The β subunit increases the ginkgolide B sensitivity of inhibitory glycine receptors
Introduction
The glycine receptor (GlyR), a member of the Cys-loop ligand-gated ion channel family, is a pentameric protein composed of homologous membrane spanning α and β subunits (Grenningloh et al., 1987, Lynch, 2004, Grudzinska et al., 2005). The GlyR mediates inhibitory neurotransmission in motor, sensory and pain pathways (Aprison, 1990, Betz et al., 1999, Harvey et al., 2004) and has been shown to be implicated in the pathogenesis of neuromotor diseases (Rajendra et al., 1997). Hence, drugs modulating GlyR function are thought to have therapeutic potential in muscle relaxation and pain relief (Laube et al., 2002, Cronin et al., 2004, Lynch, 2004). Although the GlyR is a target of convulsants such as strychnine and picrotoxin, only a few compounds are so far known to modulate GlyR function. These include zinc ions (Laube et al., 1995), neurosteroids (Maksay et al., 2001, Belelli et al., 1999), tropeines (Maksay et al., 1999), alcohols and anesthetics (Lobo et al., 2004, Davies et al., 2004).
Extracts from the leaves of the Ginkgo biloba tree, known as effective drug in ancient Chinese medicine (DeFeudis and Drieu, 2000), are becoming increasingly popular as a treatment that is claimed to reduce memory loss and other symptoms of mild cognitive disorders (Maclennan et al., 2002, Amri et al., 1996, Bastianetto et al., 2000). Ginkgolide B (GB), a terpenic trilactone component of these extracts, which is also known as a platelet activating factor antagonist, has recently been shown to inhibit GlyR mediated currents in acutely isolated hippocampal pyramidal neurons (Kondratskaya et al., 2002) and cortical slices (Ivic et al., 2003) in a non-competitive fashion (IC50 = 0.2–2 μM). Here, we made an attempt to directly determine the subunit selectivity and blocking mechanism of GB at recombinant GlyRs by expressing wild-type (wt) and mutant GlyR α1, α2 and α3 as well as β subunits in Xenopus oocytes for subsequent electrophysiological characterization. Our results show that coexpression of the β subunit confers high affinity for GB, consistent with hetero-oligomeric GlyRs being the predominant target of this component of Ginkgo extracts (Kondratskaya et al., 2004). In addition, we identify a residue in the second pore forming transmembrane domain (TM2), whose substitution alters the GB affinity of the α1 homo-oligomeric GlyR. This is, as previously proposed (Kondratskaya et al., 2004), consistent with a non-competitive block by GB of the GlyR channel.
Section snippets
Reagents
All chemicals for intra- and extracellular solutions were obtained from RBI Sigma (Taufkirchen, Germany). GB was a gift of Dr. S. Chatterjee, Willmar Schwabe GmbH, Karlsruhe (Germany).
cRNA synthesis, mutagenesis and oocyte expression
Linearized plasmid cDNAs of the human α1, α2, α3 and β GlyR subunits were used for the in vitro synthesis of cRNA (mCAP mRNA Capping Kit, Stratagene) using T7 or T3 RNA polymerases, respectively, as described previously (Laube and Betz, 1998, Laube et al., 2000, Maksay et al., 2001). The α1 mutants were generated
Homo-oligomeric α1, α2 and α3 GlyRs differ in GB sensitivity
We first examined the effect of GB on recombinant GlyRs generated by expressing the wt α subunit isoforms α1–3 in Xenopus oocytes. Voltage-clamp recordings from oocytes superfused with glycine in the absence and presence of different concentrations of GB confirmed that this compound inhibits glycine currents (Fig. 1). Fig. 1 shows the inhibitory effects of increasing concentrations of GB on currents elicited by 200 μM glycine at α1 and α2 GlyRs. When the α1 subunit was expressed, glycine-evoked
Discussion
The major findings of this study are that (i) homo-oligomeric GlyRs differ in GB sensitivity, with α1 displaying the lowest IC50 value, (ii) incorporation of the β subunit markedly increases the GB sensitivity of recombinant hetero-oligomeric GlyRs, (iii) GB inhibition is not affected by changes in agonist concentration, and (iv) a mutation within the TM2 segment of the α1 subunit reduces GB antagonism of glycine-induced currents. Notably, substitutions within the strychnine binding site and
Acknowledgements
We thank Dr. S. Chatterjee for kindly providing GB and Elena Zurkowski for excellent technical help. E.K. was supported by INTAS Young Scientist Fellowship 03-55-0599. This work was funded by the Deutsche Forschungsgemeinschaft (LA 1086/5-1) and Fonds der Chemischen Industrie (to H.B.).
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2010, Journal of Biological ChemistryCitation Excerpt :The ginkgolides are noncompetitive antagonists of GlyRs and GABAARs (18–24). Ginkgolides B, C, and J have exhibited preference for heteromeric over homomeric GlyRs in studies of cloned and native receptors (18, 19, 21). Thus, the compounds display the opposite pharmacological profile compared with that of another natural product, picrotoxin (an equimolar mixture of picrotoxinin and picrotin) (4, 25, 26).
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Present address: RIKEN Brain Research Institute (BSI), Neuronal Circuit Mechanisms Research Group, Semyanov Research Unit, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.