Genetic variants of Neuregulin 1 (NRG1) and its neuronal tyrosine kinase receptor ErbB4 are associated with risk for schizophrenia, a neurodevelopmental disorder characterized by excitatory/inhibitory imbalance and dopamine (DA) dysfunction. To date, most ErbB4 studies focused on GABAergic interneurons in the hippocampus and neocortex, particularly fast-spiking parvalbumin-positive (PV+) basket cells. However, NRG has also been shown to modulate DA levels, suggesting a role for ErbB4 signaling in dopaminergic neuron function. Here we report that ErbB4 in midbrain DAergic axonal projections regulates extracellular DA levels and relevant behaviors. Mice lacking ErbB4 in tyrosine hydroxylase-positive (TH+) neurons, but not in PV+ GABAergic interneurons, exhibit a dual imbalance of basal DA levels and fail to increase DA in response to local NRG1 infusion into the dorsal hippocampus, medial prefrontal cortex and dorsal striatum by reverse microdialysis. Using Lund Human Mesencephalic (LUHMES) cells, we show that NRG/ErbB signaling increases extracellular DA levels, at least in part, by reducing DA transporter (DAT)-dependent uptake. Interestingly, TH-Cre;ErbB4f/f mice manifest deficits in learning, spatial and working memory-related behaviors, but not in numerous other behaviors altered in PV-Cre;ErbB4f/f mice. Importantly, microinjection of a Cre-inducible ErbB4 virus (AAV-ErbB4.DIO) into the mesencephalon of TH-Cre;ErbB4f/f mice, which selectively restores ErbB4 expression in DAergic neurons, rescues DA dysfunction and ameliorates behavioral deficits. Our results indicate that NRG/ErbB4 signaling directly in DAergic axonal projections contributes to the modulation of DA homeostasis, and that NRG/ErbB4 signaling in both GABAergic interneurons and DA neurons contribute to the modulation of behaviors with relevance to psychiatric disorders.

High voltage-gated calcium channels consist of a pore-forming subunit (␣ 1 ) and three nonhomologous subunits (␣ 2 /␦, ␤, and ␥). Although it is well established that the ␤-subunit promotes traffic of channels to the plasma membrane and modifies their activity, the reversible nature of the interaction with the ␣ 1 -subunit remains controversial. Here, we address this issue by examining the effect of purified ␤ 2a protein on Ca V 1.2 and Ca V 2.3 channels expressed in Xenopus oocytes. The ␤ 2a -subunit binds to the ␣ 1 -interaction domain (AID) in vitro, and when injected into oocytes, it shifts the voltage dependence of activation and increases charge movement to ionic current coupling of Ca V 1.2 channels. This increase depended on the integrity of AID but was not abolished by bafilomycin, demonstrating that the ␣ 1 -␤ interaction through the AID site can take place at the plasma membrane. Furthermore, injection of ␤ 2a protein inhibited inactivation of Ca V 2.3 channels and converted fast inactivating Ca V 2.3/␤ 1b channels to slow inactivating channels. Inhibition of inactivation required larger concentration of ␤ 2a in oocytes expressing Ca V 2.3/␤ 1b channels than expressing Ca V 2.3 alone but reached the same maximal level as expected for a competitive interaction through a single binding site. Together, our data show that the ␣ 1 -␤ interaction is reversible in intact cells and defines calcium channels ␤-subunits as regulatory proteins rather than stoichiometric subunits.High voltage-gated calcium channels are multi-subunit proteins complexes where a pore-forming subunit combines with one or more nonhomologous auxiliary subunits (1). One of these auxiliary subunit, the ␤-subunit, is crucial for channel function, because in addition to stimulating channel activity it appears to be required for surface expression of the channel protein (2). These two effects combined result in a severalfold increase in the ionic current density in heterologous expression systems, but the relative contribution, biological relevance, and extent to which both processes are independent from each other remain elusive. Early studies show that in Xenopus oocytes, coexpression of ␤ 2a with the pore-forming ␣ 1 subunit from cardiac cells (Ca V 1.2) augments ionic currents mostly by increasing ionic current to charge movement ratio (3). Later on, it was shown that the addition of the ␤-subunit as purified protein is capable of modulating channel activity of the ␣ 1 subunit expressed in Xenopus oocytes (4, 5) and also on isolated membranes from skeletal muscle (6). These results suggest that modulation of function is separated from the effect on channel expression and predicts that binding sites remain available on the mature channel. However, the ␣ 1 -␤-subunit association depends primarily on the so-called ␣-interaction domain (AID), 2 located within the intracellular loop joining the first and second repeats of the ␣ 1 -subunit. Secondary binding sites have been identified, but they appear to be specific to certain ␣ 1 -␤ pairs, an...

