Allosteric modulators have been identified for several G protein-coupled receptors, most notably muscarinic receptors. To study their mechanism of action, we made use of a recently developed technique to generate fluorescence resonance energy transfer (FRET)-based sensors to monitor G protein-coupled receptor activation. Cyan fluorescent protein was fused to the C terminus of the M 2 muscarinic receptor, and a specific binding sequence for the small fluorescent compound fluorescein arsenical hairpin binder, FlAsH, was inserted into the third intracellular loop; the latter site was labeled in intact cells by incubation with FlAsH. We then measured FRET between the donor cyan fluorescent protein and the acceptor FlAsH in intact cells and monitored its changes in real time. Agonists such as acetylcholine and carbachol induced rapid changes in FRET, indicative of agonist-induced conformational changes. Removal of the agonists or addition of an antagonist caused a reversal of this signal with rate constants between 400 and 1100 ms. The allosteric ligands gallamine and dimethyl-W84 caused no changes in FRET when given alone, but increased FRET when given in the presence of an agonist, compatible with an inactivation of the receptors. The kinetics of these effects were very rapid, with rate constants of 80 -100 ms and ≈200 ms for saturating concentrations of gallamine and dimethyl-W84, respectively. Because these speeds are significantly faster than the responses to antagonists, these data indicate that gallamine and dimethyl-W84 are allosteric ligands and actively induce a conformation of the M 2 receptor with a reduced affinity for its agonists.

Background: Depression and anxiety are common and independent outcome predictors in patients with chronic heart failure (CHF). However, it is unclear whether CHF causes depression. Thus, we investigated whether mice develop anxiety- and depression-like behavior after induction of ischemic CHF by myocardial infarction (MI).Methods and Results: In order to assess depression-like behavior, anhedonia was investigated by repeatedly testing sucrose preference for 8 weeks after coronary artery ligation or sham operation. Mice with large MI and increased left ventricular dimensions on echocardiography (termed CHF mice) showed reduced preference for sucrose, indicating depression-like behavior. 6 weeks after MI, mice were tested for exploratory activity, anxiety-like behavior and cognitive function using the elevated plus maze (EPM), light-dark box (LDB), open field (OF), and object recognition (OR) tests. In the EPM and OF, CHF mice exhibited diminished exploratory behavior and motivation despite similar movement capability. In the OR, CHF mice had reduced preference for novelty and impaired short-term memory. On histology, CHF mice had unaltered overall cerebral morphology. However, analysis of gene expression by RNA-sequencing in prefrontal cortical, hippocampal, and left ventricular tissue revealed changes in genes related to inflammation and cofactors of neuronal signal transduction in CHF mice, with Nr4a1 being dysregulated both in prefrontal cortex and myocardium after MI.Conclusions: After induction of ischemic CHF, mice exhibited anhedonic behavior, decreased exploratory activity and interest in novelty, and cognitive impairment. Thus, ischemic CHF leads to distinct behavioral changes in mice analogous to symptoms observed in humans with CHF and comorbid depression.

The molecular genetics of panic disorder (PD) with and without agoraphobia (AG) are still largely unknown and progress is hampered by small sample sizes. We therefore performed a genome-wide association study with a dimensional, PD/AG -related anxiety phenotype based on the Agoraphobia Cognition Questionnaire (ACQ) in a sample of 1,370 healthy German volunteers of the CRC TRR58 MEGA study wave 1. A genome-wide significant association was found between ACQ and single non-coding nucleotide variants of the GLRB gene (rs78726293, p=3.3x10 -8 ; rs191260602, p=3.9x10 -8 ). We followed up on this finding in a larger dimensional ACQ sample (N=2,547) and in independent samples with a dichotomous AG phenotype based on the Symptoms Checklist (SCL-90; N=3,845) and a casecontrol sample with the categorical phenotype PD/AG (N combined =1,012) obtaining highly Partial Glrb knockout-mice demonstrated an agoraphobic phenotype. In conjunction with the clinical observation that rare coding GLRB gene mutations are associated with the neurological disorder hyperekplexia characterized by a generalized startle reaction and agoraphobic behavior, our data provide evidence that non-coding, though functional GLRB gene polymorphisms may predispose to PD by increasing startle response and agoraphobic cognitions.

