Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: (A) Confocal images of HEK293T cells expressing GFP-LRRC8B (WT) or Y380S Mutant, co-stained with ER-Tracker, Mito-Tracker, and Hoechst. Merged images show ER and mitochondrial localization. Scale bar: 10 µm. (B) Whole-cell patch clamp analysis of current density–voltage relationships under isotonic and hypotonic conditions. No significant differences at +100 mV (mean ± SEM, N = 4; one-way ANOVA, ns). (C) Cell viability (MTT) in GFP(Ctrl), LRRC8B WT, LRRC8BY380S (Mut), siControl (Scr), and siLRRC8B (KD) cells (mean ± SEM, N = 3; one-way ANOVA, ****p < 0.0001). (D) LRRC8B knockdown validated by Western blot and densitometry normalized to actin (mean ± SEM, N = 3; unpaired t-test, **p < 0.01).

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Expressing, Mutagenesis, Staining, Patch Clamp, Knockdown, Western Blot

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: (A) Representative traces of ER luminal Ca²⁺ decay after SERCA inhibition with 1 µM thapsigargin in nominally Ca²⁺-free solution, measured using R-CEPIA1er (normalized fluorescence). GFP(Ctrl) and scrambled siRNA (Scr) cells served as controls. (B) Quantification of t₁/₂ (time to 50% decay). LRRC8B Y380S (mut) cells did not reach t₁/₂ within 240 s (∼44% decay) and were excluded. Data are mean ± SEM (n = 15–30 cells, 3 independent experiments (N)); one-way ANOVA with Tukey’s test (**p < 0.01, ****p < 0.0001). (C–D) Representative CytoRCaMP traces after histamine stimulation in GFP (Ctrl), LRRC8B WT, LRRC8B Y380S (Mut) (C), and siControl (Scr) vs. siLRRC8B (KD) cells (D). Mutant cells show enhanced cytosolic Ca²⁺ peaks. (E) Peak cytosolic Ca²⁺ responses (fold change in CytoRCaMP). Mean ± SEM (n = 40–70 cells, N=4); one-way ANOVA with Tukey’s test (**p < 0.01, ***p < 0.001). (F) Basal cytosolic Ca²⁺ levels measured by fura-2 (340/380 ratio). Mean ± SEM (n = 30–80 cells, N=3); one-way ANOVA with Tukey’s test (**p < 0.01, ***p < 0.001).

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Inhibition, Fluorescence, Mutagenesis

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: (A) Representative traces of mitochondrial Ca²⁺ uptake (R-CEPIA3mt) following histamine stimulation in cells expressing GFP (Ctrl), LRRC8B WT, or LRRC8B Y380S (Mut), shown as normalized fluorescence over time. Bar graph depicts peak responses. Data are mean ± SEM (n = 12–30 cells per condition; N = 3). One-way ANOVA with Tukey’s test; **p < 0.01, ***p < 0.001. (B) Representative traces and quantification of mitochondrial Ca²⁺ uptake after histamine stimulation in cells treated with siControl (Scr) or siLRRC8B (KD). Data are mean ± SEM (n = 12–30 cells per condition; N = 3). Unpaired two-tailed Student’s t-test; **p < 0.01. (C) Representative traces and quantification of mitochondrial Ca²⁺ uptake following 5 µM ionomycin stimulation in cells expressing GFP (Ctrl), LRRC8B WT, or LRRC8B Y380S (Mut). Data are mean ± SEM (n = 30–50 cells per condition; N = 3). One-way ANOVA; *p < 0.05, **p < 0.01. (D) Representative traces and quantification of mitochondrial Ca²⁺ uptake after ionomycin stimulation in siControl (Scr) or siLRRC8B siRNA (KD)-treated cells. Data are mean ± SEM (n = 30–50 cells per condition; N = 3). Unpaired two-tailed Student’s t-test; **p < 0.01.

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Expressing, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: (A–B) Representative TMRE traces showing mitochondrial membrane potential (ΔΨ m ) after FCCP treatment in cells expressing (A) GFP (ctrl), LRRC8B wildtype (WT), or LRRC8B Y380S (Mut), and (B) siControl (Scr) or siLRRC8B (KD). (C) Percentage decrease in TMRE fluorescence following FCCP (mean ± SEM; n = 25–50 cells/condition, N = 3). (D) Representative MitoSOX images indicating mitochondrial superoxide levels in GFP (ctrl), LRRC8B WT, LRRC8B Y380S (Mut), siControl (Scr) and siLRRC8B (KD) cells. Scale bar, 20 μm. (E) Quantification of MitoSOX fluorescence (a.u.) (mean ± SEM; n = 30–100 cells/condition, N = 3). (F) RT–qPCR analysis of antioxidant genes (Catalase, SOD2, GPX1). LRRC8B WT significantly increased transcript levels compared to controls (mean ± SEM; N = 3). Statistics: One-way ANOVA with Tukey’s post hoc test; **p < 0.01, ****p < 0.0001; ns, not significant.

