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  • Previous studies have shown that zinc interacts with

    2022-01-11

    Previous studies have shown that zinc interacts with other allosteric modulators at the glycine receptor (Kirson et al., 2013, McCracken et al., 2010, McCracken et al., 2013). Of note, the chelation of zinc via tricine decreases the effects of alcohol at α1-, α2-, and α3-containing glycine receptors, essentially blocking the actions of ethanol at concentrations producing under 100% potentiation of EC5–10 glycine currents (≤100mM ethanol), while significantly inhibiting alcohol effects at higher concentrations (200mM ethanol) (McCracken et al., 2010, McCracken et al., 2013). It is possible that zinc acts as a co-factor necessary to observe appreciable modulation of receptor function by allosteric modulators at concentrations that produce little effect on their own. Nanomolar concentrations of zinc sufficient to enhance glycine receptor function are ubiquitous in vivo and in vitro, and the receptor may have evolved to respond optimally to glycine when zinc is bound. Thus it is probable that peptide selection occurs to zinc-bound glycine receptors during the panning procedure. Regardless, free or rapidly exchangeable forms of zinc are maintained in the Dexlansoprazole at levels capable of potentiating receptor currents, indicating that zinc-dependence would not necessarily hinder the potential use of these peptides in vivo (Frederickson et al., 2006, Frederickson and Bush, 2001).
    Conclusions We have extended our initial observations demonstrating the utility of using a modified phage display procedure in the discovery of novel allosteric modulators of the glycine receptor. Expression of target channels in HEK 293 cells ensured the proper conformation of multimeric channels for phage display libraries to be panned against, yielding numerous heptapeptide sequences. The addition of a negative selection step in which libraries are first washed over cells expressing receptors similar to the target can help in obtaining peptides with subunit selectivity, but may require a more stringent selection if the targets are molecularly similar. Additionally, the zinc-dependence of peptide activity seen here (Fig. 7), taken together with previous observations of the zinc-dependence of ethanol actions on the glycine receptor, indicates a possible function of zinc as an endogenous co-modulator necessary for efficient allosteric modulation of this receptor by some modulators.
    Acknowledgements This research was supported by National Institute on Alcohol Abuse & Alcoholism Grant 5P01AA020683 and by Bruce Jones & Frayne predoctoral fellowships. The zinc quantitation performed by Dr. Nathan R. Miller of the Laser Ablation and ICP-MS Facility at the University of Texas at Austin is greatly appreciated.
    Introduction N-Methyl-D-aspartic acid (NMDA) receptors are ligand-gated ion channels important for fast excitatory synaptic transmission, distributed throughout the central nervous system (Moriyoshi et al., 1991, Monyer et al., 1992, Paoletti et al., 2013). They are necessary for learning and memory and are drug targets for treating Alzheimer's disease (McKeage, 2009), depression (Moskal et al., 2014), epilepsy (Hu et al., 2016), and schizophrenia (Balu, 2016). These receptors are obligate heterotetramers, typically comprising two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits (Mayer et al., 1984). Each subunit contains an extracellular amino-terminal domain (ATD) and ligand-binding domain (LBD) in addition to a transmembrane domain (TMD) and an intracellular C-terminal domain (CTD) (Mayer, 2017). The LBDs possess an overall clamshell-like conformation in which a hinge separates two lobes of the LBD (lobes 1 and 2) (Figure 1). The TMDs of the four subunits surround a central pore to form the core of the ion channel, and in each subunit, the TMD is connected to lobe 2 of the LBD by three short linkers. Binding of two distinct agonists, glutamate and glycine (or D-serine), to the individual glutamate- and glycine-binding LBDs activates the channel. Agonist binding triggers conformational change in the LBD, causing the two lobes to close around the ligand and is thought to provide the useful work to open the channel pore, which, together with a voltage-dependent unblock of magnesium, allows the entry of calcium into the postsynaptic cell and depolarization of the membrane potential (Mayer et al., 1984, Nowak et al., 1984, MacDermott et al., 1986). Crystallographic and cryo-electron microscopic studies have captured NMDA receptor LBDs bound to glutamate and glycine, as well as various other agonists and antagonists, shedding light on the molecular interactions involved in stabilizing the ligands and conformational changes accompanying ligand binding (Inanobe et al., 2005, Furukawa et al., 2005, Yao et al., 2013, Jespersen et al., 2014, Hackos et al., 2016, Volgraf et al., 2016, Zhu et al., 2016, Tajima et al., 2016, Yi et al., 2016, Villemure et al., 2017, Lü et al., 2017, Romero-Hernandez and Furukawa, 2017, Lind et al., 2017). The precise molecular details of the dynamical processes associated with ligand binding, however, are unknown.