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

1 Intracellular Mg2+ (Mg2+i) blocks single‐channel currents and modulates the gating kinetics of NMDA receptors. However, previous data suggested that Mg2+i inhibits whole‐cell current less effectively than predicted from excised‐patch measurements. We examined the basis of this discrepancy by testing three hypothetical explanations. 2 To test the first hypothesis, that control of free Mg2+i concentration ([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+] measured in vitro, suggesting that [Mg2+]i was adequately controlled. 3 To test the second hypothesis, that open‐channel block by Mg2+i was modified by patch excision, we characterised the effects of Mg2+i using cell‐attached recordings. We found the affinity and voltage dependence of open‐channel block by Mg2+i similar in cell‐attached and outside‐out patches. Thus, the difference between Mg2+i inhibition of whole‐cell and of patch currents cannot be attributed to a difference in Mg2+i block of single‐channel current. 4 The third hypothesis tested was that the effect of Mg2+i on channel gating was modified by patch excision. Results of cell‐attached recording and modelling of whole‐cell data suggest that the Mg2+i‐induced stabilisation of the channel open state is four times weaker after patch excision than in intact cells. This differential effect of Mg2+i on channel gating explains why Mg2+i inhibits whole‐cell NMDA responses less effectively than patch responses.