(A) Decreasing the mean charge of impermeant anions from −0.85 (the default value) to −1 (orange, bottom panel), without changing the absolute number of intracellular impermeant anions caused a persistent decrease in ECl (green, top panel), EK (purple) and Vm with moderate increases in volume (middle panel) in our default single compartment model. Negligible changes in Cl- driving force (∆DF = 0.16 mV, red) were observed. (B) Analytic solution (solid lines) for different impermeant anion mean charge (z) exactly matches the steady state values from numerical, time series runs (dots) based on adjusting z as in ‘A’. Steady state ECl, EK, Vm (top panel) and [X]i (middle) increased with increasing z, while changes in z resulted in very small changes in Cl- DF (bottom, red). The vertical dashed line indicates the values at the chosen default z of −0.85. (C) Influx of a species of impermeant anions with a charge of −1.5, that decreased the mean charge z (bottom panel) from −0.85 to −1 and increased the number of impermeant anions also caused persistent decreases of ECl, EK and Vm as in ‘A’, but with larger increases in cell volume (middle panel). Again, very small persistent changes in Cl- DF were observed. The volume changes for different flux amounts and charges are illustrated in Figure 5—figure supplement 1. (D) Top, schematic of the experimental setup where whole-cell recordings were made from CA3 pyramidal cells in mouse organotypic brain slices. Impermeant anions (orange) were delivered via electroporation of the negatively charged fluorescent dextran Alexa Flour 488 via a pipette positioned near the soma of the recorded cell. GABAAR currents were elicited via photo-activation (100 ms, 470 nm LED via objective) of ChR2-expressing GAD2+ interneurons (green cells) in the presence of 5 μM CGP-35348 to block GABABRs. Lower trace, current clamp recording showing Vm changes during electroporation of anionic dextran. Confocal image demonstrating cell-localized fluorescence of the anionic dextran electroporated in ‘E’. (E) Top, widefield images with electroporation pipette (orange dashed lines) and the recording pipette (white dashed lines). Note increased fluorescence in the soma after electroporation. Below, insets show GABAAR currents evoked by photo-activation of GAD2+ interneurons at different holding potentials. Calibration: 1 s, 100 pA. Holding current (reflecting membrane current) and total current (reflecting membrane current plus the GABAAR current) were measured at the points indicated by the vertical grey and black lines, respectively. IV plots were used to calculate Vm, EGABA and DF before (left) and after (right) electroporation. (F) Top, population data showing significant decreases in mean Vm and EGABA but not DF five minutes after electroporation. Below: changes in DF over time. Point at which electroporation occurred marked with orange arrow. ‘ns’, non-significant; *p<0.05. The data for ‘D’, ‘E’ and ‘F’ is provided in Figure 5—source data 1.