Fragrant Dioxane Derivatives Identify β1-Subunit-containing GABAA Receptors

Olga A Sergeeva,Olaf Kletke,Andrea Kragler,Anja Poppek,Wiebke Fleischer,Stephan R Schubring,Boris Görg,Helmut L Haas,Xin-Ran Zhu,Hermann Lübbert,Günter Gisselmann,Hanns Hatt

doi:10.1074/jbc.m110.103309

Olga A Sergeeva, Olaf Kletke + Show 10 more

Open Access

https://doi.org/10.1074/jbc.m110.103309

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Abstract

Nineteen GABA(A) receptor (GABA(A)R) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of beta1-subunit-containing GABA(A)Rs is unknown. Here we report the discovery of a new structural class of GABA(A)R positive modulators with unique beta1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed alpha1betaxgamma2L (x-for 1,2,3) GABA(A)R FDD were 6 times more potent at beta1- versus beta2- and beta3-containing receptors. Serine at position 265 was essential for the high sensitivity of the beta1-subunit to FDD and the beta1N286W mutation nearly abolished modulation; vice versa the mutation beta3N265S shifted FDD sensitivity toward the beta1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to beta1-negative cerebellar Purkinje neurons. Immunostaining for the beta1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by beta1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of beta1-containing GABA(A)Rs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABA(A)Rs.

Highlights

TP 1, 3, and 8 and a Heisenberg stipend
Comparison of the GABA-modulatory and GABA-mimetic activities of PI 24513 across WT and point-mutated GABAA receptors expressed in Xenopus oocytes with two-electrode voltage-clamp in an assay system: the ␣1␤x␥2L (x for 1, 2, or 3) recombinant
GABA-mimetic and GABA-modulating Actions of fragrant dioxane derivatives (FDD) on Acutely Isolated Brain Neurons Differing in the Expression of the GABAA receptor (GABAAR) ␤1-Subunit—To explore the ␤1-subunit selectivity of FDDs we studied their effects on neurons acutely isolated from the adult mouse tuberomamillary nucleus (TMN) expressing the ␤1-subunit and cerebellar Purkinje neurons (PN) lacking it [22]

Summary

EXPERIMENTAL PROCEDURES

Expression of Recombinant GABAA Receptors in Xenopus Oocytes and Electrophysiology—GABAA receptor subunit cDNAs and cRNAs were obtained as follows: rat ␣1 and ␤1 cDNAs. 3– 6 days after injection of cRNA, oocytes were screened for receptor expression by two-electrode voltage-clamp recording. Whole-cell patch-clamp recordings in voltage clamp mode, fast drug application, and single cell RT-PCR procedures were performed as described previously [16, 18]. GABAAR Expression Analysis in Native Cells (Single Cell RT-PCR)—Mouse GABAAR cDNAs were amplified in a first amplification round with degenerate subfamily-specific primers, followed by the subunit-specific amplification in the second round. On day 15, patch clamp recordings were done from cultures treated in a parallel way, some cultures were used for the mRNA isolation and semiquantitative real-time RT-PCR analysis of the receptor expression (for methods see Ref. 19). Data are presented as means Ϯ S.E. of the mean (S.E.)

RESULTS

GABA mimetic action of

DISCUSSION