ABSTRACT
Recently developed pharmacogenetic and optogenetic approaches, with their own advantages and disadvantages, have become indispensable tools in modern neuroscience. Here, we employed a previously described knock-in mouse line (GABA(A)Rγ2(77I)lox) in which the γ2 subunit of the GABA(A) receptor (GABA(A)R) was mutated to become zolpidem insensitive (γ2(77I)) and used viral vectors to swap γ2(77I) with wild-type, zolpidem-sensitive γ2 subunits (γ2(77F)). The verification of unaltered density and subcellular distribution of the virally introduced γ2 subunits requires their selective labelling. For this we generated six N- and six C-terminal-tagged γ2 subunits, with which cortical cultures of GABA(A)Rγ2(−/−) mice were transduced using lentiviruses. We found that the N-terminal AU1 tag resulted in excellent immunodetection and unimpaired synaptic localization. Unaltered kinetic properties of the AU1-tagged γ2 ((AU1)γ2(77F)) channels were demonstrated with whole-cell patch-clamp recordings of spontaneous IPSCs from cultured cells. Next, we carried out stereotaxic injections of lenti- and adeno-associated viruses containing Cre-recombinase and the (AU1)γ2(77F) subunit (Cre-2A-(AU1)γ2(77F)) into the neocortex of GABA(A)Rγ2(77I)lox mice. Light microscopic immunofluorescence and electron microscopic freeze-fracture replica immunogold labelling demonstrated the efficient immunodetection of the AU1 tag and the normal enrichment of the (AU1)γ2(77F) subunits in perisomatic GABAergic synapses. In line with this,miniature and action potential-evoked IPSCs whole-cell recorded from transduced cells had unaltered amplitudes, kinetics and restored zolpidem sensitivity. Our results obtained with a wide range of structural and functional verification methods reveal unaltered subcellular distributions and functional properties of γ2(77I) and (AU1)γ2(77F) GABA(A)Rs in cortical pyramidal cells. This transgenicviral pharmacogenetic approach has the advantage that it does not require any extrinsic protein that might endow some unforeseen alterations of the genetically modified cells. In addition, this virus-based approach opens up the possibility of modifying multiple cell types in distinct brain regions and performing alternative recombination-based intersectional genetic manipulations.
Subject(s)
Adenoviridae/genetics , Lentivirus/genetics , Pyramidal Cells/physiology , Receptors, GABA-A/physiology , Animals , Cell Line , Embryo, Mammalian , Female , GABA-A Receptor Agonists/pharmacology , HEK293 Cells , Humans , Male , Mice , Mice, Knockout , Pregnancy , Pyridines/pharmacology , Recombinases/physiology , Transduction, Genetic , ZolpidemABSTRACT
The O-linked glycosylation of highly purified Drosophila 26S proteasome has been analyzed by immunological and lectin-binding methods. Five regulatory complex subunits and at least nine catalytic core subunits were recognized by two different monoclonal antibodies specific for O-linked N-acetylglucosamine-modified proteins, and by wheat germ agglutinin, which is specific for the N-acetylglucosamine sugar side-chain. The specificity of these reactions has been proved by competition studies with free N-acetylglucosamine. Three ATPase subunits of the regulatory complex, which are O-glycosylated, have previously been shown [FEBS Lett. 430 (1998) 269] to occur in phosphorylated form as well, indicating that several different post-translational modifications, with distinct regulatory potential, may be present on the same subunit.
Subject(s)
Acetylglucosamine/chemistry , Drosophila melanogaster/chemistry , Peptide Hydrolases/chemistry , Proteasome Endopeptidase Complex , Protein Processing, Post-Translational , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Binding Sites , Mass Spectrometry , Molecular Sequence Data , Oxygen/chemistry , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Subunits/chemistry , Wheat Germ Agglutinins/chemistryABSTRACT
The subunit contacts in the regulatory complex of the Drosophila 26 S proteasome were studied through the cross-linking of closely spaced subunits of the complex, and analysis of the cross-linking pattern in an immunoblot assay with the use of subunit-specific monoclonal antibodies. The cross-linking pattern of the purified 26 S proteasome exhibits significant differences as compared with that of the purified free regulatory complex. It is shown that the observed differences are due to extensive rearrangement of the subunit contacts accompanying the assembly of the 26 S proteasome from the regulatory complex and the 20 S proteasome. Cross-linking studies and electron microscopic examinations revealed that these changes are reversible and follow the assembly or the disassembly of the 26 S proteasome. Although the majority of the changes observed in the subunit contacts affected the hexameric ring of the ATPase subunits, the alterations extended over the whole of the regulatory complex, affecting subunit contacts even in the lid subcomplex. Changes in the subunit contacts, similar to those in the regulatory complex, were detected in the 20 S proteasome. These observations indicate that the assembly of the 26 S proteasome is not simply a passive docking of two rigid subcomplexes. In the course of the assembly, the interacting subcomplexes mutually rearrange their structures so as to create the optimal conformation required for the assembly and the proper functioning of the 26 S proteasome.
