The glycine receptor (GlyR) mediates fast inhibitory neurotransmission in the central nervous system (CNS). Although GlyR α1 subunits are widely distributed, α3 subunits are found only on spinal cord pain sensory neurons where they mediate central inflammatory pain sensitization. Thus, the α3 subunit is a potential therapeutic target for anti-inflammatory analgesia. It is yet to be understood why α3 subunits are represented in these synapses. Thus, α3 subunit-specific modulators are required both as therapeutic leads and as pharmacological probes for basic research. The Thesis, which consists of three independent studies, investigated the molecular pharmacology of three classes of compounds at GlyRs, especially those containing the α3 subunit. The dihydropyridines (DHPs), nifedipine and nicardipine, modulate native GlyRs at micromolar concentrations. Nicardipine has a biphasic potentiating and inhibitory effect, whereas nifedipine causes inhibition only. The first study investigated the molecular mechanism by which these compounds inhibit recombinant GlyRs. The rate of onset of inhibition in the open state was accelerated by pre-application of DHP in the closed state, with the degree of acceleration proportional to the concentration of pre-applied DHP. This implies a non-inhibitory binding site close to the DHP inhibitory site. DHP inhibition was use-dependent and independent of glycine concentration, consistent with a pore-blocking mode of action. DHP sensitivity was abolished by the G2’A mutation, providing a strong case for DHP binding site in the pore. Nifedipine exhibited an approximately 10-fold higher inhibitory potency at α1-containing relative to α3-containing receptors, whereas nicardipine was only weakly selective for α1-containing GlyRs. The differential sensitivities of nifedipine and nicardipine for different GlyR isoforms suggest that DHPs may be a useful resource to screen as pharmacological tools for selectively inhibiting different synaptic GlyR isoforms. To date there are few compounds known to pharmacologically discriminate between α1 and α3 subunit-containing GlyRs. The second study stemmed from an observation that β-alanine and taurine act as weak partial agonists of α3 GlyRs but as strong partial agonists at α1 GlyRs. Using chimeras of α1 and α3 subunits, we identified the relatively structurally divergent M4 transmembrane domain and C-terminal tail as a specific determinant of the efficacy difference. As mutation of individual non-conserved M4 residues had little influence on agonist efficacies, the reduced efficacy of α3 GlyRs is most likely a distributed effect of all non-conserved M4 residues. Given the lack of contact between M4 and other transmembrane domains, the efficacy differences are probably mediated by differential interactions between the respective M4 domains and the surrounding lipid environment. The strong influence of M4 primary structure on partial agonist efficacy suggests that the relatively poorly conserved α3 GlyR M4 domain may be a promising domain to target in the search for α3 GlyR-specific modulators. β-carbolines have recently been shown to inhibit glycine receptors in a subunit-specific manner. The third study screened four structurally similar β-carbolines, harmane (HM), tryptoline (TP), norharmane (NHM) and 6-methoxyharmalan (MH) at recombinantly expressed α1, α1β, α2 and α3 glycine receptors. The four compounds exhibited only weak subunit-specificity, rendering them unsuitable as pharmacological probes. Because they displayed competitive antagonist activity, we investigated the roles of known glycine binding residues in coordinating the four compounds. The structural similarity of the compounds, coupled with the differential effects of C-loop mutations (T204A, F207Y) on compound potency, implied direct interactions between variable β-carboline groups and mutated residues. Mutant cycle analysis employing HM and NHM revealed a strong pairwise interaction between the HM methyl group and the C-loop in the region T204 and F207. These results, which define the orientation of the bound β-carbolines, were supported by molecular docking simulations. The information may also be relevant to understanding the mechanism of β-carboline binding to GABAAR where they are potent pharmacological probes. The identification of compounds that specifically abolish α3 GlyR-mediated currents should provide a useful means to investigate the physiological roles of this subunit. Drugs that potently and selectively enhance α3-subtype GlyR function may potentially serve as lead compounds since α3-subtype GlyRs have emerged as a potential therapeutic target for pain treatment. Results from studies forming the Thesis have identified several structural elements that might be useful for developing novel α3 subunit-specific drugs in the future.
| Identifer | oai:union.ndltd.org:ADTP/254141 |
| Creators | Xuebin Chen |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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