RESUMO
The benzodiazepine binding site of GABA(A) receptors is located at the interface of the alpha and gamma subunits. Certain point mutations in these subunits have been demonstrated to dramatically reduce the affinity of benzodiazepine binding site ligands for these receptors. Recently, mice were generated with a phenylalanine (F) to isoleucine (I) substitution at position 77 in the gamma2 subunit of GABA(A) receptors. Here we tested the potency of 24 benzodiazepine binding site ligands from 16 different structural classes for inhibition of [(3)H]flunitrazepam binding to brain membranes of these gamma2F77I mice. Results indicate that the potency of the classical 1,4-benzodiazepines, of the 1,4-thienodiazepine clotiazepam, the 1,5-benzodiazepine clobazam, or the pyrazoloquinoline CGS 9896 is only 2-7-fold reduced by this gamma2F77I point mutation. The potency of the imidazopyrimidines Ru 32698, Ru 33203, and Ru 33356, of the imidazoquinoline Ru 31719, or the pyrazolopyridine CGS 20625 is reduced 10-20-fold, whereas the potency of some imidazobenzodiazepines, beta-carbolines, cyclopyrrolones, imidazopyridines, triazolopyridazines, or quinolines is 100-1000-fold reduced. Interestingly, the extent of potency reduction induced by the gamma2F77I point mutation varied within the structural classes of compounds. Results support and significantly extend previous observations indicating that the residue gamma2F77 is important for high affinity binding of some, but not all benzodiazepine site ligands.
Assuntos
Benzodiazepinas/farmacologia , Subunidades Proteicas/metabolismo , Receptores de GABA-A/metabolismo , Animais , Sítios de Ligação , Clobazam , Feminino , Flunitrazepam/farmacologia , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mutação Puntual , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/efeitos dos fármacos , Subunidades Proteicas/genética , Receptores de GABA-A/química , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/genéticaRESUMO
In cerebellum, 13 different GABA(A) receptor subunits are expressed. The number of different receptor subtypes formed in this tissue, their subunit composition and their quantitative importance so far has not been determined. In the present study, immunodepletion by immunoaffinity chromatography, as well as immunoprecipitation and western blot analysis was performed using 13 different subunit-specific antibodies to provide an overview on the subunit composition and abundance of GABA(A) receptor subtypes in mouse and rat cerebellum. Results obtained indicate that alpha1betaxgamma2, alpha1alpha6betaxgamma2, alpha6betaxgamma2, alpha6betaxdelta and alpha1alpha6betaxdelta are the major GABA(A) receptor subtypes present in the cerebellum. In addition, small amounts of alpha1betaxdelta receptors and a series of minor receptor subtypes containing alpha2, alpha3, alpha4, alpha5, gamma1 or gamma3 subunits are also present in the cerebellum. Whereas the abundance of alpha1alpha6betaxgamma2, alpha6betaxdelta and alpha1alpha6betaxdelta receptors is different in mouse and rat cerebellum, that of other receptors is quite similar in these tissues. Data obtained for the first time provide an overview on the GABA(A) receptor subtypes present in the cerebellum and represent the basis for further studies investigating changes in receptor expression and composition under pathological conditions.
