Determination of cytosolic Mg2+ activity and buffering in BC3H-1 cells with mag-fura-2

The magnesium buffer coefficient (B_Mg) was calculated for BC₃H-1 cells from the rise in cytosolic Mg²⁺ activity observed when magnesium was released from ATP after iodoacetate (IAA) and NaCN treatment. The basal cytosolic Mg²⁺ activity (0.54±0.1 mM) measured with mag-fura-2 doubled when 4.54 mM magnesium was liberated from ATP:B_Mg was 12.9 indicating that a 1 mM increase in Mg²⁺ activity requires an addition of about 13 mM magnesium. The accuracy of this value depends on these assumptions: (a) all of the magnesium released from ATP stayed in the cells; (b) the rise in Mg²⁺ was not secondary to pH-induced changes inB_Mg; (c) mag-fura-2 measured Mg²⁺ and not Ca²⁺; and (d) the accuracy of the mag-fura-2 calibration. Total magnesium did not change in response to IAA/CN treatment, thus the change in Mg²⁺ activity reflected a redistribution of cell magnesium. pH changes induced by NH₄Cl pulse and removal had little effect on Mg²⁺ activity and the changes were slower than and opposite to pH-induced changes in Ca²⁺ activity measured by fura-2. Ca²⁺ responses were temporally uncopled from Mg²⁺ responses when the cells were treated with IAA only and in no cases did Ca²⁺ levels rise above 1 M, showing that the mag-fura-2 is responding to Mg²⁺. Additional studies demonstrated that 90% of the mag-fura-2 signal was cytosolic in origin. The remaining non-diffusible mag-fura-2 either was bound to cytosolic membranes or sequestered in organelles with the fluorescence characteristics of the Mg²⁺-complexed form, even when cytosolic free Mg²⁺ activity was approximately 0.5 mM. This bound mag-fura-2 would appear to increase the K_d and thus clearly limits the accuracy of our estimmate forB_Mg. Despite this limitation, we demonstrate that Mg²⁺ is tightly regulated in face of large changes in extracellular Mg²⁺, and that the interplay observed between pH, Ca²⁺ and Mg²⁺ activities strongly supports the hypothesis that these factors interact through a shared buffer capacity of the cell.