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BIG1, a Brefeldin A-Inhibited Guanine Nucleotide-Exchange Factor, Is Required for GABA-Gated Cl– Influx Through Regulation of GABAA Receptor Trafficking

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Abstract

GABAA receptors (GABAARs) mediate the majority of fast synaptic inhibition. Trafficking regulation and protein–protein interactions that maintain the appropriate number of GABAARs at the cell surface are considered to be important mechanisms for controlling the strength of synaptic inhibition. Here, we report that BIG1, a brefeldin A (BFA)-inhibited guanine nucleotide-exchange factor (GEF) which has a known role in vesicle trafficking, is a new binding partner of GABAARs. Treatment of neurons with BFA, an uncompetitive inhibitor of BIG1 GEF activity, or depletion of BIG1 by small RNA interference (siRNA) significantly decreased GABAARs at the neuronal surface and suppressed GABA-gated influx of chloride ions. Over-expression of HA-tagged BIG1-E793K, a dominant-negative mutant, also significantly decreased GABAARs at the neuronal surface, but had no effect on the total amount of GABAARs. Inhibition of GABAAR endocytosis by muscimol increased both GABAARs and BIG1 at the neuronal surface in a time-dependent fashion, and this increase could be abolished by bicuculline. Finally, depletion of BIG1 by siRNA inhibited the muscimol-stimulated increase of GABAARs. Those data suggest an important function of BIG1 in trafficking of GABAARs to the cell surface through its GEF activity. Thus, we identify an important role of BIG1 in modulating GABA-gated Cl− influx through the regulation of cell surface expression of GABAARs.

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Acknowledgments

The anti-BIG1 antibody and BIG1 plasmids were kindly provided by Dr. Martha Vaughan and Dr. Joel Moss from the Cardiovascular-Pulmonary Branch of the National Heart, Lung, and Blood Institute, National Institutes of Health. This study was supported by National Natural Science Foundation of China (nos. 31070924 and 81173056) and the Research Fund for the Doctoral Program of Higher Education of China (no. 20100171110052).

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Authors and Affiliations

  1. Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, No. 132, Waihuan East Road, Higher Education Mega Center, Guangzhou, 510006, People’s Republic of China

    Cuixian Li, Shaorui Chen, Yang Yu, Chun Zhou, Ying Wang, Kang Le, Dong Li, Weiwei Shao, Liang Lu, Yan You, Jin Peng, Heqing Huang, Peiqing Liu & Xiaoyan Shen

  2. Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai, People’s Republic of China

    Xiaoyan Shen

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  1. Cuixian Li
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Electronic supplementary material

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The blank control was analyzed with the vehicle (Krebs-HEPES) in cultured hippocampal neurons (9 DIV) labeled with the Cl– sensitive dye MQAE. Live cell images were taken each 5 seconds. (AVI 3407 kb)

Cl– influx stimulated by GABA (100 mM) in cultured hippocampal neurons (9 DIV) labeled with MQAE was analyzed by a confocal microscope (Zeiss 710). Live cell images were taken each 5 seconds. (AVI 957 kb)

Cultured hippocampal neurons (9 DIV) were treated with 5 μg/ml BFA for 5 min, then subjected to the MQAE paradigm to monitor GABA stimulated (100 mM) Cl– influx. Live cell images were taken each 5 seconds. (AVI 5107 kb)

Cultured hippocampal neurons (9 DIV) were treated with 5 μg/ml BFA for 30 min, then subjected to the MQAE paradigm to monitor GABA stimulated (100 mM) Cl– influx. Live cell images were taken each 5 seconds. (AVI 1599 kb)

Cultured hippocampal neurons (9 DIV) were treated with 100 μM bicuculine for 15 min, and then subjected to the MQAE paradigm to monitor GABA stimulated (100 mM) Cl– influx. Live cell images were taken each 5 seconds. (AVI 1693 kb)

The blank control was analyzed with the vehicle (Krebs-HEPES) in SHSY-5Y cells transfected with negative control siRNA (NC). Live cell images were taken each 5 seconds. (AVI 61 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 each 5 seconds. (AVI 55 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 each 5 seconds. (AVI 57 kb)

Fig. S1

Cellular localization of BIG1 and GM130. Hippocampal neurons were fixed and permeabilized, followed by incubation with anti-BIG1 and anti-GM130 antibodies. Images were acquired using a laser scanning confocal microscopy (scale bar, 20 μm). (JPEG 69 kb)

High resolution image (TIFF 422 kb)

Fig. S2

Effect of BFA treatment on GABA A Rs expression in neurons. Cultured hippocampal neurons (9 DIV) were treated with vehicle (DMSO), or with 5 μg/ml BFA for 30 min. The neurons were fixed, permeabilized, and reacted with anti-GABAARβ2,3 antibodies. Images were acquired using a laser scanning confocal microscopy (scale bar, 20 μm). Fluorescent intensity was quantified with ImageJ software (NIH) in a minimum of 20 cells per slide. The experiments were repeated at least three times. (JPEG 142 kb)

High resolution image (TIFF 648 kb)

Fig. S3

Effect of BIG1 depletion on GABA A Rs expression in neurons. Hippocampal neurons (7 DIV) transfected with Cy3 labeled NC siRNA or BIG1 (G05) siRNA were fixed, permeabilized, and reacted with anti-GABAARβ2,3 antibodies. Images were acquired using a laser scanning confocal microscopy (Scale bar, 20 μm). Fluorescent intensity was quantified with ImageJ software (NIH) in a minimum of 20 cells per slide. The experiments were repeated at least three times. (JPEG 249 kb)

High resolution image (TIFF 1261 kb)

Fig. S4

Effect of muscimol, bicuculine or BFA on GABA A Rs expression in neurons. Hippocampal neurons (9 DIV) treated with vehicle (DMSO), muscimol alone (50 μM for 15 min; Musc), or bicuculline (100 μM for 10 min; Bic), or bicuculline (100 μM) plus muscimol (50 Μm for 15 min, Musc + Bic,), or BFA (5 μg/ml) plus muscimol (50 μM for 30 min, Musc + BFA). The neurons were fixed, permeabilized, and reacted with anti-GABAARβ2,3 antibodies. Images were acquired using a laser scanning confocal microscopy (Scale bar, 20 μm). Fluorescent intensity was quantified with ImageJ software (NIH) in a minimum of 20 cells per slide. The experiments were repeated at least three times. (JPEG 570 kb)

High resolution image (TIFF 2234 kb)

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Li, C., Chen, S., Yu, Y. et al. BIG1, a Brefeldin A-Inhibited Guanine Nucleotide-Exchange Factor, Is Required for GABA-Gated Cl– Influx Through Regulation of GABAA Receptor Trafficking. Mol Neurobiol 49, 808–819 (2014). https://doi.org/10.1007/s12035-013-8558-8

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