The spatiotemporal regulation of neurotransmitter transporters involves proteins that interact with their intracellular domains. Using a proteomic approach, we identified several proteins that interact with the C terminus of the serotonin transporter (SERT). These included neuronal nitric oxide synthase (nNOS), a PSD-95/ Disc large/ZO-1 (PDZ) domain-containing protein recruited by the atypical PDZ binding motif of SERT. Coexpression of nNOS with SERT in HEK293 cells decreased SERT cell surface localization and 5-hydroxytryptamine (5-HT) uptake. These effects were absent in cells transfected with SERT mutated in its PDZ motif to prevent physical association with nNOS, and 5-HT uptake was unaffected by activation or inhibition of nNOS enzymatic activity. 5-HT uptake into brain synaptosomes was increased in both nNOS-deficient and wild-type mice i.v. injected with a membrane-permeant peptidyl mimetic of SERT C terminus, which disrupted interaction between SERT and nNOS, suggesting that nNOS reduces SERT activity in vivo. Furthermore, treating cultured mesencephalic neurons with the mimetic peptide similarly increased 5-HT uptake. Reciprocally, indicating that 5-HT uptake stimulates nNOS activity, NO production was enhanced on exposure of cells cotransfected with nNOS and SERT to 5-HT. This effect was abolished by 5-HT uptake inhibitors and absent in cells expressing SERT mutated in its PDZ motif. In conclusion, physical association between nNOS and SERT provides a molecular substrate for their reciprocal functional modulation. In addition to showing that nNOS controls cell surface localization of SERT, these findings provide evidence for regulation of cellular signaling (NO production) by a substrate-carrying transporter.PDZ ͉ proteomic ͉ serotonin uptake S erotonin plays a major role in the regulation of mood, cognition, and motor behavior, and a disruption of serotonergic transmission is implicated in several pathophysiological states, including affective disorders. The activity of serotonergic pathways is critically regulated by the reuptake of 5-HT via the plasma membrane serotonin transporter (SERT), a member of the Na ϩ /Cl Ϫ -dependent transporter family (SLC6) (1, 2). SERT is of major pharmacological and clinical interest inasmuch as it represents the primary target of several widely prescribed antidepressants, including the selective [5-hydroxytryptamine (5-HT)] reuptake inhibitors, citalopram and paroxetine (2-4). Moreover, altered SERT expression or function has been suggested not only in depression, but also in anxious and obsessive-compulsive states, disorders that can likewise be improved by treatment with selective 5-HT reuptake inhibitors (4-6).Over the last 10 years, it has become evident that monoaminergic and other classes of plasma membrane transporters are not isolated proteins ''floating'' within the plasma membrane, but rather are components of protein complexes. These generally incorporate an oligomer (possibly formed of dimers) of the transporter that is physically associated with several i...

Expression of the calcium channel Ca V 2.2 is markedly suppressed by coexpression with truncated constructs of Ca V 2.2. Furthermore, a two-domain construct of Ca V 2.1 mimicking an episodic ataxia-2 mutation strongly inhibited Ca V 2.1 currents. We have now determined the specificity of this effect, identified a potential mechanism, and have shown that such constructs also inhibit endogenous calcium currents when transfected into neuronal cell lines. Suppression of calcium channel expression requires interaction between truncated and full-length channels, because there is inter-subfamily specificity. Although there is marked cross-suppression within the Ca V 2 calcium channel family, there is no cross-suppression between Ca V 2 and Ca V 3 channels. The mechanism involves activation of a component of the unfolded protein response, the endoplasmic reticulum resident RNA-dependent kinase (PERK), because it is inhibited by expression of dominant-negative constructs of this kinase. Activation of PERK has been shown previously to cause translational arrest, which has the potential to result in a generalized effect on protein synthesis. In agreement with this, coexpression of the truncated domain I of Ca V 2.2, together with full-length Ca V 2.2, reduced the level not only of Ca V 2.2 protein but also the coexpressed ␣2␦-2. Thapsigargin, which globally activates the unfolded protein response, very markedly suppressed Ca V 2.2 currents and also reduced the expression level of both Ca V 2.2 and ␣2␦-2 protein. We propose that voltage-gated calcium channels represent a class of difficult-to-fold transmembrane proteins, in this case misfolding is induced by interaction with a truncated cognate Ca V channel. This may represent a mechanism of pathology in episodic ataxia-2.

The charged multivesicular body proteins (Chmp1-7) are an evolutionarily conserved family of cytosolic proteins that transiently assembles into helical polymers that change the curvature of cellular membrane domains. Mutations in human CHMP2B cause frontotemporal dementia, suggesting that this protein may normally control some neuron-specific process. Here, we examined the function, localization, and interactions of neuronal Chmp2b. The protein was highly expressed in mouse brain and could be readily detected in neuronal dendrites and spines. Depletion of endogenous Chmp2b reduced dendritic branching of cultured hippocampal neurons, decreased excitatory synapse density in vitro and in vivo, and abolished activity-induced spine enlargement and synaptic potentiation. To understand the synaptic effects of Chmp2b, we determined its ultrastructural distribution by quantitative immuno-electron microscopy and its biochemical interactions by coimmunoprecipitation and mass spectrometry. In the hippocampus in situ, a subset of neuronal Chmp2b was shown to concentrate beneath the perisynaptic membrane of dendritic spines. In synaptoneurosome lysates, Chmp2b was stably bound to a large complex containing other members of the Chmp family, as well as postsynaptic scaffolds. The supramolecular Chmp assembly detected here corresponds to a stable form of the endosomal sorting complex required for transport-III (ESCRT-III), a ubiquitous cytoplasmic protein complex known to play a central role in remodeling of lipid membranes. We conclude that Chmp2b-containing ESCRT-III complexes are also present at dendritic spines, where they regulate synaptic plasticity. We propose that synaptic ESCRT-III filaments may function as a novel element of the submembrane cytoskeleton of spines.