Background: Viloxazine was historically described as a norepinephrine reuptake inhibitor (NRI). Since NRIs have previously demonstrated efficacy in attention deficit/hyperactivity disorder (ADHD), viloxazine underwent contemporary investigation in the treatment of ADHD. Its clinical and safety profile, however, was found to be distinct from other ADHD medications targeting norepinephrine reuptake. Considering the complexity of neuropsychiatric disorders, understanding the mechanism of action (MoA) is an important differentiating point between viloxazine and other ADHD medications and provides pharmacology-based rationale for physicians prescribing appropriate therapy. Methods: Viloxazine was evaluated in a series of in vitro binding and functional assays. Its effect on neurotransmitter levels in the brain was evaluated using microdialysis in freely moving rats. Results: We report the effects of viloxazine on serotoninergic (5-HT) system. In vitro, viloxazine demonstrated antagonistic activity at 5-HT 2B and agonistic activity at 5-HT 2C receptors, along with predicted high receptor occupancy at clinical doses. In vivo, viloxazine increased extracellular 5-HT levels in the prefrontal cortex (PFC), a brain area implicated in ADHD. Viloxazine also exhibited moderate inhibitory effects on the norepinephrine transporter (NET) in vitro and in vivo, and elicited moderate activity at noradrenergic and dopaminergic systems. Conclusion: Viloxazine's ability to increase 5-HT levels in the PFC and its agonistic and antagonistic effects on certain 5-HT receptor subtypes, which were previously shown to suppress hyperlocomotion in animals, indicate that 5-HT modulating activity of viloxazine is an important (if not the predominant) component of its MoA, complemented by moderate NET inhibition. Supported by clinical data, these findings suggest the updated psychopharmacological profile of viloxazine can be best explained by its action as a serotonin norepinephrine modulating agent (SNMA).

Eukaryotic ClC channels are dimeric proteins with each subunit forming an individual protopore. Single protopores are gated by a fast gate, whereas the slow gate is assumed to control both protopores through a cooperative movement of the two carboxy-terminal domains. We here study the role of the carboxy-terminal domain in modulating fast and slow gating of human ClC-2 channels, a ubiquitously expressed ClC-type chloride channel involved in transepithelial solute transport and in neuronal chloride homeostasis. Partial truncation of the carboxy-terminus abolishes function of ClC-2 by locking the channel in a closed position. However, unlike other isoforms, its complete removal preserves function of ClC-2. ClC-2 channels without the carboxy-terminus exhibit fast and slow gates that activate and deactivate significantly faster than in WT channels. In contrast to the prevalent view, a single carboxy-terminus suffices for normal slow gating, whereas both domains regulate fast gating of individual protopores. Our findings demonstrate that the carboxy-terminus is not strictly required for slow gating and that the cooperative gating resides in other regions of the channel protein. ClC-2 is expressed in neurons and believed to open at negative potentials and increased internal chloride concentrations after intense synaptic activity. We propose that the function of the ClC-2 carboxy-terminus is to slow down the time course of channel activation in order to stabilize neuronal excitability

Uptake through the Dopamine Transporter (DAT) is the primary mechanism of terminating dopamine signaling within the brain, thus playing an essential role in neuronal homeostasis. Deregulation of DAT function has been linked to several neurological and psychiatric disorders including ADHD, schizophrenia, Parkinson’s disease, and drug addiction. Over the last 15 years, several studies have revealed a plethora of mechanisms influencing the activity and cellular distribution of DAT; suggesting that fine-tuning of dopamine homeostasis occurs via an elaborate interplay of multiple pathways. Here, we show for the first time that the βγ subunits of G proteins regulate DAT activity. In heterologous cells and brain tissue, a physical association between Gβγ subunits and DAT was demonstrated by co-immunoprecipitation. Furthermore, in vitro pull-down assays using purified proteins established that this association occurs via a direct interaction between the intracellular carboxy-terminus of DAT and Gβγ. Functional assays performed in the presence of the non-hydrolyzable GTP analog GTP-γ-S, Gβγ subunit overexpression, or the Gβγ activator mSIRK all resulted in rapid inhibition of DAT activity in heterologous systems. Gβγ activation by mSIRK also inhibited dopamine uptake in brain synaptosomes and dopamine clearance from mouse striatum as measured by high-speed chronoamperometry in vivo. Gβγ subunits are intracellular signaling molecules that regulate a multitude of physiological processes through interactions with enzymes and ion channels. Our findings add neurotransmitter transporters to the growing list of molecules regulated by G-proteins and suggest a novel role for Gβγ signaling in the control of dopamine homeostasis.

Heterozygous mutations in the CLCN2 gene encoding the voltage-gated chloride channel CLC2 have been identified in patients with idiopathic generalized epilepsy (IGE). Yet the involvement of CLCN2 in epilepsy remains controversial. To investigate the involvement of CLCN2 in another independent sample, we screened 52 unrelated patients from IGE families and 23 patients with Doose syndrome for mutations in CLCN2. No mutations were found in patients with Doose syndrome. In three unrelated IGE families, we identified two novel missense mutations, p.Arg235Gln and p.Arg577Gln, which were absent in large ethnically-matched control populations, and one novel p.Arg644Cys variant, which was also found in five Indian controls. Functional characterization of mutant channels using heterologous expression in mammalian cells and whole-cell patch-clamp recordings revealed faster deactivation kinetics as the major phenotype of both missense mutations. This finding predicts a loss of function that may contribute to intracellular chloride accumulation or neuronal hyperexcitability. However, the incomplete segregation of the mutations among affected members and the transmission by unaffected parents suggests that these CLCN2 mutations alone are not sufficient to induce epilepsy. They may instead represent susceptibility factors among other so far undetected genetic alterations in the respective families.

