Role of PSD95 in membrane association and catalytic activity of nNOSα in nitrergic varicosities in mice gut

Abstract

We have recently shown that membrane association of neuronal nitric oxide synthase-α (nNOSα) is critical in the regulation of synthesis of NO during nitrergic neurotransmission. The purpose of this study was to examine the role of the synapse-associated proteins (SAPs) in membrane association of nNOSα. Varicosities (swellings on terminal axons) were isolated from mice gastrointestinal tract and examined for nNOSα, postsynaptic density protein 95 (PSD95), and membrane interactions by coimmunoprecipitation and SDS-PAGE. Our results show that PSD95 protein was present in the membrane fraction of the nerve varicosity, whereas both PSD95 and SAP97 were present in the cytosol. nNOSα was associated with PSD95 but not SAP97. nNOSα-PSD95 complex was bound to the membrane via palmitoylation of PSD95. Depalmitoylation of PSD95 with 2-bromopalmitate dislocates nNOSα and PSD95 from the varicosity membrane and abolishes NO production. These studies show that palmitoylation of PSD95 anchors nNOSα to the varicosity membrane and that it is obligatory for NO production by the enzyme. Palmitoylation of PSD95 may provide a novel target for regulation of nitrergic neurotransmission.