Most fast excitatory synaptic transmission in the nervous system is mediated by 
glutamate acting through ionotropic glutamate receptors (iGluRs). iGluRs (AMPA, 
kainate, and NMDA receptor subtypes) are tetrameric assemblies, formed as a 
dimer of dimers. Still, the mechanism underlying tetramerization--the necessary 
step for the formation of functional receptors that can be inserted into the 
plasma membrane--is unknown. All eukaryotic compared to prokaryotic iGluR 
subunits have an additional transmembrane segment, the M4 segment, which 
positions the physiologically critical C-terminal domain on the cytoplasmic side 
of the membrane. AMPA receptor (AMPAR) subunits lacking M4 do not express on the 
plasma membrane. Here, we show that these constructs are retained in the 
endoplasmic reticulum, the major cellular compartment mediating protein 
oligomerization. Using approaches to assay the native oligomeric state of AMPAR 
subunits, we find that subunits lacking M4 or containing single amino acid 
substitutions along an "interacting" face of the M4 helix that block surface 
expression no longer tetramerize in either homomeric or heteromeric assemblies. 
In contrast, subunit dimerization appears to be largely intact. These 
experiments define the M4 segment as a unique functional unit in AMPARs that is 
required for the critical dimer-to-tetramer transition.