Positive Modulation of the Glycine Receptor by Means of Glycine Receptor–Binding Aptamers

Abstract

According to the gate control theory of pain, the glycine receptors (GlyRs) are putative targets for development of therapeutic analgesics. A possible approach for novel analgesics is to develop a positive modulator of the glycine-activated Cl− channels. Unfortunately, there has been limited success in developing drug-like small molecules to study the impact of agonists or positive modulators on GlyRs. Eight RNA aptamers with low nanomolar affinity to GlyRα1 were generated, and their pharmacological properties analyzed. Cytochemistry using fluorescein-labeled aptamers demonstrated GlyRα1-dependent binding to the plasma membrane but also intracellular binding. Using a fluorescent membrane potential assay, we could identify five aptamers to be positive modulators. The positive modulation of one of the aptamers was confirmed by patch-clamp electrophysiology on L(tk) cells expressing GlyRα1 and/or GlyRα1β. This aptamer potentiated whole-cell Cl− currents in the presence of low concentrations of glycine. To our knowledge, this is the first demonstration ever of RNA aptamers acting as positive modulators for an ion channel. We believe that these aptamers are unique and valuable tools for further studies of GlyR biology and possibly also as tools for assay development in identifying small-molecule agonists and positive modulators.

Modulation of Glycine-Mediated Spinal Neurotransmission for the Treatment of Chronic Pain

“…In addition to the glycine transporter inhibition and GlyR PAM approaches described in this review, other methods to augment spinal glycinergic neurotransmission are also now under investigation. These include designing RNA aptamers as subtype selective GlyR PAMs, 154 developing platelet-activating factor antagonists that may reduce pain in part by increasing GlyRα3β activity, 155 targeting inflammatory mediators such as IL-1β that facilitate GlyR-mediated LTP that diminish GABAergic signaling, 78 and the utilization of gene therapy to increase GlyRα1β expression levels. 156 As further investigation of spinal glycinergic neurotransmission and GlyR pharmacology continues and new findings emerge, the field will continue to gain momentum and bolster drug discovery efforts that may someday lead to innovative therapeutics that effectively target and suppress central nociceptive signaling and provide patients with safe and effective relief from chronic pain.…”

Modulators of the Inhibitory Glycine Receptor

“…Recently, novel modulators of the GlyR were discovered through screening techniques such as phage display, , fluorescent assays, and enrichment of RNA aptamers by SELEX or from ligand libraries in search of potential analgesics …”

“…89 Tamoxifen increased the mean amplitude and frequency of glycinergic miniature currents, even in the presence of the Ca 2+ channel blocker Cd 2+ , i.e., independent of Ca 2+ channel activity. 89 Recently, novel modulators of the GlyR were discovered through screening techniques such as phage display, 90,91 fluorescent assays, 92 and enrichment of RNA aptamers by SELEX 93 or from ligand libraries in search of potential analgesics. 87 Biphasic Modulators: Modulators Described As Positive and/or Negative.…”

Preparation of Recombinant Membrane Proteins from Pichia pastoris for Molecular Investigations

“…Applying preparative procedures similar to those described in this article, a large number of these IMPs could then be extracted from P. pastoris membranes and isolated to quantity and quality levels compatible with their thorough characterization at the molecular level. A rapid, far from exhaustive, survey conducted on the last decade actually retrieved an abundance of information on a variety of IMPs produced in these conditions, covering screening and functional characterization of compounds (Wöhri et al 2013;Logez et al 2014;Scalise et al 2014;Dekki-Shalaly et al 2015;Zehnpfenning et al 2015;Zollmann et al 2015;Westh Hansen et al 2016;Igonet et al 2018), interactions with and regulation by proteins (Bornert et al 2014;Rosell et al 2014;Doshi et al 2017;Damian et al 2018) and lipids (Schölz et al 2011;Brohawn et al 2014), functional impact of critical mutations (Kapri-Pardes et al 2011;Ampah-Korsah et al 2017;Yang et al 2017;Christenson et al 2018), as well as structural insights and mechanistic details observed at the atomic level (Hino et al 2012;Kodan et al 2014;Fan et al 2011;Vinothkumar et al 2016;Wang et al 2016;Lolicato et al 2017;Deng et al 2018;Eddy et al 2018;Ye et al 2018;Garaeva et al 2019;Wang et al 2020).…”