Role of PSD 95 in membrane association and catalytic activity of nNOS α in nitrergic 1 varicosities in mice gut 2 3

23 We have recently shown that membrane association of nNOSα is critical in the regulation 24 of synthesis of nitric oxide (NO) during nitrergic neurotransmission. The purpose of this 25 study was to examine the role of the synapse associated proteins in membrane association 26 of nNOSα. Varicosities (swellings on terminal axons) were isolated from mice 27 gastrointestinal tract and examined for nNOSα, PSD95 and membrane interactions by co28 immunoprecipitation and SDS-PAGE. Our results show that PSD95 protein was present 29 in the membrane fraction of the nerve varicosity, whereas both PSD95 and SAP97 were 30 present in the cytosol. nNOSα was associated with PSD95 but not SAP97. nNOSα31 PSD95 complex was bound to the membrane via palmitoylation of PSD95. 32 Depalmitoylation of PSD95 with 2-bromopalmitate dislocates nNOSα and PSD95 from 33 the varicosity membrane and abolishes nitric oxide (NO) production. These studies show 34 that palmitoylation of PSD95 anchors nNOSα to the varicosity membrane and that is 35 obligatory for NO production by the enzyme. Palmitoylation of PSD95 may provide a 36 novel target for regulation of nitrergic neurotransmission. 37 38