Kir4.1 channels were efficiently ablated from ON OLs in cKO-1 (n = 5) and cKO-2 (n = 4) mice versus control ONs (n = 5; A). One-way ANOVA with Tukey’s multiple comparisons test was performed in A; ****p≤0.0001. Kcnj10 was upregulated during OL differentiation, and expression significantly suppressed in purified and immunopanned OPCs (ctrl: n = 3, cKO-1: n = 3) and OLs (ctrl: n = 3, cKO-1: n = 3) from cKO-1 mice (B). Conversely, Kcnj16 was not downregulated in OPCs (ctrl: n = 3, cKO-1: n = 3) and OLs (ctrl: n = 3, cKO-1: n = 3) from cKO-1 mice in vitro, however, note Kcnj16 downregulation during OPC-OL maturation (C). Cacna1c mRNA levels were increased in cultured OPCs (ctrl: n = 3, cKO-1: n = 3) but not OLs (ctrl: n = 3, cKO-1: n = 3) from cKO-1 mice suggesting a partial activation of Cav1.2 channels in OPCs (D). One-way ANOVA with Tukey’s multiple comparison tests were performed in B–D; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. No difference in outer tongue Kir4.1 IEM labeling between controls and ON cKO-1 tissue (E). Trend towards decreased Kir4.1 IEM labeling in cKO-2 ON tissue as compared to controls with respect to myelin compartments but not AS fibers (F). Background IEM labeling without primary antibody confirmed specificity of Kir4.1 IEM antibody labeling of myelin compartments and astrocyte fibers (G). Mann-Whitney tests were performed in E–G; ****p≤0.0001, p=0.8 (E, outer tongue IEM), p=0.07 (F, inner tongue IEM), p=0.6 (F, compact myelin IEM), p=0.3 (F, outer tongue IEM), p=0.81 (F, AS fiber IEM), p=0.12 (G, compact myelin IEM), p=0.08 (G, outer tongue IEM), p=0.03 (G, AS fiber IEM). Data are presented as mean ±s.e.m in A–G.