Both postsynaptic density (PSD) and postsynaptic membrane rafts (PSR) are isolated separately. However, PSD and PSR are considered to make a complex in vivo (Suzuki et al., J. Neurochem. 2011, Liu et al., J. Neurogenetics, 2013). To elucidate the more detailed structural relationship between PSD and PSR, we investigated purification process of PSD and PSR from rat forebrain synaptic plasma membrane (SPM) using three different detergents, TX-100, n-octyl beta-D-glucoside (OG) and CHAPSO at varied concentrations, and examined the distribution of subsynaptic structures and PSD proteins (both type I and type II) on sucrose density gradient by SDS-PAGE, EM and western blotting. This type of systematic examination has not been carried out before.
Three types of detergents used showed distinct separation profiles of the synaptic subdomains. After TX-100 treatment, type I PSD was recovered in two fractions: pellet (fraction 12) and insoluble fraction 8, the latter of which contained partially broken membrane raft-PSD complex. Conventional PSD prepared after TX-100 treatment was suggested to be a mixture of these two types of type I PSD pools (pellet and fraction 8). It was found that the conventional PSD does not contain inhibitory type II PSDs. Association of type I PSD with PSR was identified in the TX-100-treatment but not in the OG-treatment. Association of type II PSD with membrane rafts was suggested in OG- and CHAPSO-treatments but not in the TX-100-treatment. Treatment with relatively high concentration of detergents, in particular OG, solubilized the type I PSD proteins. CHAPSO-treatment produced various novel fractions containing unique subsynaptic structures. Besides pellet/fraction 12, novel PSD-containing structures were isolated in an insoluble fraction 11. Tow pools of GluA were identified in the synaptic region in the OG- and CHAPSO-treatments; one was associated with type I PSD and the other possibly with membrane rafts.
The knowledge obtained in this study is useful in understanding the studies using isolated PSDs and PSRs. Furthermore, novel subsynaptic structures obtained in this study may become useful materials for future analyses to understand the organization of synapses at the molecular level.