Alternate use of distinct intersubunit contacts controls GABAA receptor assembly and stoichiometry.

GABA(A) receptors are the major inhibitory transmitter receptors in the CNS. Recombinant GABA(A) receptors composed of alpha(1)beta(3)gamma(2) subunits have been demonstrated to assemble as pentamers consisting of two alpha(1), two beta(3), and one gamma(2) subunit. Using truncated and chimeric alpha(1) subunits, we identified the alpha(1)(80-100) sequence as a major binding site for gamma(2) subunits. In addition, we demonstrated its direct interaction with gamma(2)(91-104), a sequence that previously has been identified to form the contact to alpha(1) subunits. The observation that the amino acid residues alpha(1)P96 and alpha(1)H101, which can be photolabeled by [(3)H]flunitrazepam, are located within or adjacent to the alpha(1)(80-100) sequence, indicates that the benzodiazepine binding site of GABA(A) receptors is located close to this intersubunit contact. The observation that alpha(1)(80-100) interacts with gamma(2) but not with beta(3) subunits indicates the existence of an additional beta(3) binding site on alpha(1) subunits. The preferred alternate use of the gamma(2) and beta(3) binding sites in two different alpha(1) subunits of the same receptor ensures the incorporation of only a single gamma(2) subunit and thus, determines subunit stoichiometry of alpha(1)beta(3)gamma(2) receptors. Distinct binding sites and their alternate use can therefore explain how subunits of hetero-oligomeric transmembrane proteins assemble into a defined protein complex.

Typhoid and paratyphoid fever: a 10-year retrospective study of 41 cases in a Parisian hospital.

BACKGROUND: Enteric fever remains a major cause of fever in travelers. We evaluated new trends in enteric fever. METHODS: We reviewed the epidemiological, clinical, biological, bacteriological data, and outcome of all cases of typhoid and paratyphoid fever seen in our department over the last decade. The inclusion criteria were the presence of signs compatible with enteric fever and isolation of Salmonella typhi or Salmonella paratyphi A, B, or C from blood or stool cultures or any other site. RESULTS: Among the 41 patients, 38 (93%) had travel-associated enteric fever. The main geographic source of contamination was the Indian subcontinent. One patient had been vaccinated with parenteral Vi vaccine 1 year previously. Fever and headaches were the only signs which were present in more than 80% of patients. The Widal test at inclusion was positive in 27%, and a second serological test was found to be positive in 50% of evaluated cases. Blood cultures and stool cultures were positive in 34 cases and 10 cases, respectively. Salmonellae spp were isolated in both hemocultures and stool cultures in 4 cases and in urine in 1 case. Two strains of S. typhi were resistant to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. One strain of S. typhi and one of S. paratyphi B were nalidixic acid resistant. All evaluable patients were cured with the exception of 2 patients (1 failure, 1 relapse). We observed 3 toxic reactions. No patients died. CONCLUSION: The diagnosis and outcome of enteric fever are hampered by the lack of specificity of clinical and biological signs, the increasing rates of antimicrobial resistance, and the occurrence of toxic reactions during treatment.

Stoichiometry and assembly of a recombinant GABAA receptor subtype.

GABAA receptors are ligand-gated chloride ion channels that are presumed to be pentamers composed of alpha, beta, and gamma subunits. The subunit stoichiometry, however, is controversial, and the subunit arrangement presently is not known. In this study the ratio of subunits in recombinant alpha1beta3gamma2 receptors was determined in Western blots from the relative signal intensities of antibodies directed against the N terminus or the cytoplasmic loop of different subunits after the relative reactivity of these antibodies had been determined with GABAA receptor subunit chimeras composed of the N-terminal domain of one and the remaining part of the other subunit. Via this method a subunit stoichiometry of two alpha subunits, two beta subunits, and one gamma subunit was derived. Similar experiments investigating the composition of alpha1beta3 receptors expressed on the surface of human embryonic kidney (HEK) 293 cells cotransfected with alpha1 and beta3 subunits resulted in a stoichiometry of two alpha and three beta subunits. Density gradient centrifugation studies indicated that combinations of alpha1beta3gamma2 or alpha1beta3 subunits expressed in HEK 293 cells are able to form pentamers, whereas combinations of alpha1gamma2 or beta3gamma2 subunits predominantly form heterodimers. These results provide valuable information on the mechanism of GABAA receptor assembly and support the conclusion that GABAA receptors are pentamers in which a total of four alternating alpha and beta subunits are connected by a gamma subunit.

Detection and binding properties of GABA(A) receptor assembly intermediates.

Density gradient centrifugation of native and recombinant gamma-aminobutyric acid, type A (GABA(A)) receptors was used to detect assembly intermediates. No such intermediates could be identified in extracts from adult rat brain or from human embryonic kidney (HEK) 293 cells transfected with alpha(1), beta(3), and gamma(2) subunits and cultured at 37 degrees C. However, subunit dimers, trimers, tetramers, and pentamers were found in extracts from the brain of 8-10-day-old rats and from alpha(1)beta(3)gamma(2) transfected HEK cells cultured at 25 degrees C. In both systems, alpha(1), beta(3), and gamma(2) subunits could be identified in subunit dimers, indicating that different subunit dimers are formed during GABA(A) receptor assembly. Co-transfection of HEK cells with various combinations of full-length and C-terminally truncated alpha(1) and beta(3) or alpha(1) and gamma(2) subunits and co-immunoprecipitation with subunit-specific antibodies indicated that even subunits containing no transmembrane domain can assemble with each other. Whereas alpha(1)gamma(2), alpha(1)Ngamma(2), alpha(1)gamma(2)N, and alpha(1)Ngamma(2)N, combinations exhibited specific [(3)H]Ro 15-1788 binding, specific [(3)H]muscimol binding could only be found in alpha(1)beta(3) and alpha(1)beta(3)N, but not in alpha(1)Nbeta(3) or alpha(1)Nbeta(3)N combinations. This seems to indicate that a full-length alpha(1) subunit is necessary for the formation of the muscimol-binding site and for the transduction of agonist binding into channel gating.

GABA(A) receptor assembly. Identification and structure of gamma(2) sequences forming the intersubunit contacts with alpha(1) and beta(3) subunits.

GABA(A) receptors are ligand-gated chloride channels composed of five homologous subunits that specifically recognize one another and assemble around an aqueous pore. To identify domains responsible for the specificity of subunit association, we constructed C-terminal truncated gamma(2) subunits, as well as mutated and chimeric fragments. From their ability to interfere with alpha(1)beta(3)gamma(2) receptor assembly and to associate with full-length subunits, we concluded that amino acid sequences gamma(2)-(91-104) and gamma(2)-(83-90) form the sites mediating assembly with alpha(1) and beta(3) subunits, respectively. Neural network-based secondary structure prediction, Monte Carlo optimization, and hydrophobicity analysis led to the conclusion that these sites also form the intersubunit contacts in the completely assembled receptor and provided important information on the benzodiazepine-binding site and structure of GABA(A) receptors.