Pentameric assembly of a neuronal glutamate transporter.
Freeze-fracture electron microscopy was used to study the structure of a human neuronal glutamate transporter (EAAT3). EAAT3 was expressed in Xenopus laevis oocytes, and its function was correlated with the total number of transporters in the plasma membrane of the same cells. Function was assayed as the maximum charge moved in response to a series of transmembrane voltage pulses. The number of transporters in the plasma membrane was determined from the density of a distinct 10-nm freeze-fracture particle, which appeared in the protoplasmic face only after EAAT3 expression. The linear correlation between EAAT3 maximum carrier-mediated charge and the total number of the 10-nm particles suggested that this particle represented functional EAAT3 in the plasma membrane. The cross-sectional area of EAAT3 in the plasma membrane (48 +/- 5 nm(2)) predicted 35 +/- 3 transmembrane alpha-helices in the transporter complex. This information along with secondary structure models (6-10 transmembrane alpha-helices) suggested an oligomeric state for EAAT3. EAAT3 particles were pentagonal in shape in which five domains could be identified. They exhibited fivefold symmetry because they appeared as equilateral pentagons and the angle at the vertices was 110 degrees. Each domain appeared to contribute to an extracellular mass that projects approximately 3 nm into the extracellular space. Projections from all five domains taper toward an axis passing through the center of the pentagon, giving the transporter complex the appearance of a penton-based pyramid. The pentameric structure of EAAT3 offers new insights into its function as both a glutamate transporter and a glutamate-gated chloride channel.
Eskandari
S
S
Departments of Physiology and Neurobiology, University of California at Los Angeles School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095-1751, USA. seskandari@mednet.ucla.edu
Kreman
M
M
Kavanaugh
M P
MP
Wright
E M
EM
Zampighi
G A
GA
D027322:Amino Acid Transport System X-AG; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D027981:Symporters; D014982:Xenopus laevis
D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; C493400:SLC1A1 protein, human; D027981:Symporters; D018698:Glutamic Acid