MEASUREMENTS OF INTRACELLULAR MG2-MUSCLE FIBERS WITH THE FLUORESCENT INDICATOR MAG-INDO-1( CONCENTRATION IN MOUSE SKELETAL)

Measurements of intracellular free magnesium concentration ([Mg2+](i)) were performed on enzymatically isolated skeletal muscle fibers from mice, using the fluorescent ratiometric indicator mag-indo-1. An origi nal procedure was developed to calibrate the dye response within the f ibers: fibers were first permeabilized with saponin in the presence of a given extracellular magnesium concentration and were then embedded in silicone grease. The dye was then pressure microinjected into the s aponin-permeabilized silicone-embedded fibers, and fluorescence was me asured. The results show that for all tested [Mg2+], the value of the measured fluorescence ratio was higher than that found in aqueous solu tions. Furthermore, the apparent binding curve that could be fit to th e in vivo ratio data was shifted toward higher [Mg2+] by a factor of s imilar to 2, Using the in vivo calibration parameters, the mean restin g [Mg2+](i) was found to be 1.53 +/- 0.16 mM (n = 7). In an attempt to gain insight into the myoplasmic magnesium buffering capacity, we mea sured, together with mag-indo-1 fluorescence, the current elicited by the application of carbamylcholine (CCh) to the endplate of isolated f ibers, in the presence of a high extracellular magnesium concentration . The results show that, under these conditions, a change in [Mg2+](i) displaying a time course and amplitude qualitatively consistent with the CCh-induced inward current can be measured.