Fig. 2. Kir4.1 channel-mediated mechanism underlying stimulus-evoked Ca²⁺ signaling in OLs.

a, OL Ca²⁺ levels increased by increasing [K⁺]_ext with 5, 10 and 30 mM K⁺ (30-s bath application: Δ[K⁺]_bath). Left, average OL Ca²⁺ traces (mean ± s.e.m.). Right, quantification of Δ[K⁺]_bath-evoked signal amplitudes (30 mM: n = 57 cells from four mice; 10 mM: n = 52 cells from five mice; 5 mM: n = 35 cells from four mice; 5 versus 10 mM, **P = 0.0048; 5 versus 30 mM, ***P < 0.0001; 10 versus 30 mM, ***P < 0.0001; one-way ANOVA with Tukey’s multiple-comparison test). b, Left, K⁺-evoked OL Ca²⁺ response independent of axonal spiking activity, showing comparable surges with TTX. Right, box plots showing the normalized response AUCs (n = 72 cells from five mice; P = 0.8144, two-sided paired t test; NS, not significant). c, Left, barium (Ba²⁺, 100 µM) reversibly inhibited the 50-Hz-induced OL Ca²⁺ surge by 84 ± 10%. Right, box plots showing the normalized response AUCs (n = 45 cells from four mice; ***P < 0.0001, two-sided paired t test). d, Left, Ba²⁺ reduced the K⁺-evoked OL Ca²⁺ response by 88 ± 9%. Right, box plots showing the normalized response AUCs (n = 47 cells from three mice; ***P < 0.0001, two-sided paired t test). e,f, Reverse-mode NCX blocker KB-R7943 (25 μM) reduced the 50-Hz-induced Ca²⁺ increase (e) by 44 ± 11% (n = 64 cells from five mice; paired t test, ***P = 0.0002) and the K⁺-evoked Ca²⁺ response (f) by 47 ± 8% (n = 52 cells from three mice; two-sided paired t test, ***P < 0.0001). Box plots on the right show the normalized response AUCs. g, Summary of drugs tested and their inhibitory effects on 50-Hz-evoked OL Ca²⁺ surges (data are also shown as box plots including the respective P values in c and e, Fig. 1f,g, and Extended Data Figs. 2, 4 and 5): TTX (n = 82), zero Ca²⁺ (n = 44), BaCl₂ (n = 45), KB-R7943 (n = 64), SEA0400 (n = 54), CdCl₂ (n = 54), NiCl₂ (n = 60), nifedipine (n = 39), benidipine (n = 56), RuR (n = 71), bumetanide (n = 77), PPADS (n = 46), suramin (n = 33), NBQX (n = 45) and +DAP-5/7-CKA (n = 33). AMPAR, AMPA receptor; NMDAR, NMDA receptor. h, Schematic of axonal activity-mediated OL Ca²⁺ activation: high-frequency axonal activity increases [K⁺]_ext, depolarizing (Depol.) OLs through Kir4.1 and enhancing Ca²⁺ entry through NCX. Minor contributions of VGCCs, P2XR and NMDA receptors are illustrated. Box plots in a–f show the median (center line), quartiles (box bounds), mean (+) and 5th–95th percentiles (whiskers).