Molecular determinants of multiple effects of Ni2+ on NMDA receptor channels

The N-methyl-D-aspartate receptor (NR) is a ligand-gated channel with unique properties, such as requirements for two different agonists, glutamate and glycine, Mg-mediated voltage-dependence and high calcium permeability, as well as several recognition sites for endogenous and exogenous modulators. NRs form tetrameric channels containing two copies each of NR1 and NR2 (A, B, C or D) subunits, of which the commonest are NR2A and NR2B. The transition metal nickel (Ni) affects the NR channel activity in a voltage-dependent (Mg-like) manner, but also causes enhancement of activity of NR2B-containing channels and voltage-independent inhibition of those containing NR2A. This work was aimed to identify the critical residues for Ni interaction on the NR2A and NR2B subunit by expressing wild-type and mutated NR1a-NR2A and NR1a-NR2B receptors in either HEK293 cells or Xenopus laevis oocytes and measuring the response to saturating doses of agonists. The voltage-dependent block was similar in the two channel types, had an apparent IC50 between 40 and 60 µM at -60 mV, and was completely abolished by the single mutation N616G in NR2B subunit, a residue located in the narrow constriction of the channel (N+1 site). This mutation also enhanced the facilitation and unmasked a voltage-independent inhibition with IC50 > 300 µM. The voltage-independent inhibition was more prominent in NR1a-NR2A channels with IC50 close to 80 µM. When NR2 subunits deleted for their entire amino terminal domain (ATD) were expressed with wild-type NR1a subunit, voltage-independent inhibition was not modified, but, in NR1a-NR2B channels, the potentiation effect was abolished. In the latter channels, potentiation of the current was drastically reduced also by H127A, D101A, D104A point mutations and by the double mutation H127AD101A, all located in lobe I of NR2B ATD, and to a lesser extent by the point mutation T233A located in lobe II of NR2B ATD, suggesting that the interaction site that causes potentiation shares common determinants with the Zn and ifenprodil binding sites. In contrast, as the binding site responsible for voltage-independent block of NR2A-containing channels is not located in the ATD, we postulate the existence of an additional divalent cation binding site in the M3-M4 extracellular loop. Indeed, the point mutation H801A in the NR2A subunit caused an important reduction of the block, with a 8-time increase in IC50. The block was also partially decreased by H705A and H709A mutations in the same region of NR1a. This additional binding site can be responsible for specific heavy metal interaction with NR channels.