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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

Chiu, Chi-Sung and Jensen, Kimmo and Sokolova, Irina and Wang, Dan and Li, Ming and Deshpande, Purnima and Davidson, Norman and Mody, Istvan and Quick, Michael W. and Quake, Stephen R. and Lester, Henry A. (2002) 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. Journal of Neuroscience, 22 (23). pp. 10251-10266. ISSN 0270-6474. PMCID PMC6758747. doi:10.1523/jneurosci.22-23-10251.2002. https://resolver.caltech.edu/CaltechAUTHORS:20190930-133756603

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Abstract

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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1523/jneurosci.22-23-10251.2002DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6758747/PubMed CentralArticle
ORCID:
AuthorORCID
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 2002 Society for Neuroscience. Received June 4, 2002; revised Aug. 8, 2002; accepted Sept. 11, 2002. This research was supported by grants from National Institutes of Health (DA-09121, DA-010509, NS-11756, MH-49176, MH-61468, NS-030549, and National Research Service Award to M.L.) and the National Science Foundation (0119493), and by a Della Martin Fellowship (C.-S.C.). We are indebted to Cesar Labarca and other members of the Caltech and University of California Los Angeles groups for much advice, Nathan Nelson for the mGAT1 cDNA, Tau-Mu Yi and M. Simon for use and help with the confocal microscope, and Melinda Turner and Robert Farley for discussion.
Funders:
Funding AgencyGrant Number
NIHDA-09121
NIHDA-010509
NIHNS-11756
NIHMH-49176
NIHMH-61468
NIHNS-030549
NIH Predoctoral FellowshipUNSPECIFIED
NSFCBET-0119493
Della Martin FoundationUNSPECIFIED
Subject Keywords:GABA; synapse; transporter; green fluorescent protein; mouse; knock-in
Issue or Number:23
PubMed Central ID:PMC6758747
DOI:10.1523/jneurosci.22-23-10251.2002
Record Number:CaltechAUTHORS:20190930-133756603
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20190930-133756603
Official Citation: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 Chiu, Kimmo Jensen, Irina Sokolova, Dan Wang, Ming Li, Purnima Deshpande, Norman Davidson, Istvan Mody, Michael W. Quick, Stephen R. Quake, Henry A. Lester Journal of Neuroscience 1 December 2002, 22 (23) 10251-10266; DOI: 10.1523/JNEUROSCI.22-23-10251.2002
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:98955
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:30 Sep 2019 23:08
Last Modified:16 Nov 2021 17:42

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