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The Journal of Neuroscience, December 1, 2002, 22(23):10251-10266

Number, Density, and Surface/Cytoplasmic Distribution of GABA Transporters at Presynaptic Structures of Knock-In Mice Carrying GABA Transporter Subtype 1-Green Fluorescent Protein Fusions

Chi-Sung Chiu¹, Kimmo Jensen⁴, Irina Sokolova¹, Dan Wang³, Ming Li¹, Purnima Deshpande¹, Norman Davidson¹, Istvan Mody⁴, Michael W. Quick³, Stephen R. Quake², and Henry A. Lester¹

Divisions of ¹ Biology and ² Engineering and Applied Physics, California Institute of Technology, Pasadena, California 91125, ³ Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0021, and ⁴ Departments of Neurology and Physiology, University of California Los Angeles School of Medicine, Los Angeles, California 90095-1769

GABA transporter subtype 1 (GAT1) molecules were counted near GABAergic synapses, to a resolution of ~0.5 µm. Fusions between GAT1 and green fluorescent protein (GFP) were tested in heterologous expression systems, and a construct was selected that shows function, expression level, and trafficking similar to that of wild-type (WT) GAT1. A strain of knock-in mice was constructed that expresses this mGAT1-GFP fusion in place of the WT GAT1 gene. The pattern of fluorescence in brain slices agreed with previous immunocytochemical observations. [³H]GABA uptake, synaptic electrophysiology, and subcellular localization of the mGAT1-GFP construct were also compared with WT mice. Quantitative fluorescence microscopy was used to measure the density of mGAT1-GFP at presynaptic structures in CNS preparations from the knock-in mice. Fluorescence measurements were calibrated with transparent beads and gels that have known GFP densities. Surface biotinylation defined the fraction of transporters on the surface versus those in the nearby cytoplasm. The data show that the presynaptic boutons of GABAergic interneurons in cerebellum and hippocampus have a membrane density of 800-1300 GAT1 molecules per square micrometer, and the axons that connect boutons have a linear density of 640 GAT1 molecules per micrometer. A cerebellar basket cell bouton, a pinceau surrounding a Purkinje cell axon, and a cortical chandelier cell cartridge carry 9000, 7.8 million, and 430,000 GAT1 molecules, respectively; 61-63% of these molecules are on the surface membrane. In cultures from hippocampus, the set of fluorescent cells equals the set of GABAergic interneurons. Knock-in mice carrying GFP fusions of membrane proteins provide quantitative data required for understanding the details of synaptic transmission in living neurons.

Key words: GABA; synapse; transporter; green fluorescent protein; mouse; knock-in

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Copyright © 2002 Society for Neuroscience  0270-6474/02/222310251-16$05.00/0

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