Figure 1.

VGLUT-1, -2, or -3 Increase Pi Transport

(A) COS7 cells. (Left) VGLUT-1, -2, and -3 are detected in the respective COS7 subclones expressing VGLUT-IRES-EGFP constructs and not in the MOCK clone expressing only EGFP. (Insets) Twice magnified cells are labeled with DAPI (blue) and the respective VGLUT variant (white). Scale bar, 40 μm. (Middle) Pi concentrations in control condition (Pi starvation in KREBS 4.7 mM [K⁺] followed by 15 min incubation in Pi-free uptake buffer) given as pmol Pi/10³ cells were comparable between the different COS7 clones. (Right) Pi content of COS7-VGLUTs cells incubated in uptake buffer containing no or 10 mM [Pi] ± 10 μM Rose bengal (RB) is shown. Values were normalized to the controls (no Pi in all incubation steps; see values in middle panel). Mean values ± SEM; n ≥ 10.

(B) Neuronal preparations. (Left) Cultured primary neurons were preincubated for 1 h in Pi-free KREBS 4.7 mM [K⁺]. One batch of cells was then depolarized with Pi-free KREBS 30 mM [K⁺] and 80 μM Dynasore to prevent endocytosis. After washing in Pi-free KREBS 4.7 mM [K⁺], neurons were incubated for 15 min in uptake buffer ± 10 mM [Pi] ± 10 μM [RB]. The presence of Pi caused a RB-sensitive increase in cellular Pi content (light gray bars) that was augmented when cells were first depolarized to induce exocytosis (dark gray bars). Stimulation did not change resting cytoplasmic Pi concentrations (Figure S2A). (Middle) Pi content of synaptosomes from adult rat brains after successive depolarization, repolarization (Pi free buffer), and incubation in uptake buffer ± 10 mM [Pi] ± 10 μM [RB] steps (see STAR Methods). The presence of Pi increased synaptosomal Pi content in a RB-sensitive manner. (Right) Synaptosomes subjected to incubation and repolarization using KREBS buffer containing either 30 or 4.7 mM [K⁺] plus 10 mM Pi were analyzed after either 2- or 15-min incubation in Pi-free uptake buffer ± RB. Pi efflux increased with time and was enhanced by RB. The intracellular content decreased correspondingly (Figure S2B, right).

Data show mean values ± SEM given as % of Pi content observed in Pi-free control (left and middle, samples presented with no Pi) or as % of total Pi (extracellular and cytoplasmic, right) from at least 4 (left), 14 (middle), or 3 (right) preparations. Significance toward: (1) no Pi added; (2) Pi; (3) 4.7 mM [K⁺] control; (4) 30 mM [K⁺] control; and (9) MOCK.