Regular paper
Natural mutation of GABAA receptor α6 subunit alters benzodiazepine affinity but not allosteric GABA effects

https://doi.org/10.1016/0922-4106(93)90133-TGet rights and content

Abstract

The binding of the imidazobenzodiazepine, [3H]Ro 15-4513, to cerebellar granule cell-specific GABAA/benzodiazepine receptors is typically insensitive to benzodiazepine receptor agonists such as diazepam. A mutation in the α6 subunit, causing replacement of the arginine at the 100 position by glutamine (Q100), has recently been found in an alcohol- and benzodiazepine-sensitive rat line. The mutant α6(Q100)β2γ2 recombinant receptors are sensitive to diazepam. The binding of [3H]Ro 15-4513 to cerebellar diazepam-insensitive receptors is enhanced by GABA, whereas binding to diazepam-sensitive receptors is inhibited. Recombinant receptors consisting of β2 and γ2 subunits together with the wildtype α6 or mutant α6(Q100) subunit showed positive modulation of [3H]Ro 15-4513 binding by GABA, whereas α1β2γ2 receptors showed negative modulation. The picrotoxin-sensitive binding of a convulsant, t-butylbicyclophosphoro[35S]thionate ([35S]TBPS),was inhibited in the α6β2γ2 and α6(Q100)β2γ2 receptors by GABA at concentrations less than one-tenth of those required in the α1β2γ2 receptors. GABA effects on [35S]TBPS binding were only slightly affected by diazepam in the α6(Q100)β2γ2 receptors, while profound effects were seen in the α1β2γ2 receptors in the presence of diazepam. The results with the mutant receptor suggest that the α1 and α6 subunits are responsible for differential allosteric actions by GABA on other binding sites, independently of the structures defining the benzodiazepine binding pharmacology.

References (27)

  • W. Wisden et al.

    GABAA receptor channels: from subunits to functional entities

    Curr. Opin. Neurobiol.

    (1992)
  • E.P. Bonetti et al.

    Ro 15-4513: Partial inverse agonism at the BZR and interaction with ethanol

    Pharmacol. Biochem. Behav.

    (1989)
  • C. Chen et al.

    High-efficiency transformation of mammalian cells by plasmid DNA

    Mol. Cell. Biol.

    (1987)
  • Cited by (39)

    • The neurobiology of alcohol consumption and alcoholism: An integrative history

      2013, Pharmacology Biochemistry and Behavior
      Citation Excerpt :

      These rats have been selectively bred for many generations to demonstrate genetically determined differences in response to the incoordinating effects of ethanol (AT rats show little effect, ANT rats are highly sensitive Eriksson and Rossi, 1981). Esa Korpi and his colleagues (Malminen and Korpi, 1988; Uusi-Oukari and Korpi, 1989) discovered an interesting mutation (Korpi and Seeburg, 1993) in the α6 subunit that distinguished the AT from the ANT rats. This mutation in the α6 subunit in the ANT rats resulted in GABA-A receptors which were significantly more sensitive to benzodiazepines (Korpi and Seeburg, 1993), and significantly different in the characteristics for the binding of the purported (Suzdak et al., 1986a) “alcohol antagonist”, Ro-15-4513 (Uusi-Oukari and Korpi, 1990).

    • Does ethanol act preferentially via selected brain GABA<inf>A</inf> receptor subtypes? the current evidence is ambiguous

      2007, Alcohol
      Citation Excerpt :

      The density of high-affinity [3H]muscimol binding sites is lower in cerebellar membranes of ANT rats than in those of AT rats (Mäkelä et al., 1996; Uusi-Oukari and Korpi, 1989), although there is no indication that these binding properties differ between the α6β2γ2 and α6(R100Q)β2γ2 receptors. The KD values of about 3 nM, that is, in the same range as for α1β2γ2 receptors (KD, 8 nM; Korpi and Seeburg, 1993). The high-affinity [3H]muscimol binding in the cerebellum is dependent on the expression of α6 and δ subunit-containing receptors (Korpi et al., 2002b).

    • Modulation of GABA<inf>A</inf> receptors in cerebellar granule neurons by ethanol: a review of genetic and electrophysiological studies

      2007, Alcohol
      Citation Excerpt :

      It was subsequently shown that the binding difference was mediated by the α6-R100Q single nucleotide polymorphism (SNP) (Korpi et al., 1993; also see Korpi et al., this issue). It is likely that this point mutation accounts for at least part of the genetic difference in benzodiazepine sensitivity in the AT and ANT rats (Korpi and Seeburg, 1993). Given the known interaction between EtOH and GABAA-Rs, it has been speculated by many investigators that the α6-R100Q mutation might be at least partially responsible for the differential EtOH sensitivity of the AT and ANT selected lines, although in the original report it was noted that the mutation did not affect EtOH modulation of α6β2γ2 receptors expressed in a cell culture system (Korpi et al., 1993).

    View all citing articles on Scopus
    View full text