Topic: synaptic membranes

α2δ proteins serve as auxiliary subunits of voltage-gated calcium channels (CaV) and regulate the formation and differentiation of synapses. To comparatively study the neuronal localization of the four α2δ isoforms (α2δ-1 to -4), we previously established hemagglutinin (HA) tagging, which allows the surface detection with anti-HA antibody labeling. However, localizing α2δ proteins in synaptic membranes is limited because the synaptic cleft is densely packed with trans-synaptic and extracellular matrix proteins. Thus, classical antibodies have restricted access to extracellular epitopes. Here,

We have developed a protocol to isolate synaptic and extrasynaptic membranes from post-mortem human cortex. This allowed us to characterize the distribution of the major brain NMDAR subunits, GluN2B, GluN2A, GluN1 and GluN3A in controls and AD samples. We used specific antibodies and western blots to analyse the level and phosphorylation of the NMDAR subunits. We have found that GluN2B, GluN2A, GluN1 are predominantly at synaptic membranes, while GluN3A is more abundant at extrasynaptic membranes. When the synaptic distribution was analysed in AD fractions, GluN2B and GluN2A showed lower level