Permeation and block of N‐methyl‐D‐aspartic acid receptor channels by divalent cations in mouse cultured central neurones.

Abstract

1. Spinal cord and hippocampal neurones in cell culture were voltage clamped using the tight-seal, whole-cell recording technique. The concentration of sodium and a series of divalent cations in the extracellular media was varied to study permeation through excitatory amino acid receptor channels activated by the selective agonists N-methyl-D-aspartic acid (NMDA), kainic acid and quisqualic acid. 2. On raising the extracellular calcium concentration, with [Na+]o held constant at 105 mM, the reversal potential of responses to NMDA shifted in the depolarizing direction. This shift was adequately described by the extended constant-field equation over the range 0.3-50 mM-calcium. Using ionic activity coefficients we calculate a value of PCa/PNa = 10.6. Under the same experimental conditions the reversal potential of responses to kainic and quisqualic acids was much less affected by raising the calcium concentration, such that PCa/PNa = 0.15. A depolarizing shift of the NMDA reversal potential was also recorded during application of 20 mM-barium, strontium or manganese, suggesting permeation of these ions. The permeability sequence was Ca2+ > Ba2+ > Sr2+ .mchgt. Mn2+. No depolarizing shift of the NMDA reversal potential occurred during application of 20 mM-cobalt, magnesium or nickel. 3. In experiments in which the extracellular Na+ concentration was varied the extended constant-field equation was adequate in predicting shifts of the NMDA reversal potential recorded on varying [Na+]o over the range 50-150 mM, but failed to accurately predict the reversal potential of responses to NMDA with 10 mM-[Ca2+]o and only 10 or 20 mM-[Na+]o. These results imply an apparent increase in PCa/PNa on lowering [Na+]o and may result from interaction of permeant ions within the channel. Barium and to a lesser extent calcium, but not strontium (all 20 mM), reduced the slope conductance of responses to NMDA recorded within .+-. 15 mV of the reversal potential; over this limited range of membrane potential the current-voltage relationship remained linear in the presence of each of these ions. In contrast manganese produced a strong, voltage-dependent block of responses to NMDA, similar to that produced by magnesium, such that even close to the reversal potential the NMDA current-voltage relationship was highly non-linear. Thus manganese both permeates and blocks the NMDA receptor channel. 5. Raising the extracellular calcium concentration, from 0. 1 to 5 mM, had two effects on the conductance mechanism activated by NMDA. Over the membrane potential range -10 to -100 mV the NMDA-activated conductance was reduced; however, at hyperpolarized membrane potentials, from -100 to -200 mV, this increase in [Ca2+]o produced a voltage-dependent relief of the blocking action of Mg2+. 6. An N-shaped current-voltage relationship for responses to NMDA, with a limb of positive slope conductance from -150 to -200 mV, recorded in the presence of 0.1 mM-Ca2+ and micromolar amounts of extracellular Mg2+, suggests that even magnesium can permeate through NMDA receptor channels under conditions of extreme hyperpolarization. 7. Our results suggest that there is a much larger calcium flux through NMDA-than through kainate- or quisqualate-activated receptor channels. The interaction of Ca2+ and Mg2+ ions, as well as interactions between these divalent cations and permeant monovalent ions, are likely to be important factors in determining the physiological role of NMDA receptors in central synaptic transmission.