Fig 2: AS Kir4.1 Is Required for FaMN Function, Fast-Twitch Muscle Fiber Size, and Peak Force(A) Breeding scheme and electrophysiology recording schematic.(B) Two representative current steps at 3× rheobase (RB).(C–E) Rheobase (C), input resistance (D), and steady-state (SS) firing frequency (at 3× rheobase) (E) demonstrate altered intrinsic electrophysiological deficits in MNs from AS-Kir4.1cKO-ChAT-GFP animals at P12–P15 (n = 12 control MNs, n = 14 AS-Kir4.1cKO MNs from at least 3 animals per group, boxplot, Mann-Whitney test).(F) Input resistance versus rheobase scatterplot shows shift in electrophysiological properties from fast-like to slow-like in AS-Kir4.1cKO compared to control MNs.(G) Cross sections of TA muscle fibers immunolabeled for marker of fast-twitch muscle (myosin type 2) and laminin in P30 AS-Kir4.1cKO and cre-negative control animals (scale bar, 50 µm).(H) Quantification of TA muscle fiber cross-sectional area (n = 3 mice/group, >100 muscle fibers/animal, boxplot, Mann-Whitney test).(I–K) Abnormal muscle strength behavior in AS-Kir4.1cKO mice. Adult AS-Kir4.1cKO animals generate less peak force (>P50, n = 14–15 mice/group, boxplot, Welch’s t test) (I). AS-Kir4.1cKO animals have slower front and hindlimb movements as assessed by gait analysis with the catwalk behavioral test. Swing speed corresponds to the limb speed while in the air (same animals as in I, boxplot, Welch’s t test) (J). AS-Kir4.1cKO animals display a shorter latency to fall on the accelerating rotarod at P30–P35 (n = 7 mice/group, mean ± SEM, two-way ANOVA, Bonferroni post hoc test) (K). Mice performed three trials (T, T2, and T3) per day on 3 consecutive days (D1, D2, and D3). *p < 0.05, **p < 0.01. Edges of boxplots denote interquartile range (25th–75th percentile) with whiskers denoting 1.5 times the interquartile range and black line denoting the median value.

Fig 3: Kir4.1 Upregulation in Ventral Horn AS around Large FaMN MNs(A and B) Kir4.1 mRNA is enriched in spinal cord compared to other CNS regions in humans (GTEx-Consortium, 2015) (A) and mice (Kasukawa et al., 2011) (B). Data are represented as mean ± SEM.(C and D) Perineuronal enrichment of Kir4.1 expression in human and mouse ventral spinal cord.(C) KIR4.1 is expressed around SMI32+ neurons in adult human ventral spinal cord (scale bars, 1 mm, left; 50 µm, right). Representative image of n = 3 control human spinal cord. Patient data are provided in Table S1.(D) Kir4.1 is expressed around ChAT+ MNs in mouse ventral lumbar spinal cord at P16 (scale bars, 200 µm, left; 50 µm, right). Dotted line denotes gray/white matter boundary. WM, white matter. Arrowheads denote Kir4.1 enrichment around ventral horn neurons. High-resolution images are single-plane confocal images.(E) Increased ventral (V) compared to dorsal (D) Kir4.1 protein by western blot from mouse lumbar spinal cord at P30 (n = 4 mice, mean ± SEM, Welch’s t test).(F) Left: fold change of Kir4.1 mRNA levels between ventral and dorsal samples from cultured (n = 6 mice, mean ± SEM, one-sample t test) neonatal mouse spinal cord AS. Right: FACS-purified AS from P5 Aldh1l1-GFP+ (n = 3 mice, mean ± SEM, one-sample t test) mouse spinal cord.(G) Kir4.1 mRNA levels in P5 Aldh1l1-GFP+ FACS-purified AS compared to Aldh1l1-GFP- non-AS cells from mouse ventral spinal cord (n = 3 mice, mean ± SEM, one-sample t test).(H) Kir4.1 protein is preferentially found around larger MMP-9+ FaMNs (white arrowheads) compared to smaller MMP-9- SaMNs (yellow arrowheads) at P30 (scale bar, 50 µm).(I) Quantification of Kir4.1 signal intensity around individual MMP-9+ or MMP-9- MN (n = 4 mice, >100 MN counts/animal, boxplot, Mann-Whitney test).(J) Kir4.1 loss in AS from VGLUT1 KO animals at P26. VGLUT1 KO mice were crossed with EAAT2-td-Tomato reporter for AS visualization.(K) Quantification of Kir4.1 immunofluorescence intensity per AS (EAAT2-td-tomato+) (n = 2 mice, >50 AS counts/animal, boxplot, Mann-Whitney test; scale bar, 40 µm, insert: 20 µm). *p < 0.05, **p < 0.01, ***p < 0.001. Edges of boxplots denote interquartile range (25th–75th percentile) with whiskers denoting 1.5 times the interquartile range and the black line denoting the median value.

Fig 4: AS Kir4.1 Regulates MN Size through PI3K/mTOR/pS6 Pathway(A and C) Immunofluorescence co-staining of mTOR downstream effector pS6 and MMP-9+ MNs in P30 AS-Kir4.1cKO LOF mice (A) and in P60 AAV-Kir4.1 GOF mice (C).(B and D) Quantification of pS6 fluorescence intensity per MMP-9+ MN (B, LOF: n = 3 mice/group, 80 MNs counts/animal; D, GOF: n = 5 mice/group, 200 MN counts/animal, boxplot, Mann-Whitney test, scale bar, 25 µm).(E) Rapamycin treatment in AAV-Kir4.1 GOF mice.(F) Immunofluorescent co-staining of ChAT (left) and MMP-9 (right) in vehicle-treated AAV-td-Tomato and AAV-Kir4.1-eGFP mice (top two panels) or rapamycin-treated AAV-Kir4.1-eGFP mice (lower panel).(G and H) Quantification of ChAT+ (G) and MMP-9+ (H) MN size in vehicle-treated AAV-td-Tomato and AAV-Kir4.1-eGFP and rapamycin-treated AAV-Kir4.1-eGFP mice (n = 3–4 mice/group, 100 MN counts/animal, boxplot, Kruskal-Wallis test, scale bar, 30 µm).(I) Incubation of acute spinal cord slices in solutions with high K+ concentration.(J) Detection of ChAT-GFP+ MNs in the ventral horn of P14 mice after 2 hr incubation in 3 mM KCl, 12 mM KCl, or 5 mM mannitol ACSF.(K) Quantification of MN soma area in the corresponding conditions (n = 4 mice/group, average 250 MN counts/animal, mean ± SEM, Kruskal-Wallis test, scale bar, 25 µm).

Fig 5: Immunohistochemistry of the mouse cochlea. A, Bdp1 protein expression in the cochlea at postnatal day 5. Bdp1 shows expression in the stria vascularis (red arrow), Reissner's membrane (red arrowhead), basilar membrane (black arrowhead), and spiral ligament (black arrow). B–D, Bdp1, Kcnj10 and laminin expression in the stria vascularis. Our immunohistochemistry detected Bdp1 protein in the proximity of blood vessels (example of the same blood vessel in adjacent sections is indicated with an arrow in B, C, D). We used Kcnj10 as a marker of intermediate cells (C) and laminin as a marker of the basal lamina surrounding blood vessels (D) in the stria vascularis in the sections shown in A. The expression pattern suggests that Bdp1 is expressed in endothelial cells of the stria vascularis. Scale bars: A: 50 µm; B–D: 10 µm. sv: stria vascularis.