The dual role of chloride in synaptic vesicle glutamate transport
- UCSF School of Medicine, United States
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 …
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 …
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…
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 = …
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 …
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.
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), …
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 …
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 …
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 …
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 …
Figure 4
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 …
Tables
Key resources table
| Reagent type | Designation | Source or reference | Identifiers | Additional |
|---|---|---|---|---|
| Gene (Rattus norvegicus) | VGLUT1 | Edwards lab | NM_053859.2 | |
| Gene (Rattus norvegicus) | VGLUT2 | Edwards lab | NM_053427.1 | |
| Gene (Rattus norvegicus) | VGLUT3 | Edwards lab | NM_153725.1 | |
| Cell line (Homo sapiens) | HEK293T cells | UCSF Cell Culture Fac. | ||
| Transfected construct | pEGFP-N1 | Clontech | ||
| Antibody | Mouse anti-actin | Sigma | MAB1501R | (1:3000) |
| Antibody | Guinea pig anti-VGLUT1 | Millipore Sigma | AB5905 | (1:2000) |
| Recombinant DNA reagent | PolyJet | SignaGen | SL100688 | |
| Recombinant DNA reagent | Lipofectamine 2000 | Gibco Life Technologies | ||
| Software, algorithm | Prism 5.0 | GraphPad |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.34896.014
