Molecular determinants of multiple effects of nickel on NMDA receptor channels.
Nickel (Ni2+) is a toxic metal that affects the function of several neuronal ionic channels. Ni2+ inhibits N-methyl-D: -aspartate receptor (NR) channel in a voltage-dependent manner, but also causes enhancement of NR2B-containing channel activity and voltage-independent inhibition of those containing NR2A. The present work was aimed to find the sites of Ni2+ interaction on the NR2A and NR2B subunits by expressing wild-type and mutated NRs in either HEK293 cells or Xenopus laevis oocytes. The point mutation N616G in the pore region of the NR2B subunit completely removed the voltage-dependent block. In NR2 subunits deleted for their entire amino terminal domain (ATD) and expressed with wild-type NR1 subunit, voltage-independent inhibition of NR2A-containing channels was not modified, but the potentiation effect was abolished in NR2B-containing channels. In the latter channels, potentiation of the current was also removed by H127A, D101A, D104A point mutations and by the double mutation H127AD101A, all located in lobe I of ATD, and reduced by the point mutation T233A in lobe II, suggesting that the interaction site that causes potentiation shares common determinants with the Zn2+ and ifenprodil binding sites. In contrast, in NR2A-containing channels, we postulate the existence of an additional divalent cation binding site in the M3-M4 extracellular loop. In these channels, the point mutation H801A in the NR2A subunit caused an important reduction of the voltage-independent block, with a 7-time increase in IC(50). The block was also partially, but not as prominently, reduced by the double mutation H705AH709A in the same region of NR1. This additional binding site can be responsible of specific heavy metal interaction with NR channels.
Gavazzo
Paola
P
Istituto di Biofisica, Consiglio Nazionale delle Ricerche, via De Marini, 6, 16149 Genoa, Italy.
Guida
Patrizia
P
Zanardi
Ilaria
I
Marchetti
Carla
C
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D055592:Biophysical Phenomena / Q000187:drug effects; D002461:Cell Line, Transformed; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation / Q000379:methods; D018698:Glutamic Acid / Q000494:pharmacology; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects* / Q000235:genetics; D008958:Models, Molecular; D009532:Nickel / Q000494:pharmacology*; D009865:Oocytes; D018408:Patch-Clamp Techniques / Q000379:methods; D010880:Piperidines / Q000378:metabolism / Q000494:pharmacology; D017354:Point Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics* / Q000378:metabolism; D013096:Spermine / Q000494:pharmacology; D014131:Trace Elements / Q000494:pharmacology*; D014162:Transfection / Q000379:methods; D014981:Xenopus; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
C120997:NR2A NMDA receptor; C121001:NR2B NMDA receptor; D010880:Piperidines; D016194:Receptors, N-Methyl-D-Aspartate; D014131:Trace Elements; D013096:Spermine; D018698:Glutamic Acid; D009532:Nickel; D015032:Zinc; C010739:ifenprodil