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), 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, deact 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) 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 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), 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 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), 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 receptors-TARP γ-2 complexes in HEK 293T cells, 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 (black), GluA1i/GluA2(R)i-G + ABHD6 (orange), GluA1i/GluA2(R)i-G + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G + 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 (black), GluA1i/GluA2(R)i-G + ABHD6 (orange), GluA1i/GluA2(R)i-G + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G + TARP γ-2 + ABHD6 (red).

(C) Glutamate (Glu, 10 mM) induced currents in an outside-out patch excised from an HEK 293T cell transfected with GluA1i/GluA2(R)i-G (black), GluA1i/GluA2(R)i-G + ABHD6 (orange), GluA1i/GluA2(R)i-G + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G + 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 (C1), and the summary bar graphs of the τ w, rec (C2). 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)

Schematic illustration of the AMPAR subunit and the Sequence alignment of RNA splice variants and editing of AMPAR.

(A) Topology of a single AMPAR subunit in the plasma membrane. Each subunit consists of an extracellular N-terminal domain (NTD), a ligand-binding domains (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.

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 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 (black), GluA1i/GluA2(R)i-G + ABHD6 (orange), GluA1i/GluA2(R)i-G + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G + 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 (black), GluA1i/GluA2(R)i-G + ABHD6 (orange), GluA1i/GluA2(R)i-G + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G + 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 (black), GluA1i/GluA2(R)i-G + ABHD6 (orange), GluA1i/GluA2(R)i-G + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G + 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.

Pearson’s correlation between natural logarithm peak amplitude (pA) and the τ w, deact and τ w, des coexpression with various GluA subunits.

(A-J) 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.

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.