Fig 2: Autophagy is required for Kir2 degradation.(A–C) There was no difference in Kir2.1 or Kir2.3 mRNA expression in SPNs of iSPNAtg7cKO 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 iSPNCtrl or iSPNAtg7cKO 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: iSPNctrl: n = 11, iSPNAtg7cKO: n = 11. DARPP32: iSPNctrl: n = 14, iSPNAtg7cKO: n = 11. Kir2.1: iSPNctrl: n = 7, iSPNAtg7cKO: n = 9. Kir2.3: iSPNctrl: n = 7, iSPNAtg7cKO: n = 9. Data analyzed by two-tailed, unpaired t test. (I-J) Kir2.1 is localized in LC3-GFP+ puncta in Atg5WT but not Atg5KO MEFs. BafilomycinA1 (BafA1; 100 nM 2 hr) treatment increases the number of LC3/Kir2.1-double labeled puncta in Atg5WT 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 Atg5KO 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 Atg5KO 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: F4,335 = 7.880, p<0.0001. (N) No significant difference in the internalization of antibody-labeled surface Kir2.1 channels in Atg5KO 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: F3,362 = 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—source data 1.Source data for Figure 5.

Fig 3: Hyperacetylation of Kir2.1 at K334 in the absence of autophagy inhibits channel activity.(A) Immunoprecipitation of Kir2.1 reveals elevated levels of acetylated lysines on Kir2.1 in Atg5KOMEFs without a change in ubiquitination. Representative blots shown from at least three independent replicates. (B) A conserved motif in the C-terminal tail of Kir2.1 and Kir2.3 contains three modifiable lysines and has previously been implicated in Kir2 channel degradation. (C) A degradation screen in which K334, K338 and K346 were mutated to the unmodifiable residue, arginine, reveals that K334 is required for Kir2.1 degradation in Atg5WT MEFs. N: (T=0min, T = 120 min) WT (52,79), K334R (27,51), K338R (40,41), K346R (23,11). Data analyzed by two-way ANOVA followed by Bonferroni post-hoc test. Kir genotype x time: F(3,390) = 3.211, p = 0.0230. (D-E) Kir2.1 K334R current is normalized in Atg5KO MEFs but does not affect Kir2.1 current in Atg5WT MEFs. Data analyzed by two-way ANOVA followed by Bonferroni post-hoc test. Cell genotype x Kir genotype: F(1,42) = 9.603, p = 0.0035. (F-G) Kir2.1 K334Q, with an acetylation-mimic at K334, has reduced current in Atg5WT MEFs. Voltage step protocol is the same as in (D). Data analyzed by two-tailed unpaired t test. t24=2.707, p=0.0123. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, † p<0.0001. Experiments in C, E and G were combined from at least three independent experiments.Figure 8—source data 1.Source Data for Figure 8.

Fig 4: Autophagy is not required for Kir2 trafficking.(A) Sample traces from MEFs transfected with Kir2.1 demonstrate an inwardly rectifying, barium sensitive current that is absent in untransfected cells. (B) Atg5KO MEFs have reduced Kir2.1 current compared to Atg5WT MEFs. WT: N = 8, KO: N = 12. Voltage step x genotype: F(14, 252)=8.985, p<0.0001. (C) Representative micrographs of Atg5WT and Atg5KO MEFs. On right, the Kir2.1 channel in the WT image has been contrasted to compare staining pattern. Scale bar 20 µm. (D-F) Elevated levels of both surface and total levels of Kir2.1 in Atg5KO MEFs. Scale bar 20 µm, inset scale bar 1 µm. (E) t147=4.511, p<0.0001. (F) t147=4.511, p<0.0001. (G-J) Subcellular fractionation reveals elevated levels of Kir2.1 and Kir2.3 in all fractions of iSPNAtg7cKO mice compared to controls but no change in the relative distribution of Kir2.1 or Kir2.3 between genotypes. (H) p62 is elevated in the total homogenate of iSPNAtg7KO mice. (I) No change in distribution or level of Kv1.2 between genotypes. iSPNCtrl: N = 5. iSPNAtg7cKO: N = 3. TH, total homogenate; PNS, post-nuclear supernatant; S2, 20,000xg supernatant; P2, 20,000xg pellet; P3, resuspended P2 spun at 100,000xg; SPM, synaptic plasma membranes isolated from 1.0M and 1.2M sucrose interface. Data were analyzed by two-way ANOVA for each analyzed protein with fraction and genotype as factors. No significant interaction between fraction and genotype was found for any protein. Fraction was significant for each protein. Genotype was only significant for Kir2.1 and Kir2.3. Kir2.1: Genotype: F(1,6)=9.373, p = 0.0222. Kir2.3: Genotype: F(1,6)=6.615, p = 0.0422. See Table 1 for detailed statistics. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, † p<0.0001. Experiments in B, E, and F were combined from at least three independent experiments.Figure 7—source data 1.Source Data for Figure 7.