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    • Although glycine receptors predominate in the brain

    2021-10-08

    Although glycine receptors predominate in the brain stem and spinal cord, they are also expressed in higher brain regions such as the nucleus accumbens, frontal cortex, and hippocampus (Jonsson et al., 2012, Jonsson et al., 2009, Molander and Söderpalm, 2005b). A variety of drugs of abuse, including alcohol, inhalants and volatile anesthetics enhance glycine receptor function at concentrations that are achieved in vivo (Lynch, 2004, Molander et al., 2005, Xiong et al., 2009). Individual receptor subunits of this Cys-loop superfamily form pentamers around central ion-conducting pores. In humans, there are 4 glycine receptor subunits (α1, α2, α3, and β) that can form α-homomeric or αβ-heteromeric channels. Enhanced activation of these channels through positive allosteric modulation effectively treats inflammatory pain in rodents and may aid in the treatment of addictions such as alcoholism, through modulation of dopamine release (Molander et al., 2005; Molander and Söderpalm, 2005a, Molander and Söderpalm, 2005b; Xiong et al., 2009). However, while α3-containing receptors are thought to predominate in glycine receptor-mediated analgesia, it is not entirely clear which subtypes are responsible for the behavioral and dopamine-modulating effects of alcohol. While α1− and α2−containing receptors seem to be the most likely in vivo targets of alcohol (Blednov et al., 2015), there would be considerable utility in the development of glycine receptor modulators with subunit selectivity, to definitively determine the relative contributions of each particular subtype to alcohol's pharmacological effects. Our previous work validated the use of phage display for the discovery of novel peptide modulators of the glycine receptor (Tipps et al., 2010). In the present report, we expand on these studies by identifying and characterizing the action of peptides with selectivity for α1β and α3β over α2β−containing glycine receptors and identify several possible amino Radezolid consensus sequences within the heptapeptides. Interestingly, the actions of these peptides appear to be zinc-dependent. This zinc dependence was also previously shown to affect alcohol modulation of the glycine receptor (McCracken et al., 2010), suggesting that the presence of zinc may be necessary for efficient modulation of glycine receptor activity by allosteric modulators.
    Materials and methods
    Results
    Discussion The glycine receptor is a putative target for the treatment of inflammatory pain and alcoholism (Blednov et al., 2015; Jonsson et al., 2009; Molander et al., 2007, Molander et al., 2005, Molander and Söderpalm, 2005a, Tipps et al., 2010; Xiong et al., 2009); novel specific modulators of glycine receptor function could thus have potential therapeutic applications. Of particular interest would be modulators capable of enhancing receptor activity, as several studies have shown that glycine receptor activation in the nucleus accumbens and ventral tegmental area decreases ethanol drinking in rodents (Molander and Söderpalm , 2005a; Molander et al., 2005, Li et al., 2012). Tipps et al. (2010) demonstrated the feasibility of utilizing phage display to identify novel peptide modulators of the glycine receptor. In the present report, we expand on these studies, identifying heptapeptides acting on the glycine receptor with subunit selectivity (Fig. 1, Fig. 5), and characterizing peptide actions on receptor function. To achieve this, we incorporated a negative selection step into the phage selection process, in which the phage library was first washed over HEK 293 cells expressing glycine receptor subunits we wished to select against before panning against the glycine receptor target of interest. Peptides that bound non-specifically to the endogenous membrane components of HEK 293 cells, or to our expressed negative selection receptor subunits would, in principle, be eliminated from the population of the library upon transfer of the supernatant to the positive selection plates containing HEK 293 cells expressing the α1β glycine receptor. Through this negative selection step peptides capable of acting selectively on α1β compared to α2β glycine receptors were identified. However, we did not identify peptides that differentiated between α1β and α3β glycine receptors (Fig. 5) despite the inclusion of the latter in the negative selection procedure of panning series D7.2. This may be due to a higher level of similarity between α1- and α3-containing glycine receptors at the peptide binding areas. Why then did any α3β-binding peptides survive the panning procedure if they were negatively selected against? One reason could be that the panning is somewhat 'leaky’. If some phage capable of binding in the negative selection do not bind, but do bind in the subsequent positive selection, they would be amplified and re-introduced into the next round of panning.