Splice‐isoform specific immunolocalization of neuronal nitric oxide synthase in mouse and rat brain reveals that the PDZ‐complex‐building nNOSα β‐finger is largely exposed to antibodies

Abstract

Knock out mice deficient for the splice-isoform alphaalpha of neuronal nitric oxide synthase (nNOSalphaalpha) display residual nitric oxide synthase activity and immunosignal. To attribute this signal to the two minor neuronal nitric oxide synthase splice variants, betabeta and gammagamma, we generated isoform-specific anti-peptide antibodies against the nNOSalphaalpha specific betabeta-finger motif involved in PDZ domain scaffolding and the nNOSbetabeta specific N-terminus. The nNOSalphaalpha betabeta-finger-specific antibody clearly recognized the 160-kDa band of recombinant nNOSalphaalpha on Western blots. Using immunocytochemistry, this antibody displayed, in rats and wild-type mice, a labeling pattern similar to but not identical with that obtained using a commercial pan-nNOS antibody. This similarity indicates that the majority of immunocytochemically detectable nNOS is not likely to be complexed with PDZ-domain proteins via the betabeta-finger motif. This conclusion was confirmed by the inhibition of PSD-95/nNOS interaction by the nNOSalphaalpha betabeta-finger antibody in pull-down assays. By contrast, nNOSalphaalpha betabeta-finger labeling was clearly reduced in hippocampal and cortical neuropil areas enriched in NMDA receptor complex containing spine synapses. In nNOSalphaalpha knock out mice, nNOSalphaalpha was not detectable, whereas the pan-nNOS antibody showed a distinct labeling of cell bodies throughout the brain, most likely reflecting betabeta/gammagamma-isoforms in these cells. The nNOSbetabeta antibody clearly detected bacterial expressed nNOSbetabeta fusion protein and nNOSbetabeta in overexpressing HEK cells by Western blotting. Immunocytochemically, individual cell bodies in striatum, cerebral cortex, and in some brain stem nuclei were labeled in knock out but not in wild-type mice, indicating an upregulation of nNOSbetabeta in nNOSalphaalpha deficient animals.

Identification of alternate transcripts of neuronal nitric oxide synthase in the mouse retina

“…Furthermore, the large increase in lightstimulated NO-induced fluorescence in the IPL was seen in apparent synaptic boutons, and nNOSa is associated with PSD-95 at the synapse by virtue of its PDZ domain (Brenman et al, 1996), suggesting that nNOSa was responsible for the NO-induced fluorescence seen in puncta of the IPL. In addition, much of the somatic NO-induced fluorescence could also have come from nNOSa, in that a more recent study found that only 10% of nNOSa is actually associated with PSD-95 (Langnaese et al, 2007). However, it is still possible that nNOSg was contributing to the observed NO-induced fluorescence for several reasons.…”

Concepts of neural nitric oxide‐mediated transmission

“…Other splice variants also exist, namely nNOSb and nNOSc, both of which lack the amino terminal PDZ domain (Brenman et al, 1996). nNOSc has little or no enzymatic activity but nNOSb is active and is upregulated in the striatum and cortex in mice lacking the nNOSa isoform (Eliasson et al, 1997;Langnaese et al, 2007), which probably accounts for the relatively mild phenotype of such animals compared to ones lacking the b and c variants as well (Gyurko et al, 2002).…”

PDZ Domains as Drug Targets

“…c) In ZO-1 PDZ2, the domain folds from two nonfunctional monomeric domains where βB and βC of the two domains swap in order to complete the fold into the functional dimeric PDZ tandem. [44,45] In addition to the carboxylate-dependent binding of PDZ domains, it has more recently been demonstrated that PDZ domains can also bind internal peptide motifs [42,[46][47][48][49] and phospholipids. e) PDZ domain ligands can also bind using internal motifs where it usually still relies on the insertion of a hydrophobic residue into the binding pocket and either hydrophobic or hydrogen bond interaction coordination of the P −2 , but the interaction also relies on the P +1 or P +2 residue being negatively charged (Glu/Asp), using the carboxylic acid as substitute for the C-terminal.…”