Distribution of an NMDA receptor : GFP fusion protein in sensory neurons is altered by a C-terminal construct

The NMDA receptor plays an important role in mediating sensory input to the spinal cord. Domains within the C-terminus of the NMDA receptor bind to cy toskeletal proteins and facilitate membrane targeting and synaptic clusteri ng, and may participate in regulation of receptor function. One strategy to manipulate NMDA receptor function is to express C-terminal constructs in n eurons to disrupt synaptic clustering via competition for binding motifs in cytosketetal proteins and postsynaptic densities. Biolistic particle-media ted gene transfer was used to deliver plasmid DNA into organotypic cultures of dorsal root ganglia (DRG). Fusion proteins consisting of recombinant (r )NMDA receptor subunit 1-1 (rNRI-I) deletion constructs and enhanced green fluorescent protein (GFP) were expressed in sensory neurons and demonstrate d unique distribution patterns within the cell. Expression of the full leng th rNR1-1:GFP construct was cytosolic and localized to membranous patches s imilar to endogenous NR1-1 protein expression in sensory neurons. Expressio n of a construct containing only the C-terminus, GFP:C0C1C2, demonstrated n uclear and membranous. localization. When the GFP:C0C1C2 construct was co e xpressed with rNR1-1 in sensory neurons, membranous localization of rNR1-1 was disrupted. In contrast, co-expression of a C-terminal cassette lacking the C1 exon cassette, GFP:C0C2, with rNRI-I did not alter the membranous di stribution of rNR1-1. This observation verifies the utility of a gene trans fer strategy to diminish membranous NR1-1 content by expressing a construct containing the C1 exon cassette.