Probing the pore region of recombinant N-methyl-D-aspartate channels using external and internal magnesium block.
Mg2+ ions block N-methyl-D-aspartate (NMDA) channels by entering the pore from either the extracellular or the cytoplasmic side of the membrane in a voltage-dependent manner. We have used these two different block phenomena to probe the structure of the subunits forming NMDA channels. We have made several amino acid substitutions downstream of the Q/R/N site in the TMII region of both NR1 and NR2A subunits. Mutant NR1 subunits were coexpressed with wild-type NR2A subunits and vice versa in Xenopus oocytes. We found that individually mutating the first two amino acid residues downstream to the Q/R/N site affects mostly the block by external Mg2+. Mutations of residues five to seven positions downstream of the Q/R/N site do not influence the external Mg2+ block, but clearly influence the block by internal Mg2+. These data add support to the hypothesis that there are two separate binding sites for external and internal Mg2+ block. They also indicate that the C-terminal end of TMII contributes to the inner vestibule of the pore of NMDA channels and thus provide additional evidence that TMII forms a loop that reemerges toward the cytoplasmic side of the membrane.
Kupper
J
J
Laboratoire de Neurobiologie, Centre National de la Recherche Scientifique Unité de Recherche Associée 1857, Ecole Normale Supéricure, Paris, France.
Ascher
P
P
Neyton
J
J
D000595:Amino Acid Sequence; D000818:Animals; D015640:Ion Channel Gating / Q000187:drug effects*; D007473:Ion Channels / Q000737:chemistry / Q000502:physiology*; D008274:Magnesium / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009865:Oocytes; D018408:Patch-Clamp Techniques; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry* / Q000502:physiology; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D007473:Ion Channels; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins; D008274:Magnesium