Voltage-gated calcium channels mediate the influx of Ca 2؉ ions into eukaryotic cells in response to membrane depolarization. They are hetero-multimer membrane proteins formed by at least three subunits, the poreforming ␣ 1 -subunit and the auxiliary ␤-and ␣ 2 ␦-subunits. The ␤-subunit is essential for channel performance because it regulates two distinct features of voltage-gated calcium channels, the surface expression and the channel activity. Four ␤-subunit genes have been cloned, ␤ 1-4 , with molecular masses ranging from 52 to 78 kDa, and several splice variants have been identified. The ␤ 1b -subunit, expressed at high levels in mammalian brain, has been used extensively to study the interaction between the pore forming ␣ 1 -and the regulatory ␤-subunit. However, structural characterization has been impaired for its tendency to form aggregates when expressed in bacteria. We applied an on-column refolding procedure based on size exclusion chromatography to fold the ␤ 1b -subunit of the voltage gated-calcium channels from Escherichia coli inclusion bodies. The ␤ 1b -subunit refolds into monomers, as shown by sucrose gradient analysis, and binds to a glutathione S-transferase protein fused to the known target in the ␣ 1 -subunit (the ␣-interaction domain). Using the cutopen oocyte voltage clamp technique, we measured gating and ionic currents in Xenopus oocytes expressing cardiac ␣ 1 -subunit (␣ 1C ) co-injected with folded-␤ 1b -protein or ␤ 1b -cRNA. We demonstrate that the co-expression of the ␣ 1C -subunit with either folded-␤ 1b -protein or ␤ 1b -cRNA increases ionic currents to a similar extent and with no changes in charge movement, indicating that the ␤ 1b -subunit primarily modulates channel activity, rather than expression.Changes in the intracellular calcium concentration regulate a variety of cellular functions such as neurotransmission, muscle contraction, hormone secretion, and gene expression. High threshold voltage-activated calcium channels are the main route for calcium entry in electrically excitable cells. They are membrane protein complexes composed of at least three nonhomologous subunits, the ␣ 1 -, ␤-, and the ␣ 2 /␦-subunit. Through molecular cloning, at least 10 genes encoding mammalian ␣ 1 -subunits (␣ 1A-I and ␣ 1S ) have been identified in different cell types (1). Although the ␣ 1 -subunit encompasses all the structural elements of a functional voltage-activated calcium channel, such as the ion-conduction pathway, the voltage sensor, and drug-binding sites, the ␤-subunit seems to be essential for channel performance (2) and to be acting at two levels: (i) channel expression by interfering with the ␣ 1 -subunit endoplasmic reticulum (ER) 1 -retention signal to facilitate intracellular trafficking (3, 4), and (ii) channel activity by modifying the electrophysiological properties of the channel (5-7).Two highly conserved sequences have been identified as the primary interaction site between the ␣ 1 -and the ␤-subunit, the ␣ 1 -subunit interaction domain (AID) that lies within the c...

The dopamine transporter (DAT) is an important regulator of brain dopamine (DA) homeostasis, controlling the intensity and duration of DA signaling. DAT is the target for psychostimulants—like cocaine and amphetamine—and plays an important role in neuropsychiatric disorders, including attention-deficit hyperactivity disorder and drug addiction. Thus, a thorough understanding of the mechanisms that regulate DAT function is necessary for the development of clinical interventions to treat DA-related brain disorders. Previous studies have revealed a plethora of protein–protein interactions influencing DAT cellular localization and activity, suggesting that the fine-tuning of DA homeostasis involves multiple mechanisms. We recently reported that G-protein beta-gamma (Gβγ) subunits bind directly to DAT and decrease DA clearance. Here we show that Gβγ induces the release of DA through DAT. Specifically, a Gβγ-binding/activating peptide, mSIRK, increases DA efflux through DAT in heterologous cells and primary dopaminergic neurons in culture. Addition of the Gβγ inhibitor gallein or DAT inhibitors prevents this effect. Residues 582 to 596 in the DAT carboxy terminus were identified as the primary binding site of Gβγ. A TAT peptide containing the Gβγ-interacting domain of DAT blocked the ability of mSIRK to induce DA efflux, consistent with a direct interaction of Gβγ with the transporter. Finally, activation of a G-protein-coupled receptor, the muscarinic M5R, results in DAT-mediated DA efflux through a Gβγ-dependent mechanism. Collectively, our data show that Gβγ interacts with DAT to promote DA efflux. This novel mechanism may have important implications in the regulation of brain DA homeostasis.