L-type voltage-gated calcium channels (VGCCs) are heteromultimeric protein complexes composed of a pore-forming α1 subunit and two auxiliary subunits: cytoplasmic β subunit and extracellular α 2/δ subunit. However, the neuronal function of Ahnak and the functional significance of its interaction with the p11/Anxa2 complex in the brain have not yet been investigated. Previous reports showed Ahnak expression in endothelial cells in the blood brain barrier, epithelial cells in the choroid plexus and ependymal cells in the ventricular wall of the adult mouse brain, in which a role for Ahnak in the formation of tight junctions was proposed. The interaction of Ahnak with the p11/Anxa2 protein complex was first demonstrated in a canine kidney cell line Madin-Darby Canine Kidney (MDCK), in which p11 and Anxa2 were required for recruitment of Ahnak to the plasma membrane. Ahnak is an extremely large protein with a molecular weight of 680 kDa. Using this binding assay, we also identified Ahnak as a binding partner of the p11/Anxa2 complex. SMARCA3 constitutive knockout (KO) did not cause depression-like behaviors but it abolished behavioral and neurogenic responses to SSRIs. We identified a chromatin-remodeling factor, named SMARCA3, as a binding partner of the p11/Anxa2 complex from HEK293 cells. p11 forms a heterotetrameric protein complex with Anxa2. p11 increases the surface expression of 5HT1B and 5HT4, thereby potentiating serotonergic signaling and facilitating the actions of antidepressants such as selective serotonin reuptake inhibitors (SSRIs). In an initial study in our laboratory, p11 was identified as a binding partner of several subtypes of serotonin receptors such as 5HT1B, 5HT1D, and 5HT4 by yeast two-hybrid assays. Thus, it is critical to identify binding partners and downstream effectors of p11 and characterize their role in depression-like behaviors in order to fully understand the mechanism by which p11 controls depression-like behaviors. Because p11 is an adaptor-like small protein with a molecular weight of 11 kDa, its function is likely mediated by its interacting partners. Conversely, p11 overexpression in mice leads to an antidepressant-like behavioral phenotype. p11 null mice exhibit depression-like behaviors and abolished behavioral responses to antidepressants. The levels of p11 mRNA and protein in the brain are downregulated in depressed humans, suicide victims, and a mouse model of depression, suggesting an important role for p11 in depression pathophysiology. Alterations of p11 are implicated in the etiology of major depressive disorder (MDD) and in the therapeutic actions of antidepressants. S100A10 (p11) is a member of the S100 protein family. Our results demonstrate L-type VGCC as an effector of the Ahnak/p11/Anxa2 complex, revealing a novel molecular connection involved in the control of depressive behavior. In contrast, PV interneuron-selective Ahnak KO mice display an antidepressant-like behavioral phenotype. Constitutive Ahnak KO mice or forebrain glutamatergic neuron-selective Ahnak KO mice display a depression-like behavioral phenotype similar to that of constitutive p11 KO mice. A decrease in the L-type calcium influx is observed in both glutamatergic neurons and parvalbumin (PV) GABAergic interneurons of Ahnak KO mice. Cell surface expression of the α1 subunits and L-type calcium current are significantly reduced in primary cultures of Ahnak knockout (KO) neurons compared to wild-type controls. Ahnak, through its N-terminal region, scaffolds the L-type pore-forming α1 subunit and, through its C-terminal region, scaffolds the β subunit of VGCC and the p11/Anxa2 complex. Ahnak is expressed in p11-positive as well as p11-negative neurons. Together these data suggest the existence of an Ahnak/p11/Anxa2 protein complex. Protein levels of Ahnak, p11, and Anxa2 are highly and positively correlated in the brain. Here we report that Ahnak, whose function in the brain has been obscure, stabilizes p11 and Anxa2 proteins in the hippocampus and prefrontal cortex in the rodent brain. However, the existence of an endogenous regulator in the brain regulating p11, L-type VGCC, and depressive behavior has not been known. Alterations of S100A10 (p11) level are also implicated in the etiology of major depressive disorder. Genetic polymorphisms of the L-type voltage-gated calcium channel (VGCC) are associated with psychiatric disorders including major depressive disorder.
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