Figure 8. 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 Atg5^KOMEFs 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 Atg5^WT 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 Atg5^KO MEFs but does not affect Kir2.1 current in Atg5^WT 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 Atg5^WT 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—figure supplement 1. Reduced acetylation of Kir2.1 K334R in Atg5^KO MEFs but acetylation status at K334 does not affect surface residence of Kir2.1.

(A) Representative western blots of immunoprecipitated WT Kir2.1^{extHA-Flag-SNAP} or Kir2.1^{extHA-Flag-SNAP} K334R. Immunoblots for acetylated lysines (AceK) or Flag. (B) Quantification of acetylated lysine signal normalized to WT or K334R Kir2.1^{extHA-Flag-SNAP} in immunoprecipiates from Atg5^KO MEFs shows a reduction in total Kir2.1 acetylation when K334 is mutated to arginine. Data analyzed with two-tailed, unpaired t test. t₁₀ = 2.452; p = 0.0341. (C) Surface staining for WT Kir2.1, Kir2.1 K334R, and Kir2.1 K334Q in Atg5^WT or Atg5^KO MEFs reveals that mutations at K334 does not affect surface localization of Kir2.1 in either MEF cell line. Data analyzed with two-way ANOVA. Cell genotype x Channel genotype: F_(2,142)=0.04505, p = 0.9560; Cell genotype: F_(1,142)=75.66, p<0.0001; Channel Genotype: F_(2,142)=0.6539, p = 0.5216. *p<0.05, † p<0.0001.