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14-3-3ζ Mediates GABAAR Activation by Interacting with BIG1

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Abstract

Most fast synaptic inhibitions in the mammalian brain are mediated by GABAA receptors (GABAARs). An appropriate level of GABAAR expression at the cell surface is essential for neurodevelopment and the efficacy of GABAergic synaptic transmission. We previously reported that brefeldin A–inhibited GDP/GTP exchange factor 1 (BIG1), a binding partner of GABAARs, plays an important role in trafficking GABAARs to the cell surface. However, its regulatory mechanisms remain unknown. In the present study, we identified a new cellular protein, 14-3-3ζ, which can interact with the β subunit of GABAARs and BIG1 both in vitro and in vivo and colocalizes in the soma, dendrites, and axons of hippocampal neurons. Overexpression of 14-3-3ζ-WT increased the surface expression of BIG1 in dendrites and axons, as well as the binding of BIG1 with GABAAR. Depleted 14-3-3ζ with efficacious siRNA attenuated the interaction between BIG1 and GABAARs and resulted in significant decreases in the surface expression levels of BIG1 and GABAAR. GABAAR agonist treatment increased the expression levels of BIG1 and 14-3-3ζ on the surface, indicating that 14-3-3ζ is involved in regulating BIG1-mediated GABAAR surface expression. Depletion of BIG1 or 14-3-3ζ significantly decreased GABAAR expression at the cell surface and suppressed the GABA-gated influx of chloride ions. These data indicate that the combination of 14-3-3ζ and BIG1 is required for GABAAR membrane expression. Our results provide a potential promising therapeutic target for neurological disorders involving GABAergic synaptic transmission.

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Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgments

This work was supported by the "technology innovation 2030-major projects" on brain science and brain-like computing of the Ministry of Science and Technology of China (2021ZD0202603), the Guangdong Basic and Applied Basic Research Foundation (2022A1515012036), and the National Natural Science Foundation of China (31500841).

Funding

This work was supported by the “technology innovation 2030-major projects” on brain science and brain-like computing of the Ministry of Science and Technology of China (No. 2021ZD0202603), and the National Natural Science Foundation of China (grant number 31500841).

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Authors and Affiliations

  1. Experiment Teaching & Administration Center, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China

    Cuixian Li, Shen Huang & Tianguo Hong

  2. Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510515, China

    Jin Peng

  3. Laboratory of Immunopharmacology, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, China

    Chun Zhou

  4. SMU-KI United Medical Inflammatory Center, School of Pharmaceutical Sciences; Guangdong Provincial Key Laboratory of Shock and Microcirculation, Southern Medical University, Guangzhou, 510515, China

    Chun Zhou

  5. Key Laboratory of Mental Health of the Ministry of Education, Southern Medical University, Guangzhou, 510515, China

    Jie Tang

  6. Department of Physiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China

    Jie Tang

  7. Department of Otolaryngology Head & Neck Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China

    Jie Tang

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  1. Cuixian Li
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  2. Shen Huang
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  3. Jin Peng
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Contributions

Cuixian Li, Chun Zhou, and Jie Tang contributed to the study conception and design. Material preparation, data collection, and analysis were performed by all authors. The first draft of the manuscript was written by Cuixian Li and Jie Tang. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Chun Zhou or Jie Tang.

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This study was approved by the Institutional Animal Care and Use Committee (IACUC) at Southern Medical University. Principles established by the Committee of Southern Medical University on Animal Care were followed at all times.

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No human subject was involved in this study. Procedures involving animal subjects were approved by the Institutional Animal Care and Use Committee (IACUC) at Southern Medical University.

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Supplementary Information

Fig. S1.

BIG1 immunoprecipitation (IP) and Coomassie blue staining. Products of IP with control IgG or anti-BIG1 antibodies from 1 mg of fresh rat hippocampus homogenate were separated by 10% SDS–PAGE gel and stained with Coomassie R250 blue. After destaining, the gel was divided into four pieces for mass spectrometry analysis according to the centers of molecular weight marker bands from top to bottom (>170, ~55, ~50, 36-26 kDa). (PNG 181 kb)

High Resolution Image (TIF 370 kb)

Table S1.

Proteins from 36-26 kDa gel segments identified by more than two peptides in LC-MS/MS analyses. Proteins in gel block (36-26 kDa) were subjected to in-gel trypsin digestion and analyzed by LC–MS/MS. Normalized to the IgG pull down results, proteins pulled down by BIG1 with more than two unique peptides that represent the same target protein were shown. (DOCX 15 kb)

The blank control was analyzed with the vehicle (Krebs-HEPES) in SH-SY5Y cells labeled with the Cl– sensitive dye MQAE. Live cell images were taken every 5 seconds. (AVI 1166 kb)

Cl– influx stimulated by GABA (100 mM) in SHSY-5Y cells transfected with negative control siRNA (NC) was analyzed by a confocal microscope (Zeiss 710). Live cell images were taken every 5 seconds. (AVI 1278 kb)

Cl– influx stimulated by GABA (100 mM) in SHSY-5Y cells transfected with BIG1 siRNA (G05) was analyzed by a confocal microscope (Zeiss 710). Live cell images were taken every 5 seconds. (AVI 2598 kb)

Cl– influx stimulated by GABA (100 mM) in SHSY-5Y cells transfected with 14-3-3ζ siRNA (F06) was analyzed by a confocal microscope (Zeiss 710). Live cell images were taken every 5 seconds. (AVI 2823 kb)

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Li, C., Huang, S., Peng, J. et al. 14-3-3ζ Mediates GABAAR Activation by Interacting with BIG1. Mol Neurobiol 60, 1721–1732 (2023). https://doi.org/10.1007/s12035-022-03172-z

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