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Membrane, Expressing, Fluorescence, Quantitative RT-PCR

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: (A) OCR traces following sequential injections of oligomycin, FCCP, and rotenone/antimycin A in control(ctrl), LRRC8B wildtype (WT), and LRRC8B Y380S (Mut) expressing cells. Quantification shows that LRRC8B Mutant reduces basal respiration, ATP production, and maximal respiration, while increasing non-mitochondrial oxygen consumption. (B) OCR traces in siControl(scr) and siLRRC8B (KD) cells with corresponding quantification. Knockdown does not affect basal respiration or ATP production, but decreases maximal respiration and increases non-mitochondrial oxygen consumption. Data are mean ± SEM (N = 3). ns, not significant; *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Control, Expressing, Mutagenesis, Knockdown

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: Diagram illustrating the membrane topology of LRRC8B, comprising 803 amino acids and four transmembrane domains (TM1: residues 26–46; TM2: 120–140; TM3: 262–282; TM4: 308–328). Both N- and C-termini are oriented toward the cytosol. The Y380S mutation (indicated by a red star) is located in the cytosolic region, proximal to the leucine-rich repeat domain (LRRD; residues 463–803), which contains 13 leucine-rich repeats. (Created in https://BioRender.com )

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Membrane, Mutagenesis

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: Multiple sequence alignment showing conservation of the tyrosine residue (Y380) in LRRC8B across human LRRC8 paralogs (LRRC8A, LRRC8C, LRRC8D, and LRRC8E) and among different species, including human, mouse, rat, and bovine. The conserved tyrosine residue is highlighted in red. Asterisks indicate fully conserved residues across sequences.

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Sequencing, Residue

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: Volcano plot showing LC–MS/MS–based proteomic analysis of GFP pull-down samples from HEK293T cells expressing GFP-tagged wild-type (WT) or mutant LRRC8B. The x-axis represents log₂ fold change (mutant vs WT), and the y-axis shows −log₁₀ adjusted p-value. Proteins enriched in WT samples are shown on the left, while those enriched in mutant samples are shown on the right. Selected significantly enriched proteins are labeled. VDAC1 is identified as a WT-enriched interactor.

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Expressing, Mutagenesis, Labeling

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: (A–B) Immunoblot analysis of VDAC in whole-cell lysates (A) and mitochondrial fractions (B) from cells expressing LRRC8B wildtype (WT) or LRRC8B Y380S (Mut). (C) Densitometric quantification (normalized to actin) shows no difference in VDAC levels between conditions (mean ± SEM, N = 3; unpaired two-tailed t-test, ns). (D) Input blots confirm comparable expression of LRRC8B WT and LRRC8B Mutant. (E–F) Co-immunoprecipitation of LRRC8B-GFP (WT or mutant) followed by VDAC immunoblotting shows reduced VDAC association with LRRC8B Mutant. Quantification normalized to immunoprecipitated LRRC8B-GFP confirms this decrease (mean ± SEM, N = 3–4; unpaired two-tailed t-test, ***p < 0.001).

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Western Blot, Expressing, Two Tailed Test, Mutagenesis, Immunoprecipitation

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: Representative immunoblot images showing VDAC protein levels in total cell lysates and isolated mitochondrial fractions from cells transfected with GFP-tagged LRRC8B WT (W) or mutant Y380S (M) constructs. Actin (∼42 kDa) is included as a loading control for total lysates. Red boxes indicate the regions that were cropped and presented in the main figures. Molecular weight markers are shown where applicable.

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Western Blot, Isolation, Transfection, Mutagenesis, Construct, Control, Molecular Weight

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: (A) Representative immunoblot images showing protein levels of GFP-tagged LRRC8B (WT and mutant) and VDAC in input lysates used for pull-down assays. (B–C) Immunoblot analysis of four independent immunoprecipitation (IP) experiments. Bands at ∼120 kDa confirm successful pull-down of GFP-tagged LRRC8B (WT and mutant) using an anti-GFP antibody. VDAC (∼32 kDa) bands indicate co-precipitation of VDAC with LRRC8B WT and mutant proteins. VDAC levels were normalized to the corresponding LRRC8B (WT or mutant) levels for quantification. Red boxes highlight the regions that were cropped and presented in the main figures. Molecular weight markers are shown where applicable.

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Western Blot, Mutagenesis, Immunoprecipitation, Molecular Weight

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: Volcano plot showing LC–MS/MS–based proteomic analysis of GFP pull-down samples from HEK293T cells expressing GFP-tagged wild-type (WT) or mutant LRRC8B. The x-axis represents log₂ fold change (mutant vs WT), and the y-axis shows −log₁₀ adjusted p-value. Proteins enriched in WT samples are shown on the left, while those enriched in mutant samples are shown on the right. Selected significantly enriched proteins are labeled. VDAC1 is identified as a WT-enriched interactor.

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Expressing, Mutagenesis, Labeling

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: Representative immunoblot images showing VDAC protein levels in total cell lysates and isolated mitochondrial fractions from cells transfected with GFP-tagged LRRC8B WT (W) or mutant Y380S (M) constructs. Actin (∼42 kDa) is included as a loading control for total lysates. Red boxes indicate the regions that were cropped and presented in the main figures. Molecular weight markers are shown where applicable.

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Western Blot, Isolation, Transfection, Mutagenesis, Construct, Control, Molecular Weight

Journal: bioRxiv

Article Title: A neuropsychiatric disease-associated mutation in LRRC8B disrupts cellular Ca²⁺ signaling, mitochondrial function, and bioenergetics

doi: 10.64898/2026.04.16.718892

Figure Lengend Snippet: (A) Representative immunoblot images showing protein levels of GFP-tagged LRRC8B (WT and mutant) and VDAC in input lysates used for pull-down assays. (B–C) Immunoblot analysis of four independent immunoprecipitation (IP) experiments. Bands at ∼120 kDa confirm successful pull-down of GFP-tagged LRRC8B (WT and mutant) using an anti-GFP antibody. VDAC (∼32 kDa) bands indicate co-precipitation of VDAC with LRRC8B WT and mutant proteins. VDAC levels were normalized to the corresponding LRRC8B (WT or mutant) levels for quantification. Red boxes highlight the regions that were cropped and presented in the main figures. Molecular weight markers are shown where applicable.

Article Snippet: The plasmid encoding GFP-tagged human LRRC8B (HG24935-ACG; Sino Biological Inc, USA).

Techniques: Western Blot, Mutagenesis, Immunoprecipitation, Molecular Weight