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.
GABA transporter subtype 1 (GAT1) molecules were counted near GABAergic synapses, to a resolution of approximately 0.5 microm. 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.
Chiu
Chi-Sung
CS
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
Jensen
Kimmo
K
Sokolova
Irina
I
Wang
Dan
D
Li
Ming
M
Deshpande
Purnima
P
Davidson
Norman
N
Mody
Istvan
I
Quick
Michael W
MW
Quake
Stephen R
SR
Lester
Henry A
HA
D000818:Animals; D019921:Biotinylation; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D050485:GABA Plasma Membrane Transport Proteins; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D026901:Membrane Transport Proteins; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009928:Organ Specificity; D027361:Organic Anion Transporters; D018408:Patch-Clamp Techniques; D017729:Presynaptic Terminals / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013234:Stem Cells / Q000166:cytology; D009435:Synaptic Transmission / Q000502:physiology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D002352:Carrier Proteins; D018076:DNA, Complementary; D050485:GABA Plasma Membrane Transport Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D011993:Recombinant Fusion Proteins; C493413:SLC6A1 protein, human; C493414:Slc6a1 protein, mouse; D049452:Green Fluorescent Proteins; D005680:gamma-Aminobutyric Acid