Background

Second-generation antipsychotics (SGAs) are frontline treatments for serious mental illness. Often, individual patients benefit only from some SGAs and not others. The mechanisms underlying this unpredictability in treatment efficacy remain unclear. All SGAs bind the dopamine D₃ receptor (D3R) and are traditionally considered antagonists for dopamine receptor signaling.

Methods

Here, we used a combination of two-photon calcium imaging, in vitro signaling assays, and mouse behavior to assess signaling by SGAs at D3R.

Results

We report that some clinically important SGAs function as arrestin-3 agonists at D3R, resulting in modulation of calcium channels localized to the site of action potential initiation in prefrontal cortex pyramidal neurons. We further show that chronic treatment with an arrestin-3 agonist SGA, but not an antagonist SGA, abolishes D3R function through postendocytic receptor degradation by GASP1 (G protein-coupled receptor-associated sorting protein-1).

Conclusions

These results implicate D3R-arrestin-3 signaling as a source of SGA variability, highlighting the importance of including arrestin-3 signaling in characterizations of drug action. Furthermore, they suggest that postendocytic receptor trafficking that occurs during chronic SGA treatment may contribute to treatment efficacy.

The voltage-gated L-type Ca²⁺ channel Ca_V1.2 is crucial for initiating heartbeat and control of a number of neuronal functions such as neuronal excitability and long-term potentiation. Mutations of Ca_V1.2 subunits result in serious health problems, including arrhythmia, autism spectrum disorders, immunodeficiency, and hypoglycemia. Thus, precise control of Ca_V1.2 surface expression and localization is essential. We previously reported that α-actinin associates and colocalizes with neuronal Ca_V1.2 channels and that shRNA-mediated depletion of α-actinin significantly reduces localization of endogenous Ca_V1.2 in dendritic spines in hippocampal neurons. Here we investigated the hypothesis that direct binding of α-actinin to Ca_V1.2 supports its surface expression. Using two-hybrid screens and pull-down assays, we identified three point mutations (K1647A, Y1649A, and I1654A) in the central, pore-forming α₁1.2 subunit of Ca_V1.2 that individually impaired α-actinin binding. Surface biotinylation and flow cytometry assays revealed that Ca_V1.2 channels composed of the corresponding α-actinin-binding-deficient mutants result in a 35-40% reduction in surface expression compared to that of wild-type channels. Moreover, the mutant Ca_V1.2 channels expressed in HEK293 cells exhibit a 60-75% decrease in current density. The larger decrease in current density as compared to surface expression imparted by these α₁1.2 subunit mutations hints at the possibility that α-actinin not only stabilizes surface localization of Ca_V1.2 but also augments its ion conducting activity.

Postsynaptic density protein-95 (PSD-95) localizes AMPA-type glutamate receptors (AMPARs) to postsynaptic sites of glutamatergic synapses. Its postsynaptic displacement is necessary for loss of AMPARs during homeostatic scaling down of synapses. Here, we demonstrate that upon Ca²⁺ influx, Ca²⁺/calmodulin (Ca²⁺/CaM) binding to the N-terminus of PSD-95 mediates postsynaptic loss of PSD-95 and AMPARs during homeostatic scaling down. Our NMR structural analysis identified E17 within the PSD-95 N-terminus as important for binding to Ca²⁺/CaM by interacting with R126 on CaM. Mutating E17 to R prevented homeostatic scaling down in primary hippocampal neurons, which is rescued via charge inversion by ectopic expression of CaM^R126E, as determined by analysis of miniature excitatory postsynaptic currents. Accordingly, increased binding of Ca²⁺/CaM to PSD-95 induced by a chronic increase in Ca²⁺ influx is a critical molecular event in homeostatic downscaling of glutamatergic synaptic transmission.

Despite the central role PSD-95 plays in anchoring postsynaptic AMPARs, how PSD-95 itself is tethered to postsynaptic sites is not well understood. Here we show that the F-actin binding protein α-actinin binds to the very N terminus of PSD-95. Knockdown (KD) of α-actinin phenocopies KD of PSD-95. Mutating lysine at position 10 or lysine at position 11 of PSD-95 to glutamate, or glutamate at position 53 or glutamate and aspartate at positions 213 and 217 of α-actinin, respectively, to lysine impairs, in parallel, PSD-95 binding to α-actinin and postsynaptic localization of PSD-95 and AMPARs. These experiments identify α-actinin as a critical PSD-95 anchor tethering the AMPAR-PSD-95 complex to postsynaptic sites.