Pentameric assembly of a neuronal glutamate transporter

Freeze-fracture electron microscopy was used to study the structure of a hu man neuronal glutamate transporter (EAAT3). EAAT3 was expressed in Xenopus laevis oocytes, and its function was correlated with the total number of tr ansporters in the plasma membrane of the same cells. Function was assayed a s the maximum charge moved in response to a series of transmembrane voltage pulses. The number of transporters in the plasma membrane was determined f rom the density of a distinct 10-nm freeze-fracture particle, which appeare d in the protoplasmic face only after EAAT3 expression. The linear correlat ion between EAAT3 maximum carrier-mediated charge and the total number of t he 10-nm particles suggested that this particle represented functional EAAT 3 in the plasma membrane. The cross-sectional area of EAAT3 in the plasma m embrane (48 +/- 5 nm(2)) predicted 35 +/- 3 transmembrane alpha-helices in the transporter complex. This information along with secondary structure mo dels (6-10 transmembrane alpha-helices) suggested an oligomeric state for E AAT3, EAAT3 particles were pentagonal in shape in which five domains could be identified. They exhibited fivefold symmetry because they appeared as eq uilateral pentagons and the angle at the vertices was 110 degrees. Each dom ain appeared to contribute to an extracellular mass that projects approxima te to 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 pentamer ic structure of EAAT3 offers new insights into its function as both a gluta mate transporter and a glutamate-gated chloride channel.