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Direct visualization of glutamate transporter elevator mechanism by high-speed AFM.


ABSTRACT: Glutamate transporters are essential for recovery of the neurotransmitter glutamate from the synaptic cleft. Crystal structures in the outward- and inward-facing conformations of a glutamate transporter homolog from archaebacterium Pyrococcus horikoshii, sodium/aspartate symporter GltPh, suggested the molecular basis of the transporter cycle. However, dynamic studies of the transport mechanism have been sparse and indirect. Here we present high-speed atomic force microscopy (HS-AFM) observations of membrane-reconstituted GltPh at work. HS-AFM movies provide unprecedented real-space and real-time visualization of the transport dynamics. Our results show transport mediated by large amplitude 1.85-nm "elevator" movements of the transport domains consistent with previous crystallographic and spectroscopic studies. Elevator dynamics occur in the absence and presence of sodium ions and aspartate, but stall in sodium alone, providing a direct visualization of the ion and substrate symport mechanism. We show unambiguously that individual protomers within the trimeric transporter function fully independently.

SUBMITTER: Ruan Y 

PROVIDER: S-EPMC5320997 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Direct visualization of glutamate transporter elevator mechanism by high-speed AFM.

Ruan Yi Y   Miyagi Atsushi A   Wang Xiaoyu X   Chami Mohamed M   Boudker Olga O   Scheuring Simon S  

Proceedings of the National Academy of Sciences of the United States of America 20170130 7


Glutamate transporters are essential for recovery of the neurotransmitter glutamate from the synaptic cleft. Crystal structures in the outward- and inward-facing conformations of a glutamate transporter homolog from archaebacterium <i>Pyrococcus horikoshii</i>, sodium/aspartate symporter Glt<sub>Ph</sub>, suggested the molecular basis of the transporter cycle. However, dynamic studies of the transport mechanism have been sparse and indirect. Here we present high-speed atomic force microscopy (HS  ...[more]

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