EtOH modifies the biophysical properties of cellular membranes and redistributes GluR1 to microdomains. (
a) Representative immunofluorescent images showing EtOH facilitated a rapid (2 min) redistribution of the microdomains (GM1+, red) into enlarged clusters along dendrites, simultaneously resulted in redistributions of GluR1 (green) with clusters located to GM1+ microdomains (arrowheads indicate GM1/GluR1-co-localized microdomains). ( b) Size of individual GM1+ microdomains, and ( c) number of GluR1 located to GM1+ microdomains ( n=276–440 microdomains from a minimum of 21 dendrites and 3 separate cultures per condition). ( d) Representative immunoblots showing detergent-resistant membrane microdomains isolated by density centrifugation containing a membrane microdomain-enriched protein Flotillin (largely located to the more buoyant lipid-rich fractions, 1–3), a non-microdomain protein Transferrin (largely located outside of membrane microdomains, fractions 7–9) and GluR1. EtOH (2 min) induced a redistribution of GluR1 to membrane microdomains. ( e) Representative immunoblots of surface biotin protein labeling and immunoprecipitation with subsequent immunoblotting for GluR1 or GluR2. ( f) Quantitative analysis of immunoblots showing that EtOH specifically increased surface GluR1 but not total GluR1. Data are presented as mean±s.d. EtOH, ethanol. * P<0.05, *** P<0.001 compared with vehicle; # P<0.05, ### P<0.001 compared with EtOH. Scale bar, 20 μm.