Free intracellular Mg2+ concentration and inhibition of NMDA responses in cultured rat neurons

1. Intracellular Mg2+ (Mg-i(2+)) blocks single-channel currents and modulat es the gating kinetics of NMDA receptors. However, previous data suggested that Mg-i(2+) inhibits whole-cell current less effectively than predicted f rom excised-patch measurements. We examined the basis of this discrepancy b y testing three hypothetical explanations. 2. To test the first hypothesis, that control of free Mg-i(2+) concentratio n ([Mg2+](i)) during whole-cell recording was inadequate, we measured [Mg2](i) using mag-indo-1 microfluorometry. The [Mg2+](i) measured in cultured neurons during whole-cell recording was similar to the pipette [Mg2+] measu red in vitro, suggesting that [Mg2+](i) was adequately controlled. 3. To test the second hypothesis, that open-channel block by Mg-i(2+) was m odified by patch excision, we characterised the effects of Mg-i(2+) using c ell-attached recordings, me found the affinity and voltage dependence of op en-channel block by Mg-i(2+) similar in cell-attached and outside-out patch es. Thus, the difference between Mg-i(2+) inhibition of whole-cell and of p atch currents cannot be attributed to a difference in Mg-i(2+) block of sin gle-channel current. 4. The third hypothesis tested was that the effect of Mg-i(2+) on channel g ating was modified by patch excision. Results of cell-attached recording an d modelling of whole-cell data suggest that the Mg-i(2+)-induced stabilisat ion of the channel open state is four times weaker after patch excision tha n in intact cells. This differential effect of Mg-i(2+) on channel gating e xplains why Mg-i(2+) inhibits whole-cell NMDA responses less effectively th an patch responses.