Figure 5.

Lack of effect of cell volume on KCC in Mg²⁺-depleted cells. Cells (not density separated) were suspended in 160 mM NaCl, 10 mM HEPES, 1 mM EDTA, pH 7.5, 0.2% ethanol ± 10 μM A23187, and incubated 10 min at 30°C to deplete Mg²⁺. Each suspension was then washed once in the above medium (without ethanol and A23187), split in half, and washed once in either the same medium or in medium with 160 mM NaNO₃ instead of 160 mM NaCl. Cells were then resuspended in HEPES-buffered NaCl or NaNO₃ media with osmolality varied from 185 to 410 mosmol/kg H₂O by adding either water or 1 M NaCl (or NaNO₃). Suspensions were incubated 20 min at 30°C before adding 1 μCi ⁸⁶Rb⁺. Influx was measured for 30 min at 30°C. All flux solutions contained 10⁻⁴ M ouabain and 5 mM K⁺ (added as KCl or KNO₃). (Top) No A23187. (Bottom) Mg²⁺ depletion with A23187. Flux in Cl⁻ media (•). Flux in NO₃ ⁻ media (▪).