Regulation and functional consequences of mGlu4 RNA editing.

Metabotropic glutamate receptor 4 (mGlu4) is one of eight mGlu receptors within the Class C G protein-coupled receptor superfamily. mGlu4 is primarily localized to the presynaptic membrane of neurons where it functions as an auto and heteroreceptor controlling synaptic release of neurotransmitter. mGlu4 is implicated in numerous disorders and is a promising drug target; however, more remains to be understood about its regulation and pharmacology. Using high-throughput sequencing, we have validated and quantified an adenosine-to-inosine (A-to-I) RNA editing event that converts glutamine 124 to arginine in mGlu4; additionally, we have identified a rare but novel K129R site. Using an in vitro editing assay, we then validated the pre-mRNA duplex that allows for editing by ADAR enzymes and predicted its conservation across the mammalian species. Structural modeling of the mGlu4 protein predicts the Q124R substitution to occur in the B helix of the receptor that is critical for receptor dimerization and activation. Interestingly, editing of a receptor homodimer does not disrupt G protein activation in response to the endogenous agonist, glutamate. Using an assay designed to specifically measure heterodimer populations at the surface, however, we found that Q124R substitution decreased the propensity of mGlu4 to heterodimerize with mGlu2 and mGlu7 Our study is the first to extensively describe the extent and regulatory factors of RNA editing of mGlu4 mRNA transcripts. In addition, we have proposed a novel functional consequence of this editing event that provides insights regarding its effects in vivo and expands the regulatory capacity for mGlu receptors.

Generation and characterization of human induced pluripotent stem cells (iPSCs) from three male and three female patients with CDKL5 Deficiency Disorder (CDD).

CDKL5 Deficiency Disorder (CDD) is a rare X-linked monogenic developmental encephalopathy that is estimated to affect 1:42,000 live births. CDD is caused by pathogenic variants in the CDKL5 gene and is observed in both male and female patients. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from fibroblasts of six unrelated CDD patients-three males and three females. These patients are clinically diagnosed to present with classic CDD phenotypes, including refractory epilepsy and global developmental delay, and are being followed in a longitudinal clinical study.

G protein-coupled receptors differentially regulate glycosylation and activity of the inwardly rectifying potassium channel Kir7.1.

Kir7.1 is an inwardly rectifying potassium channel with important roles in the regulation of the membrane potential in retinal pigment epithelium, uterine smooth muscle, and hypothalamic neurons. Regulation of G protein-coupled inwardly rectifying potassium (GIRK) channels by G protein-coupled receptors (GPCRs) via the G protein ßγ subunits has been well characterized. However, how Kir channels are regulated is incompletely understood. We report here that Kir7.1 is also regulated by GPCRs, but through a different mechanism. Using Western blotting analysis, we observed that multiple GPCRs tested caused a striking reduction in the complex glycosylation of Kir7.1. Further, GPCR-mediated reduction of Kir7.1 glycosylation in HEK293T cells did not alter its expression at the cell surface but decreased channel activity. Of note, mutagenesis of the sole Kir7.1 glycosylation site reduced conductance and open probability, as indicated by single-channel recording. Additionally, we report that the L241P mutation of Kir7.1 associated with Lebers congenital amaurosis (LCA), an inherited retinal degenerative disease, has significantly reduced complex glycosylation. Collectively, these results suggest that Kir7.1 channel glycosylation is essential for function, and this activity within cells is suppressed by most GPCRs. The melanocortin-4 receptor (MC4R), a GPCR previously reported to induce ligand-regulated activity of this channel, is the only GPCR tested that does not have this effect on Kir7.1.

60 YEARS OF POMC: Regulation of feeding and energy homeostasis by α-MSH.

The melanocortin peptides derived from pro-opiomelanocortin (POMC) were originally understood in terms of the biological actions of α-melanocyte-stimulating hormone (α-MSH) on pigmentation and adrenocorticotrophic hormone on adrenocortical glucocorticoid production. However, the discovery of POMC mRNA and melanocortin peptides in the CNS generated activities directed at understanding the direct biological actions of melanocortins in the brain. Ultimately, discovery of unique melanocortin receptors expressed in the CNS, the melanocortin-3 (MC3R) and melanocortin-4 (MC4R) receptors, led to the development of pharmacological tools and genetic models leading to the demonstration that the central melanocortin system plays a critical role in the regulation of energy homeostasis. Indeed, mutations in MC4R are now known to be the most common cause of early onset syndromic obesity, accounting for 2-5% of all cases. This review discusses the history of these discoveries, as well as the latest work attempting to understand the molecular and cellular basis of regulation of feeding and energy homeostasis by the predominant melanocortin peptide in the CNS, α-MSH.

Further exploration of M1 allosteric agonists: subtle structural changes abolish M1 allosteric agonism and result in pan-mAChR orthosteric antagonism.

This letter describes the further exploration of two series of M(1) allosteric agonists, TBPB and VU0357017, previously reported from our lab. Within the TPBP scaffold, either electronic or steric perturbations to the central piperidine ring led to a loss of selective M(1) allosteric agonism and afforded pan-mAChR antagonism, which was demonstrated to be mediated via the orthosteric site. Additional SAR around a related M(1) allosteric agonist family (VU0357017) identified similar, subtle 'molecular switches' that modulated modes of pharmacology from allosteric agonism to pan-mAChR orthosteric antagonism. Therefore, all of these ligands are best classified as bi-topic ligands that possess high affinity binding at an allosteric site to engender selective M(1) activation, but all bind, at higher concentrations, to the orthosteric ACh site, leading to non-selective orthosteric site binding and mAChR antagonism.

Development of a novel, CNS-penetrant, metabotropic glutamate receptor 3 (mGlu3) NAM probe (ML289) derived from a closely related mGlu5 PAM.

Herein we report the discovery and SAR of a novel metabotropic glutamate receptor 3 (mGlu(3)) NAM probe (ML289) with 15-fold selectivity versus mGlu(2). The mGlu(3) NAM was discovered via a 'molecular switch' from a closely related, potent mGlu(5) positive allosteric modulator (PAM), VU0092273. This NAM (VU0463597, ML289) displays an IC(50) value of 0.66 µM and is inactive against mGlu(5).