Rules of engagement for NMDA receptor subunits.
Canonical NMDA receptors assemble from two glycine-binding NR1 subunits with two glutamate-binding NR2 subunits to form glutamate-gated excitatory receptors that mediate synaptic transmission and plasticity. The role of glycine-binding NR3 subunits is less clear. Whereas in Xenopus laevis oocytes, two NR3 subunits coassemble with two NR1 subunits to form a glycine-gated receptor, such a receptor has yet to be found in mammalian cells. Meanwhile, NR1, NR2, and NR3 appear to coassemble into triheteromeric receptors in neurons, but it is not clear whether this occurs in oocytes. To test the rules that govern subunit assembly in NMDA receptors, we developed a single-molecule fluorescence colocalization method. The method focuses selectively on the plasma membrane and simultaneously determines the subunit composition of hundreds of individual protein complexes within an optical patch on a live cell. We find that NR1, NR2, and NR3 follow an exclusion rule that yields separate populations of NR1/NR2 and NR1/NR3 receptors on the surface of oocytes. In contrast, coexpression of NR1, NR3A, and NR3B yields triheteromeric receptors with a fixed stoichiometry of two NR1 subunits with one NR3A and one NR3B. At least part of this regulation of subunit stoichiometry appears to be caused by internal retention. Thus, depending on the mixture of subunits, functional receptors on the cell surface may follow either an exclusion rule or a stoichiometric combination rule, providing an important constraint on functional diversity. Cell-to-cell differences in the rules may help sculpt distinct physiological properties.
Ulbrich
Maximilian H
MH
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
Isacoff
Ehud Y
EY
D000818:Animals; D001369:Axons / Q000378:metabolism; D005260:Female; D017930:Genes, Reporter / Q000235:genetics; D009010:Monte Carlo Method; D009865:Oocytes; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate
Brain Res Mol Brain Res. 2002 Apr 30;100(1-2):43-52
12008020
J Neurosci. 1995 Oct;15(10):6509-20
7472413
J Pharmacol Exp Ther. 2007 Aug;322(2):739-48
17502428
Nature. 2002 Feb 14;415(6873):793-8
11823786
J Neurosci. 2006 Apr 26;26(17):4559-66
16641235
Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12759-64
12228726
J Neurosci. 2003 Nov 5;23(31):10064-73
14602821
J Gen Physiol. 1997 Nov;110(5):485-502
9348322
Nature. 2005 Nov 10;438(7065):185-92
16281028
J Neurosci. 1995 Oct;15(10):6498-508
7472412
Sci STKE. 2004 Oct 19;2004(255):re16
15494561
J Neurosci. 2001 Feb 15;21(4):1228-37
11160393
Nat Methods. 2007 Apr;4(4):319-21
17369835
Nature. 1998 May 28;393(6683):377-81
9620802
J Neurosci. 2001 Dec 1;21(23):RC185
11717388
Nat Biotechnol. 2004 Dec;22(12):1567-72
15558047
J Neurophysiol. 2002 Apr;87(4):2052-63
11929923
J Neurosci. 1998 Apr 15;18(8):2954-61
9526012
Nature. 1987 Feb 5-11;325(6104):529-31
2433595