Direct measurement of intracellular free magnesium in frog skeletal muscle using magnesium-selective microelectrodes

Abstract

Mg 2+-selective microelectrodes have been used to measure the intracellular free Mg 2+ concentration in frog skeletal muscle fibers. Glass capillaries with a tip diameter of less than 0.4 μm were backfilled with the Mg 2+ sensor, ETH 1117. In the absence of interfering ions, they gave Nernstian responses between 1 and 10 mM free Mg 2+. In the presence of an ionic environment resembling the myoplasm, the microelectrode response was sub Nernstian (18–24 mV) but still useful. The electrodes were calibrated before and after muscle-fiber impalements. In quiescent fibers from sartorius muscle ( Rana pipiens), with resting membrane potentials not less than −82 mV, the intracellular free Mg 2+ concentration was 3.8±0.41 (S.E.) mM ( n=58) at 22°C. No significant change in the intracellular free Mg 2+ was observed following extensive (approx. 6 h) incubation in Mg 2+-free media. Increasing the external concentration of magnesium from 4 to 20 mM (approx. 15 min) produced a slow and small enhancement (1.8 mM) of [Mg 2+] i, which was fully reverted when the divalent cation was removed from the bathing solution. No change in ionic magnesium resting concentration was observed when the muscle fibers were treated either with caffeine 3 mM or with Na +-free solutions. In depolarized muscle fibers (−23±2.7 mV) treated with 100 mM K +, the myoplasmic [Mg 2+] was 3.7±0.45 (S.E.) mM, n=6, immediately after the spontaneous relaxation of the contracture. Similar determinations in muscle fibers during stimulation at low frequency (5 Hz), and after fatigue development, showed no changes in the concentration of free cytosolic Mg 2+. These results point out that [Mg 2+] i is not modified under these three different experimental conditions.