The transport of all classical transmitters into synaptic vesicles depends on an outwardly directed H+ electrochemical driving force (?H+) produced by the vacuolar H+-ATPase. However, vesicular glutamate transport differs from the vesicular transport of other classical transmitters, and relies almost entirely on the electrical component o this gradient (??) rather than the chemical gradient (?pH). Indeed, it remains unclear whethe the vesicular glutamate transporters (VGLUTs) mediate H+ exchange at all. They may simply catalyze facilitated diffusion, or even function as anion channels. In contrast, the closely relate transporter sialin catalyzes the electroneutral cotransport of H+ with sialic acid, and it remains unknown how two members of the SLC17 family can mediate such apparently different activities. However, sialin has also been reported to mediate vesicular glutamate transport, suggesting that the two different activities reflect a common underlying mechanism. The long-term objective of this program is to understand how the SLC17 family confers both ??-driven diffusion and H+ cotransport. The strategy is to determine the structure of proteins in this family and use this information to guide studies of mechanism. Screening a number of bacterial proteins related to the VGLUTs, we have identified one that can be crystallized under a number of different conditions, and that diffracts to 3.7 in the lipidic cubic phase. We have also reconstituted the recombinant protein into artificial membranes and shown that it catalyzes the cotransport of an organic anion with H+, similar to sialin. We now propose to 1) refine the structure of DgoT at atomic resolution; 2) determine the structure of DgoT in different functional states, including substrate-bound; 3) test the role of specific residues implicated by the structur in substrate recognition and H+ movement; and 4) determine the structure of a metazoan VGLUT. The results will help us to understand how one class of transport proteins and perhaps even one protein can couple in apparently different ways to the H+ electrochemical driving force. At the same time, structural analysis should illuminate the mechanism for allosteric regulation of the VGLUTs by chloride, which remains poorly understood, and by H+, which we have recently discovered. The identification of mutants with altered properties also provides us with tools to test the physiological role of these properties by genetic manipulation in vitro and n vivo.

Public Health Relevance

Synaptic transmission involves the regulated exocytosis of vesicles filled with transmitter, but we still understand little about the basic mechanisms that transport classical neurotransmitters into synaptic vesicles or their potential for regulation. In contrast to other vesicular neurotransmitter transporters, the vesicular glutamate transporters rely primarily on membrane potential (rather than pH gradient) as the driving force, and the mechanism that underlies this coupling remains unknown. It also remains unclear how the closely related sialic acid transporter sialin catalyzes H+ cotransport with sialic acid and the relationship between these two activities. By determining the structure of proteins in this family, we thus hope to elucidate the mechanistic basis for these different activities, which will help to understand their role in synaptic transmission, their potential for regulation, and their contribution to neuropsychiatric disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS089713-01A1
Application #
8964141
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Silberberg, Shai D
Project Start
2015-08-01
Project End
2019-04-30
Budget Start
2015-08-01
Budget End
2016-04-30
Support Year
1
Fiscal Year
2015
Total Cost
$651,321
Indirect Cost
$240,393
Name
University of California San Francisco
Department
Physiology
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Chang, Roger; Eriksen, Jacob; Edwards, Robert H (2018) The dual role of chloride in synaptic vesicle glutamate transport. Elife 7: