Functional expression of distinct NMDA channel subunits tagged with green fluorescent protein in hippocampal neurons in culture.
We generated expression vectors for N-terminally green fluorescent protein -tagged NR2A and NR2B subunits (GFP-NR2A and GFP-NR2B). Both constructs expressed GFP and formed functional NMDA channels with similar properties to untagged controls when co-transfected with NR1 subunit partner in HEK293 cells. Primary cultured hippocampal neurons were transfected at five days in vitro with these vectors. Fifteen days after transfection, well-defined GFP clusters were observed for both GFP-NR2A and GFP-NR2B subunits being co-localized with endogenous NR1 subunit. Whole-cell recordings showed that the GFP-NR2A subunit determined the decay of NMDA-mediated miniature spontaneous excitatory postsynaptic currents (NMDA-mEPSCs) in transfected neurons. Live staining with anti-GFP antibody demonstrated the surface expression of GFP-NR2A and GFP-NR2B subunits that was partly co-localized a presynaptic marker. Localization of NMDA receptor clusters in dendrites was studied by co-transfection of CFP-actin and GFP-NR2 subunits followed by anti-GFP surface staining. Within one week after plating most surface NMDAR clusters were distributed on dendritic shafts. Later in development, a large portion of surface clusters for both GFP-NR2A and GFP-NR2B subunits were clearly localized at dendritic spines. Our report provides the basis for studies of NMDA receptor location together with dendritic dynamics in living neurons during synaptogenesis in vitro.
Luo
J-H
JH
Department of Physiology and Biophysics, Georgetown University School of Medicine, 3900 Reservoir Road NW, Washington, DC 20007, USA.
Fu
Z-Y
ZY
Losi
G
G
Kim
B G
BG
Prybylowski
K
K
Vissel
B
B
Vicini
S
S
D000818:Animals; D000831:Animals, Newborn; D002460:Cell Line; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents / Q000032:analysis; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics*; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D013194:Staining and Labeling; D014162:Transfection / Q000379:methods
D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D016194:Receptors, N-Methyl-D-Aspartate; C121001:NR2B NMDA receptor