Fragrant dioxane derivatives identify beta1-subunit-containing GABAA receptors

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.

FIGURE 1.

PI24513 modulates GABA-mediated currents in recombinant GABA_A receptors expressed in Xenopus oocytes. A, chemical structure of PI24513 (R₁ = CH₃), a stereoisomer mix of 80% (−)-2S,4R,6S-trimethyl-4-(4′-methylphenyl)-[1,3]-dioxane) and 20% (−)-2S,4S,6S-trimethyl-4-(4′-methylphenyl)-[1,3]-dioxane) and VC (R₁ = H) with the same stereoisomer ratio. B, concentration-response curves for the GABA-modulating action of PI24513 at α₁β_1–3γ_2L GABA_A receptors. Values are shown as means of 7–8 oocytes. C, example of response to PI24513 in the absence of GABA compared with the maximal GABA-evoked response in the α₁β₁γ_2L receptor. D and E, PI24513 affects the affinity of the α₁β₁γ_2L receptor for GABA but not the maximal evoked current. 100 μm PI 24513 lowered the EC₅₀ value for GABA from 11.2 ± 0.3 μm to 2.2 ± 0.2 μm (n = 4) but had virtually no effect on the maximal evoked current at saturating GABA concentrations. F, potentiation of GABA-evoked current by 100 μm PI24513 in oocytes expressing homomeric β₁ GABA_A receptors. G, example of 100 μm PI 24513 action on GABA-evoked responses in oocytes expressing α₁β₁γ_2L (co-applied with 3 μm of GABA) or α₁β_1(M286W)γ_2L (co-applied with 1 μm of GABA).

FIGURE 2.

Pharmacological, immunohistochemical, and single cell RT-PCR analysis of GABA_AR expression in hypothalamic TMN and cerebellar Purkinje neurons. A, photographs of acutely isolated TMN and Purkinje neurons (left) and gels illustrating RT-PCR analysis of GABA_AR expression in the same neurons (middle) and in positive controls (right). M: DNA size marker (100 bp ladder). B, SCS inhibits GABA-evoked responses in TMN but does not affect them in Purkinje neurons. C, dose-response curves illustrating the difference between PN and TMN neurons in FDD modulation. D, HEK293 cells expressing recombinant GABA_AR containing different β-subunit types stained with β1-antisera. Scale bar, 20 μm. E, extracted co-localized points (left) and original image of rat TMN neurons in slice (right) stained with histidine decarboxylase (HDC, cy3) and β1 (AF488) antibodies. Scale bar, 10 μm.

FIGURE 3.

Spontaneous inhibitory postsynaptic currents (sIPSC) are modulated by FDD differently in TMN and Purkinje neurons. A, summary histograms illustrating change (% of control) in time to decay, amplitude and area of sIPSCs in the presence of VC or PI24513 taken at 20 μm in rat TMN neurons. Number of investigated neurons (n) is given above the plot. B, representative traces of sIPSC recordings in mouse TMN neuron in control and in the presence of different concentrations of VC. On the left side, cumulative decay time fraction plots are given comparing whole control and FDD periods. Kolmogorov-Smirnov Z = 0.54, 3.7, and 4.45 for the upper, middle, and lower plots, respectively, p values are given next to the plots. C, average values for the time to decay in control and in the presence of different concentrations of FDD in two neuronal groups (values for each concentration are compared with their own controls measured in the same experiment, p < 0.05(*), p < 0.01(**), p < 0.005(***)). Number of cells is indicated on gray bars. D, examples of averaged sIPSCs (56–315 events averaged for each picture) obtained in one experiment either in TMN (left) or in Purkinje neuron (right).

FIGURE 4.

Hypothalamic neurons functionally express the β1-subunit of GABA_AR. A, colocalization of MAP2 (blue, AF350) and β1 (green, AF488) proteins in control. Asterisks indicate staining of the nuclei of glial (MAP2-negative) cell. B, distribution of β1 immunoreactivity (AF488) in neurons after patch-clamp recordings (filled with biocytin, in red). Ten-day-old cultures were grown further either in transfection medium without siRNA (control) or with β1-siRNA for 5 days. Left: β1-subunit immunoreactivity, right: co-localization of biocytin and β1 immunoreactivities. Scale bars in A and B, 20 μm. C, dose-response curves for PI 24513-modulation of GABA responses (EC_10–20) differ between control and siRNA-treated hypothalamic neurons. Averaged (4–9 cells) EC₅₀ values are indicated. D, firing frequency of total population of hypothalamic neurons, normalized to the corresponding control value, measured with MEAs as total number of spikes (NoS) per min, is dose-dependently reduced by FDD or propofol. Reduced sensitivity to FDD is seen after β1-siRNA treatment. Open symbols indicate control measurements, filled symbols the measurements in cultures treated with β1-siRNA. Significance levels for the difference in modulatory action of FDD are indicated with stars. *, p < 0.05; **, p < 0.01; ***, p < 0.005.