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Persistance of some polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans of pentachlorophenol in human adipose tissue

100%

Gorski T. , Konopka L. , Brodzki M.

tom 35

nr 4

Different pharmacological profile in alpha1-gamma2 and alpha1-beta2-gamma2 GABAA receptors

75%

Brodzki M. , Czyzewska M. M. , Rutkowski R. , Kisiel M. , Jatczak M. , Mozrzymas J. W.

nr Supl.

BACKGROUND AND AIMS: GABAA receptors (GABAAR) mediate the main component of ionotropic inhibitory transmission in adult mammalian brain. These receptors are heteropentamers and are strongly diversified throughout the CNS but the most frequent subunit composition is alpha1-beta2-gamma2. It has been reported that alpha1-gamma2 receptors can be potently expressed in recombinant GABAAR model (Verdoorn et al. 1990). In this study we aimed to characterize the kinetic and pharmacological profile of these receptors in comparison to alpha1-beta2-gamma2 ones. METHODS: We used patch-clamp technique with ultrafast (~10e–4 s) solution exchange based on theta-glass capillaries driven by a piezoelectric translator. We used HEK293 cells which were transiently transfected by GABAAR subunit cDNA using standard calcium phosphate method. Modulators used were zinc ions, low extracellular pH, flurazepam and pentobarbital. RESULTS: Zinc ions inhibited current responses more strongly for alpha1-gamma2 GABAARs, however, zinc effect on desensitization onset was observed only for alpha1-beta2-gamma2 receptors. In the case of responses mediated by alpha1-beta2-gamma2 receptors, lowering extracellular pH enhanced current amplitudes and prolonged deactivation time course of currents elicited by short and saturating GABA pulses to a much larger extent than for alpha1-gamma2 ones. Saturating GABA elicited responses mediated by alpha1-beta2-gamma2 receptors were slightly inhibited by flurazepam, whereas in the case of alpha1-gamma2 receptors, currents were significantly potentiated. Activation by high concentrations of pentobarbital yielded similar rebound current amplitudes in both receptor subtypes. CONCLUSIONS: We found that although alpha1-gamma2 receptors show a similar kinetic profile to alpha1-beta2-gamma2 receptors they are characterized by pharmacological properties that are substantially different. Supported by NCN grant DEC-2013/11/B/NZ3/00983.

GABAA receptor binding site residue Beta2 glutamate 155:possible role in channel preactivation

75%

Kisiel M. , Jatczak M. , Czyzewska M. M. , Brodzki M. , Cajkowski C. , Mozrzymas J. W.

nr Supl.

BACKGROUND AND AIMS: The GABAA receptor is the main mediator responsible for inhibitory transmission in the brain. In our previous work (Szczot et al. 2014), we demonstrated for α1β2γ2 receptors that “classical” channel gating (opening/closing and desensitization) is preceded by a preactivation step, which is most likely initiated at the agonist-binding site. Here, we investigated the role of β2E155 residue in channel gating focusing on preactivation. Residue β2E155 is located in the GABA-binding site and may directly interact with agonist. Moreover, agonist induced local motions near this residue suggests it is an initial trigger that couples agonist binding to channel gating. METHODS: In this study, we combined ultrafast solution exchange with patch-clamp electrophysiology to record macroscopic currents mediated by wild-type and mutant (β2E155C) α1β2γ2 and α1β2 receptors. RESULTS: Cysteine substitution of β2E155 caused a large right-shift of the dose–response curves for GABA-elicited currents, which was independent of the presence of γ2 subunit. Furthermore, especially for α1β2γ2 receptors, β2E155C slowed down macroscopic desensitization kinetics. The mutant receptors also exhibited spontaneous channel activity. Taken together, the data suggest this mutation alters not only GABA binding but also GABA-mediated gating transitions. Nonstationary noise analysis of variance showed that for α1β2γ2 receptors, the β2E155C mutation significantly decreased maximal open probability without affecting single channel conductance. CONCLUSIONS: Model kinetic simulations of our data indicate that β2E155 is likely involved in preactivation transitions that precede channel opening supporting its role as an initial trigger for coupling binding to gating. This research has been financially supported by grant National Centre of Science grant: DEC-2013/11/B/NZ3/00983 and by ministry grant Pbmn135.