Fig 2: Co-IP Western blots showing degree of association of Cav3 and chloride channels ClC-2, ClC-3, and SWELL1 in rat pulmonary vein (PV; n=2). IB indicates immunoblot; IP, immunoprecipitation. Cav3 indicates caveolin 3; ClC, chloride channel; SWELL1, also known as LRRC8A (leucine rich repeat containing protein 8A).

Fig 3: Suppressing neonatal epileptiform activity through inhibiting CLC-3 channels. a Field potential recording showing strong inhibition of epileptiform activity (0 Mg2+) by NaGluc (20 mM) in the CA3 pyramidal layer of hippocampal slices from neonatal mice (P12). b Power spectra of epileptiform activity (5-min time windows) before (red), during (green), and after (black) NaGluc application. The amplitude of power (integrative area under the power spectrum trace) was significantly reduced after NaGluc application. c, d In P26 hippocampal slices, however, NaGluc only showed modest inhibitory effect on the epileptiform activity. e Normalized power showing the time course of NaGluc inhibition on the epileptiform activity induced by 0 Mg2+ aCSF. NaGluc dramatically reduced the power of epileptiform activity in the P6–8 and P10–12 neonatal slices (P < 0.001, paired Student’s t-test). As a control, the gray line represents the effect of adding 20 mM NaCl on neonatal (P8–12) epileptiform activity, there was no significant change (P > 0.1, paired Student’s t-test). f Dose-dependent inhibition of NaGluc on the power (left) and paroxysmal discharges (right) in the neonatal brain slices. The percentage change of power and paroxysmal discharges were normalized to the 20 min of stable recordings before NaCl or NaGluc application. Control (Ctrl) represents the effect of 20 mM NaCl. Compared to control, the power and paroxysmal discharges were significantly reduced in the presence of 10 mM or 20 mM NaGluc. Data are shown as mean ± s.e.m. *** P < 0.001

Fig 4: CLC-3 Cl- channels regulate EGABA during epileptic stimulation in neonatal neurons. a Gramicidin-perforated recordings revealed a depolarizing shift in the GABAA-R reversal potential (EGABA) in CA3 neurons after induction of epileptiform activity in 0 Mg2+ aCSF. b Bath application of NKCC1 inhibitor bumetanide induced a hyperpolarizing shift in EGABA in normal aCSF, but did not abolish the depolarizing shift of EGABA induced by 0 Mg2+ aCSF. c KCC2 inhibitor VU0240551 had no effect on the EGABA under normal aCSF, and showed no effect on the depolarizing shift induced by 0 Mg2+ aCSF. d Gluconate strongly inhibited the depolarizing EGABA shift induced by 0 Mg2+ aCSF. Gluconate itself did not affect the EGABA in normal aCSF. e In CLC-3 KO mice, EGABA did not change when treated with 0 Mg2+ aCSF. The dash line in panels b-e shows the control I-V plot in normal aCSF in A (black line). (F) Quantified data showing the EGABA changes under various conditions in neonatal CA3 pyramidal neurons (P8–9). g Bar graphs showing EGABA changes in adult CA3 pyramidal neurons. Note that NaGluc (20 mM) could not abolish the EGABA shift induced by 0 Mg2+ aCSF in adult animals. Data are shown as mean ± s.e.m. ** P < 0.01, *** P < 0.001

Fig 5: The expression of ClC-3 was increased in the ipsilateral hippocampus of chronic TLE mice.(a) Representative double immunofluorescence images showing the neuronal co-expression of ClC-3 and NeuN in the hippocampal CA1 region of control and chronic TLE mice. Scale bar, 20 µm. Images were selected from three mice for each group. (b) The ClC-3 protein in the homogenates of hippocampus from control and TLE mice was detected at 72-95 kDa by Western blot. (c) Densitometric analysis results showing the increased expression of ClC-3 in the hippocampus of the chronic TLE mice. (**P < 0.05, unpaired two-tailed t-test, n = 6 mice for each group). (d-f) Spearman's rank correlation between the expression of ClC-3 in the hippocampus and the average absolute power of ictal HFOs (d, rho = 0.9429, *P < 0.05), the number of seizures per day (e, rho = 0.7409, P > 0.05) and average seizure duration (f, rho = 0.4857, P > 0.05). n = 6 mice for each analysis.