Demonstration of functional coupling between γ-aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles

doi:10.1073/pnas.0730698100

Demonstration of functional coupling between γ-aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles

Departments of ^*Molecular Biosciences and ^‡Medicinal Chemistry, University of Kansas, Lawrence, KS 66045; ^†Department of Biomedical Science, Florida Atlantic University, Boca Raton, FL 33431; ^§Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037; and ^¶Department of Otolaryngology, Naval Medical Center, San Diego, CA 92134-5000

Communicated by Herbert Weissbach, Florida Atlantic University, Boca Raton, FL (received for review October 30, 2002)

Abstract

l-Glutamic acid decarboxylase (GAD) exists as both membrane-associated and soluble forms in the mammalian brain. Here, we propose that there is a functional and structural coupling between the synthesis of γ-aminobutyric acid (GABA) by membrane-associated GAD and its packaging into synaptic vesicles (SVs) by vesicular GABA transporter (VGAT). This notion is supported by the following observations. First, newly synthesized [³H]GABA from [³H]l-glutamate by membrane-associated GAD is taken up preferentially over preexisting GABA by using immunoaffinity-purified GABAergic SVs. Second, the activity of SV-associated GAD and VGAT seems to be coupled because inhibition of GAD also decreases VGAT activity. Third, VGAT and SV-associated Ca²⁺/calmodulin-dependent kinase II have been found to form a protein complex with GAD. A model is also proposed to link the neuronal stimulation to enhanced synthesis and packaging of GABA into SVs.

Footnotes

↵ ‖ To whom correspondence should be addressed. E-mail: jwu{at}fau.edu.
↵ ** Chen, R., Di, S., Wei, J.-N. & Wu, J. Y. (2001) Soc. Neurosci. Abstr. 27, 124.8.
Abbreviations:

GAD,

l-glutamic acid decarboxylase;

GABA,

γ-aminobutyric acid;

MGAD,

membrane-associated GAD;

SGAD,

soluble GAD;

HGAD,

human GAD;

VGAT,

vesicular GABA transporter;

CaMKII,

Ca2+/calmodulin-dependent kinase II;

SV,

synaptic vesicle;

HGAD65,

human GAD65;

HSC70,

heat shock cognate 70;

CSP,

cysteine-string protein;

GSH,

glutathione