G␤␥ subunits are known to bind to and activate Gprotein-activated inwardly rectifying K ؉ channels (GIRK) by regulating their open probability and bursting behavior. Studying G-protein regulation of either native GIRK (I KACh ) channels in feline atrial myocytes or heterologously expressed GIRK1/4 channels in Chinese hamster ovary cells and HEK 293 cells uncovered a novel G␤␥ subunit mediated regulation of the inwardly rectifying properties of these channels. I KACh activated by submaximal concentrations of acetylcholine exhibited a ϳ2.5-fold stronger inward rectification than I KACh activated by saturating concentrations of acetylcholine. Similarly, the inward rectification of currents through GIRK1/4 channels expressed in HEK cells was substantially weakened upon maximal stimulation with co-expressed G␤␥ subunits. Analysis of the outward current block underlying inward rectification demonstrated that the fraction of instantaneously blocked channels was reduced when G␤␥ was over-expressed. The G␤␥ induced weakening of inward rectification was associated with reduced potencies for Ba 2؉ and Cs ؉ to block channels from the extracellular side. Based on these results we propose that saturation of the channel with G␤␥ leads to a conformational change within the pore of the channel that reduced the potency of extracellular cations to block the pore and increased the fraction of channels inert to a pore block in outward direction.G-protein-activated inwardly rectifying K ϩ channels (GIRKs) 1 are expressed in many areas of the brain and in supraventricular myocytes of the heart (1, 2). Activation of G-protein-coupled receptors that couple to G i -proteins such as the M 2 muscarinic acetylcholine receptors (M 2 -mAChRs) lead to a dissociation of heterotrimeric G-proteins into activated ␣ subunits and ␤␥ dimers. G␤␥ subunits are known to bind to GIRK channels and increase the open probability of these channels (3, 4). Cardiac I KACh channels are formed by heteromultimers of GIRK1 and GIRK4 subunits (4). The binding site of G␤␥ subunits to GIRK channels was mapped primarily to the C terminus of GIRK1 and GIRK4 (4 -8). Cross-linking studies have demonstrated that the heterotetrameric channel can bind up to 4 G␤␥ subunits (9). However, despite much experimental effort the mechanism by which G␤␥ activates these channels is not well understood.GIRK channels belong to the family of strong inwardly rectifying K ϩ channels, which are characterized by their strong inwardly rectifying current-voltage relationships. The inward rectification has been linked to the presence of intracellular Mg 2ϩ and polyamines (10 -12). These positively charged cytoplasmic ions are thought to block outward K ϩ currents by blocking the pore of channels from the inside (10 -13); however, for a related inwardly rectifying channel Kir2.1 this hypothesis has recently been questioned (14). Inward rectification of K ϩ channels is not only voltage-dependent but also dependent on the extracellular K ϩ concentration (11). The inward rectification of these K ...

MicroRNAs are about 22 nucleotide long single-stranded RNA molecules, negatively regulating gene expression of a single gene or a gene network. In neural tissues, they have been implicated in developmental and neuroplasticity-related processes, such as neurogenesis, differentiation, apoptosis and long-term potentiation. Their molecular mode of action is reminiscent of findings of genome-wide association studies in mental disorders, unable to attribute the risk of disease to a specific gene, but rather to multiple genes, gene-networks and gene-environment interaction. As such, microRNAs are an attractive target for research. Here, we review clinical studies conducted in humans on microRNAs in mental disorders with a particular focus on schizophrenia, bipolar disorder, major depressive disorder and anxiety disorders. The majority of clinical studies have focused on schizophrenia. The most robust finding has been reported for rs1625579 located in MIR137HG, which was associated with schizophrenia on a genome-wide level. Concerning bipolar disorder, major depression and anxiety disorders, promising results have been published, but only a considerably smaller number of clinical studies is available and genome-wide association studies did not suggest a direct link to microRNAs so far. Expression of microRNAs as biomarkers of mental disorders and treatment response is currently emerging with preliminary results. Larger-scaled genetic and functional studies along with translational research are needed to enhance our understanding of microRNAs in mental disorders. These studies will aid in disentangling the complex genetic nature of these disorders and possibly contribute to the development of novel, individualized diagnostic and therapeutic approaches.