Figure 5. Autophagy is required for Kir2 degradation.

(A–C) There was no difference in Kir2.1 or Kir2.3 mRNA expression in SPNs of iSPN^Atg7cKO mice in an RNAscope assay (N = 77–173 cells from three mice per group). Inset shows just RNAscope and DAPI. Scale bar 30 µm. Data analyzed by the Kolmogorov-Smirnov test, Genotype effect: p>0.05 for Kir2.1 and Kir2.3. (D) Representative western blots of specified proteins from total striatal lysates from iSPN^Ctrl or iSPN^Atg7cKO mice. Quantifications of (E) p62 (t₂₀ = 3.551, p = 0.0020), (F) DARPP32 (t₂₃ = 1.984, p = 0.0593), (G) Kir2.1 (t₁₄ = 3.435, p = 0.0040), and (H) Kir2.3 (t₁₄ = 4.492, p = 0.0005) relative to actin. p62: iSPN^ctrl: n = 11, iSPN^Atg7cKO: n = 11. DARPP32: iSPN^ctrl: n = 14, iSPN^Atg7cKO: n = 11. Kir2.1: iSPN^ctrl: n = 7, iSPN^Atg7cKO: n = 9. Kir2.3: iSPN^ctrl: n = 7, iSPN^Atg7cKO: n = 9. Data analyzed by two-tailed, unpaired t test. (I-J) Kir2.1 is localized in LC3-GFP+ puncta in Atg5^WT but not Atg5^KO MEFs. BafilomycinA1 (BafA1; 100 nM 2 hr) treatment increases the number of LC3/Kir2.1-double labeled puncta in Atg5^WT MEFs. Scale bar 20 µm. Inset scale bar 1 µm. Analyzed by one-way ANOVA followed by Bonferroni post-hoc test. F_(2,26)=25.64, p<0.0001. (K-L) A reduction of Lamp1⁺Kir2.1⁺ puncta in Atg5^KO MEFs. Scale bar 20 µm, inset scale bar 1 µm. Data analyzed by two-tailed, unpaired t test. t₂₃ = 3.083, p = 0.0053. (M) Reduced degradation of SNAP-tag labeled Kir2.1 in Atg5^KO MEFs. N: (WT,KO): T = 0 min (35,30), T = 30 min (30,27), T = 60 min (25,38), T = 180 min (33,67), T = 360 min (40,20). Data analyzed by two-way ANOVA. Genotype x time: F_4,335 = 7.880, p<0.0001. (N) No significant difference in the internalization of antibody-labeled surface Kir2.1 channels in Atg5^KO MEFs. N: (WT,KO): T = 0 min (76,56), T = 30 min (52,37), T = 60 min (41,42), T = 120 min (28,38). Data analyzed by two-way ANOVA. Genotype x time: F_3,362 = 0.8038, ns; Time: F_(3,362) = 25.88, p = 0.0001; Genotype: F_(1,362) = 1.877, ns. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, † p<0.0001.

Figure 5—figure supplement 1. Loss of autophagy does not affect Kir2.1 expression in dSPNAtg7cKO mice.

(A) Representative western blots from control or dSPN^Atg7cKO mice. (B-G) Quantification of relative protein levels in control and dSPN^Atg7cKO mice shows no significant difference between (B) Kir2.1 [dSPN^Ctrl: n = 10, dSPN^Atg7cKO: n = 11; t₁₉ = 0.5816, p = 0.5677], (C) Kir2.3 [dSPN^Ctrl: n = 12, dSPN^Atg7cKO: n = 10; t₂₀ = 0.08775, p = 0.9309], (D) Kv1.2 [dSPN^Ctrl: n = 10, dSPN^Atg7cKO: n = 11; t₁₉ = 0.04627, p = 0.9636], (E) PSD95 [dSPN^Ctrl: n = 11, dSPN^Atg7cKO: n = 12; t₂₁ = 0.3730, p = 0.7129], or (G) DARPP32 [dSPN^Ctrl: n = 6, dSPN^Atg7cKO: n=5; t₉ = 0.6178, p = 0.5520], but a significant increase in (F) p62 [dSPN^Ctrl: n = 11, dSPN^Atg7cKO: n = 11; t₂₀=6.936, p<0.0001]. (H) Representative western blots for Kv1.2 and PSD95 in control or iSPN^Atg7cKO mice. (I-J) Quantification of Kv1.2 [iSPN^Ctrl: n = 11, iSPN^Atg7cKO: n = 11; t₂₀ = 0.3213, p = 0.7513] and PSD95 [iSPN^Ctrl: n = 12, iSPN^Atg7cKO: n = 11; t₂₁ = 1.264, p = 0.2202] shows no difference between control and iSPNAtg7cKO mice. Data analyzed by two-tailed unpaired t test. ns p>0.05, *** p<0.0001.

Figure 5—figure supplement 2. Higher steady-state levels of Kir2.1 and reduced degradation of Kir2.1 in Atg5^KO MEFs.

(A) Schematic of Kir2.1-externalHA-Flag-SNAP (abb. Kir2.1 below) construct. (B) Representative western blots and (C) quantification for Flag, Kir2.1, LC3B and actin show elevated steady state levels of Kir2.1 in transiently transfected in Atg5^KO MEFs compared to Atg5^WT MEFs. N = 3 per genotype. Data analyzed with two-tailed unpaired t test, t₄ = 3.390, p = 0.0275. (D and E) Representative blots and aggregate data from six independent replicates per timepoint treated with cycloheximide at t = 0 min. Data were analyzed with a two-way ANOVA followed by Bonferroni post-hoc test. Time x genotype: F_(4,44)=2.764, p = 0.0391. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, † p<0.0001.

Figure 5—figure supplement 3. Kir2.1 degradation is disrupted in Atg7^KO MEFs.

(A) Representative western blot showing the absence of Atg7 in Atg7^KO primary MEFs. (B) Representative images of primary MEFs transfected with Kir2.1-Flag-SNAP and labeled with SNAPcell ligand. (C) Snap-labeled Kir2.1 is significantly reduced in Atg7^WT cells but not Atg7^KO cells. N: (0,120 min) WT: (7,6), KO: (7,6). Data analyzed by two-way ANOVA. Genotype x time: F_(1,22)=2.323, p = 0.1417; Genotype: F_(1,22)=2.323, p = 0.1417. Time: F_(1,22)=7.675, p = 0.0112. (D) The distribution of Kir2.1 is not different between Atg7^WT and Atg7^KO cells. Data are combined from two independent replicates. *p<0.05.