Mechanisms of glutamate‐stimulated Mg2+ influx and subsequent Mg2+ efflux in rat forebrain neurones in culture.
Authors: Stout A K , Li-Smerin Y , Johnson J W , Reynolds I J 
Published: December 2014

1. Mag‐fura‐2 fluorescence microscopy and whole‐cell patch‐clamp recordings were used to measure glutamate‐induced changes in the intracellular free Mg2+ concentration ([Mg2+]i) and Mg2+ currents, respectively, in cultured forebrain neurones from fetal rats in the absence of extracellular Na+ (Nao+) and Ca2+ (Cao2+). 2. Increasing the extracellular Mg2+ concentration ([Mg2+]o) from 9 to 70 mM significantly enhanced the maximum [Mg2+]i induced by a 5 min 100 microM glutamate plus 1 microM glycine stimulation ([Mg2+]i,5 min) from 2.04 +/‐ 0.07 to 2.98 +/‐ 0.20 mM. Increasing [Mg2+]o from 9 to 70 mM also significantly enhanced the initial rate of rise in [Mg2+]i upon glutamate stimulation from 0.41 +/‐ 0.02 to 0.81 +/‐ 0.08 mM min‐1. 3. The glutamate‐stimulated increase in [Mg2+]i was not altered by prior depletion of intracellular free Na+ (Nai+). For paired stimulations in single neurones, the mean [Mg2+]i,5 min was 1.95 +/‐ 0.17 mM under Na(+)‐depleted conditions and 1.94 +/‐ 0.16 mM under control conditions. 4. The glutamate‐stimulated increase in [Mg2+]i was significantly reduced when NMDA channel‐permeant Cs+ or K+ ions were used as the Na+ substitute instead of the presumably NMDA channel‐impermeant ions N‐methyl‐D‐glucamine (NMDG), Tris or sucrose. The mean [Mg2+]i,5 min was 0.56 +/‐ 0.06 and 0.74 +/‐ 0.08 mM in the presence of Cs+ or K+, respectively, compared with 2.13 +/‐ 0.10, 1.93 +/‐ 0.11 and 2.07 +/‐ 0.22 mM in the presence of NMDG, Tris or sucrose, respectively. 5. In whole‐cell recordings performed with Cs+ as the primary intracellular cation, application of 100 microM NMDA plus 10 microM glycine induced inward currents that reversed around ‐55 mV in an extracellular solution containing 70 mM Mg2+ and 31 mM NMDG as the only cations. The currents were reversibly inhibited by DL‐2‐amino‐5‐phosphonovaleric acid (APV). In an extracellular solution containing 2 mM Mg2+ and 140 mM NMDG, NMDA plus glycine activated outward currents at potentials more depolarized than ‐90 mV. 6. In whole‐cell recordings made with NMDG as the principal cation in the patch pipette, application of NMDA plus glycine in the 70 mM Mg2+ extracellular solution induced inward currents at voltages more negative than +15 mV. The ratio of the current measured under these conditions to the current measured in an extracellular solution containing Na+ as the principal cation (0.073:1) was nearly constant from cell to cell. 7. Following a 5 min glutamate stimulation in the presence of 9 mM Mg2+, [Mg2+]i returned to basal levels at a mean rate of 58.1 +/‐ 2.1 microM min‐1. Complete removal of Nao+ significantly inhibited the rate of recovery to 31% of control. Raising [Mg2+]o to 30 mM in combination with removal of Nao+ did not inhibit recovery significantly more than either manipulation alone (28% of control). 8. These results suggest that glutamate‐stimulated increases in [Mg2+]i that occur in the absence of Nao+ and Cao2+ result from Mg2+ entry through NMDA‐activated ion channels. Furthermore, recovery from a glutamate‐induced Mg2+ load appears to be primarily due to Mg2+ efflux via a mechanism whose characteristics are consistent with Na(+)‐Mg2+ exchange.