(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 …
(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 …
(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…
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 = …
(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 …
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.
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), …
(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 …
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 …
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 …
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 …
(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 …
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 |