of Glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch from HEK 293T cells transfected with various GluA subunits.

Average traces of deactivation of AMPAR with overexpression of ABHD6 in HEK 293T cells.

The average traces of Glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch from HEK 293T cells transfected with GluA (black), GluA + ABHD6 (orange), GluA + TARP γ-2 (blue), and GluA + TARP γ-2 + ABHD6 (red). (A) GluA1i. (B) GluA1o. (C) GluA2(Q)i-R. (D) GluA2(Q)o-R. (E) GluA2(Q)i-G. (F) GluA2(Q)o-G. (G, H) GluA2(Q)i-R-TARP γ-2 tandem. (I, J) GluA1i-TARP γ-2 tandem.

Average traces of desensitization of AMPAR with overexpression of ABHD6 in HEK 293T cells.

The average traces of Glutamate (10 mM Glu, 500 ms) induced currents in the outside-out patch from HEK 293T cells transfected with GluA (black), GluA + ABHD6 (orange), GluA + TARP γ-2 (blue), and GluA + TARP γ-2 + ABHD6 (red). (A) GluA1i. (B) GluA1o. (C) GluA2(Q)i-R. (D) GluA2(Q)o-R. (E) GluA2(Q)i-G. (F) GluA2(Q)o-G. (G, H) GluA2(Q)i-R-TARP γ-2 tandem. (I, J) GluA1i-TARP γ-2 tandem.

Typical traces of the recovery from desensitization of AMPAR in HEK 293T cells.

(A-F) Glutamate (Glu, 10 mM) induced currents in an outside-out patch excised from an HEK 293T cell transfected with GluA (black), GluA + ABHD6 (orange), GluA + TARP γ-2 (blue), and GluA+ TARP γ-2 + ABHD6 (red). The first application of 100 ms glutamate was followed by a second glutamate application at increasing pulse intervals at-60 mV. The typical traces from a cell are normalized and aligned to the peak. The typical traces of the recovery from desensitization. (A) GluA1i. (B) GluA1o. (C) GluA2(Q)i-R. (D) GluA2(Q)o-R. (E) GluA2(Q)i-G. (F) GluA2(Q)o-G. (G) GluA2(Q)i-R-TARP γ-2 tandem. (H) GluA1i-TARP γ-2 tandem.

Average traces of the deactivation, desensitization, and recovery from desensitization of GluA1i/GluA2(R)i-G (or GluA2(R)i-G/GluA3(R)i) receptors-TARP

γ-2 complexes in HEK 293T cells. (A) The average traces of the τ w, deact of glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch recorded at-60 mV from HEK 293T cells transfected with GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i (black), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + ABHD6 (orange), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 + ABHD6 (red). (B) The average traces of the τ w, des, and peak amplitude of glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch recorded at-60 mV from HEK 293T cells transfected with GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i (black), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + ABHD6 (orange), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 + ABHD6 (red). (C) The typical trace of recovery from desensitization. Glutamate (Glu, 10 mM) induced currents in an outside-out patch excised from an HEK 293T cell transfected with GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i (black), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + ABHD6 (orange), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 + ABHD6 (red). The first application of 100 ms glutamate was followed by a second glutamate application at increasing pulse intervals at-60 mV.

Average traces of the deactivation, desensitization, and recovery from desensitization of GluA4i-TARP

γ-2 complexes and GluA1i-TARP γ-8 complexes in HEK 293T cells. (A) The average traces of the τ w, deact of glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch recorded at-60 mV from HEK 293T cells transfected with GluA4i or GluA1i (black), GluA4i or GluA1i (black) + ABHD6 (orange), GluA4i or GluA1i + TARP γ-2 (blue), or GluA4i or GluA1i + TARP γ-2 + ABHD6 (red). (B) The average traces of the τ w, des, and peak amplitude of glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch recorded at-60 mV from HEK 293T cells transfected with GluA4i or GluA1i (black), GluA4i or GluA1i (black) + ABHD6 (orange), GluA4i or GluA1i + TARP γ-2 (blue), or GluA4i or GluA1i + TARP γ-2 + ABHD6 (red). (C) The typical trace of recovery from desensitization. glutamate (Glu, 10 mM) induced currents in an outside-out patch excised from an HEK 293T cell transfected with GluA4i or GluA1i (black), GluA4i or GluA1i (black) + ABHD6 (orange), GluA4i or GluA1i + TARP γ-2 (blue), or GluA4i or GluA1i + TARP γ-2 + ABHD6 (red). The first application of 100 ms glutamate was followed by a second glutamate application at increasing pulse intervals at-60 mV.

Summary of peak amplitude (pA) of GluAs when co-transfected with/without γ-2 or/and ABHD6.

Summary of P values for comparison of peak amplitude.

Summary of τ w, deact (ms) of GluAs when co-transfected with/without γ-2 or/and ABHD6.

Summary of P values for comparison of τ w, deact.

Summary of τ w, des (ms) of GluAs when co-transfected with/without γ-2 or/and ABHD6.

Summary of P values for comparison of τ w, des.

Summary of τ w, rec (ms) of GluAs when co-transfected with/without γ-2 or/and ABHD6.

Summary of P values for comparison of τ w, rec.

Summary of GluA1i/GluA2(R)i-G receptors when co-transfected with/without γ-2 or/and ABHD6.

Summary of P values for comparison of GluA1i/GluA2(R)i-G receptors when co-transfected with/without γ-2 or/and ABHD6.

Summary of GluA2(R)i-G/GluA3(R)i receptors when co-transfected with/without γ-2 or/and ABHD6.

Summary of P values for comparison of GluA2(R)i-G/ GluA3(R)i receptors when co-transfected with/without γ-2 or/and ABHD6.

Summary of wild-type primary hippocampal neurons and ABHD6 knockout primary hippocampal neurons.

Summary of P values for comparison of wild-type primary hippocampal neuron and ABHD6 knockout primary hippocampal neuron.

Summary of GluA4i receptors when co-transfected with/without γ-2 or/and ABHD6.

Summary of P values for comparison of GluA4i receptors when co-transfected with/without γ-2 or/and ABHD6.

Summary of GluA1i receptors when co-transfected with/without γ-8 or/and ABHD6.

Summary of P values for comparison of GluA1i receptors when co-transfected with/without γ-8 or/and ABHD6.