The dual role of chloride in synaptic vesicle glutamate transport

  1. Roger Chang
  2. Jacob Eriksen
  3. Robert H Edwards  Is a corresponding author
  1. UCSF School of Medicine, United States
4 figures, 1 table and 1 additional file

Figures

Figure 1 with 3 supplements
Chloride and glutamate currents in endosomes expressing VGLUT1.

(A,D) Whole endosome patch clamp recording configuration, with 140 mM Cl- at pH 5.0 and NPPB in the pipette, showing outward currents at positive lumenal potentials and inward currents at negative …

https://doi.org/10.7554/eLife.34896.002
Figure 1—figure supplement 1
Chloride currents in endosomes without lumenal NPPB.

(A) Whole endosome recording conditions with 140 mM Cl- at pH 5.0 but no NPPB in the pipette (lumen). Representative recordings from cells expressing only rab Q79L (B), VGLUT1 (D), VGLUT2 (E) and …

https://doi.org/10.7554/eLife.34896.003
Figure 1—figure supplement 2
Chloride and glutamate currents (with lumenal NPPB) in VGLUT2+ and VGLUT3+ endosomes.

(A) Whole endosome recording conditions with 140 mM Cl- at pH 5.0 and 200 µM NPPB in the pipette (lumen). Representative recordings from endosomes expressing VGLUT2 (B,E) and VGLUT3 (C,F). The top (A…

https://doi.org/10.7554/eLife.34896.004
Figure 1—figure supplement 3
Sensitivity of VGLUT1 Cl- and glutamate currents to Evans Blue.

Representative endosome recordings with 140 mM Cl- at pH 5.0 in the patch pipette, either Cl- (A) or glutamate (D) in the bath solution, with increasing concentrations of Evans Blue in the bath (n = …

https://doi.org/10.7554/eLife.34896.005
Figure 2 with 2 supplements
Role of lumenal and cytoplasmic Cl- in endosomes expressing VGLUT1.

(A–C) Representative whole endosome recordings under the same conditions as in Figure 1 but in the presence of 10 mM (A), 30 mM (B) and 50 mM lumenal Cl- (C) (n = 4 each). (D) To assess …

https://doi.org/10.7554/eLife.34896.006
Figure 2—figure supplement 1
Dependence of VGLUT1 currents on lumenal Cl-.

The compiled inward and outward (Cl- and glutamate) VGLUT1 currents from Figure 2 are replotted as a function of lumenal Cl- concentration. Bar graph indicates mean ± SEM. n = 4.

https://doi.org/10.7554/eLife.34896.007
Figure 2—figure supplement 2
Effects of Br- and PO43- on VGLUT1+ endosomes.

Representative recordings (A–C) and compiled data (D) for VGLUT1+ (A), VGLUT2+ (B) and VGLUT3+ (C) endosomes with 140 mM Br- at pH 5 in the pipette and 140 mM NMDG gluconate (0 Cl-), Br- (140 mM), …

https://doi.org/10.7554/eLife.34896.008
Figure 3 with 3 supplements
Transmembrane arginine residues control allosteric activation and permeation by Cl-.

(A) Sequence alignment of transmembrane domains TM 7, 1 and 4 from the related VGLUT isoforms and species with highly conserved arginine highlighted (red). (B) Representative whole endosome …

https://doi.org/10.7554/eLife.34896.009
Figure 3—figure supplement 1
Chloride and glutamate currents in endosomes expressing mutant VGLUTs.

Compiled data (A–C) and sample traces (D–I) for alanine substitutions at conserved arginine residues in TM7, 1 and 4 of VGLUT1 (A), VGLUT2 (B,D–F) and VGLUT3 (C,G–I). All endosomes were recorded …

https://doi.org/10.7554/eLife.34896.010
Figure 3—figure supplement 2
Chloride and glutamate currents in endosomes expressing TM4 arginine mutants.

Representative recordings with 140 mM Cl- in the pipette from endosomes expressing VGLUT1 R176K (B) and R176Q (n = 3) (C). Representative recordings with 0 mM Cl- in the pipette of endosomes …

https://doi.org/10.7554/eLife.34896.011
Figure 3—figure supplement 3
R184A eliminates the requirement for external Cl- to activate the inward Cl- conductance of VGLUT2 misexpressed at the plasma membrane.

Whole cell recording of VGLUT2 misexpressed at the plasma membrane (pmVGLUT2) of HEK293T cells. (A) Representative ramp currents from HEK293T cells expressing wild type pmVGLUT2 (left), R184A …

https://doi.org/10.7554/eLife.34896.012
Relationship between Cl- and glutamate conductances.

(A–C) Dose-response of outward currents to external Cl- (B) or glutamate in the presence of either 1 mM or 10 mM external Cl- (C), all at lumenal pH 5.0. The EC50 for glutamate (C) was 376 µM in 10 …

https://doi.org/10.7554/eLife.34896.013

Tables

Key resources table
Reagent typeDesignationSource or referenceIdentifiersAdditional
Gene (Rattus norvegicus)VGLUT1Edwards labNM_053859.2
Gene (Rattus norvegicus)VGLUT2Edwards labNM_053427.1
Gene (Rattus norvegicus)VGLUT3Edwards labNM_153725.1
Cell line (Homo sapiens)HEK293T cellsUCSF Cell Culture Fac.
Transfected constructpEGFP-N1Clontech
AntibodyMouse anti-actinSigmaMAB1501R(1:3000)
AntibodyGuinea pig anti-VGLUT1Millipore SigmaAB5905(1:2000)
Recombinant DNA reagentPolyJetSignaGenSL100688
Recombinant DNA reagentLipofectamine 2000Gibco Life Technologies
Software, algorithmPrism 5.0GraphPad

Additional files

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