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. 2014 Sep;231(17):3479-91.
doi: 10.1007/s00213-014-3568-4. Epub 2014 Apr 23.

11-trifluoromethyl-phenyldiazirinyl Neurosteroid Analogues: Potent General Anesthetics and Photolabeling Reagents for GABAA Receptors

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Free PMC article

11-trifluoromethyl-phenyldiazirinyl Neurosteroid Analogues: Potent General Anesthetics and Photolabeling Reagents for GABAA Receptors

Zi-Wei Chen et al. Psychopharmacology (Berl). .
Free PMC article

Abstract

Rationale: While neurosteroids are well-described positive allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptors, the binding sites that mediate these actions have not been definitively identified.

Objectives: This study was conducted to synthesize neurosteroid analogue photolabeling reagents that closely mimic the biological effects of endogenous neurosteroids and have photochemical properties that will facilitate their use as tools for identifying the binding sites for neurosteroids on GABAA receptors.

Results: Two neurosteroid analogues containing a trifluromethyl-phenyldiazirine group linked to the steroid C11 position were synthesized. These reagents, CW12 and CW14, are analogues of allopregnanolone (5α-reduced steroid) and pregnanolone (5β-reduced steroid), respectively. Both reagents were shown to have favorable photochemical properties with efficient insertion into the C-H bonds of cyclohexane. They also effectively replicated the actions of allopregnanolone and pregnanolone on GABAA receptor functions: they potentiated GABA-induced currents in Xenopus laevis oocytes transfected with α1β2γ2L subunits, modulated [(35)S]t-butylbicyclophosphorothionate binding in rat brain membranes, and were effective anesthetics in Xenopus tadpoles. Studies using [(3)H]CW12 and [(3)H]CW14 showed that these reagents covalently label GABAA receptors in both rat brain membranes and in a transformed human embryonal kidney (TSA) cells expressing either α1 and β2 subunits or β3 subunits of the GABAA receptor. Photolabeling of rat brain GABAA receptors was shown to be both concentration-dependent and stereospecific.

Conclusions: CW12 and CW14 have the appropriate photochemical and pharmacological properties for use as photolabeling reagents to identify specific neurosteroid-binding sites on GABAA receptors.

Figures

Figure 1
Figure 1
Structures of CW14 and CW12.
Figure 2
Figure 2. Photolysis products of CW14, [3 H]CW14 and [3H]3β-CW14
The figure shows a reverse-phase TLC (RP-TLC) plate with the origin at the bottom. More polar compounds migrate further up the plate. CW14 (1), [3H]-CW14 (2), and the 3β-epimer of [3H]CW14 (3) irradiated with ultraviolet light (UV) in either ethanol (ETOH, center) or cyclohexane (right) were analyzed by RP-TLC. Non UV-irradiated compounds 1, 2 and 3 were also analyzed by RP-TLC (left). An autoradiogram of the plate was produced to visualize radiolabeled compounds, followed by charring of the plate to visualize non-radiolabeled compounds. The image is an overlay of the autoradiogram with the charred plates (the black dots indicate the charred spots). The photolysis product slightly more polar than the parent compound (presumed intramolecular reaction) is denoted by *. The CW14-ethanol adduct is denoted by ** and the CW14-cyclohexane adduct is denoted by #.
Figure 3
Figure 3. CW14 and CW12 modulate GABAA receptor function
A. CW14 and CW12 potentiate GABA-elicited currents in Xenopus laevis oocytes expressing α1β2γ2 GABAA receptors. Representative traces of currents elicited by 2 μM GABA in the absence and presence of 0.5 μM pregnanolone (3α5βP) and CW14 (panel A1), or 0.5 μM allopregnanolone (3α5αP) and CW12 (panel A2). Membrane potential was −70 mV. B. CW14 and CW12 have minimal effect on GABA-induced currents in oocytes expressing α1(Q241L)β2γ2L GABAA receptors. Representative traces of currents elicited by 0.2 μM GABA in the absence and presence of 100 μM pentobarbital (PB, positive control) and 0.5 μM CW14 (panel B1) or 100 μM PB and 0.5 μM CW12 (panel B2). Summary graphs (right) show the effects (mean ± SEM) of PB and CW14 of (panel B1; n =5 oocytes) or PB and CW12 (panel B2; n =4 oocytes) relative to the current produced by 0.2 μM GABA alone. * = P< 0.05 compared to GABA alone; **P< 0.01 compared to GABA alone; ns = not significant compared to GABA alone. C. Direct activation of GABAA receptors by CW12 and CW14 in oocytes expressing α1β2γ2L GABAA receptors. Representative traces (left) of currents elicited by 0.1, 0.5, 1.0 and 10 μM CW12 and CW14 or 2 μM GABA (grey trace). Bar graph shows concentration-dependent activation of current by CW12 and CW14 (n=4 for each concentration) expressed as a fraction of the current elicited by 2 μM GABA.
Figure 4
Figure 4. Biological actions of CW12 and CW14
A. CW12 (panel A1) and CW14 (panel A2) modulation of [35S]TBPS binding in rat brain membranes. Each curve represents the mean ± SEM of three experiments with each point performed in triplicate. Closed circles (continuous lines) are from experiments performed in the absence of GABA and open circles with dashed lines from experiments performed in the presence of 5 μM GABA. B. Anesthetic effects of CW12 and CW14 in tadpoles. Loss of righting reflex (LRR) and loss of swimming reflex (LSR) in tadpoles induced by CW12 (B1) and CW14 (B2). The EC50 values for LRR by CW12 and CW14 were 110 ± 21 nM and 112 ± 14 nM respectively. The EC50 values for LSR by CW12 and CW14 were 655 ± 43 nM and 747 ± 364 nM, respectively.
Figure 5
Figure 5. SDS-PAGE autoradiograms of [3H]CW12 and [3H]CW14 photolabeled rat brain membranes
Rat brain membranes (400 μg) were photolabeled with either: (A) 1 μM [3H]CW12 or; (B) 0.3, 1 and 3 μM [3H]CW14. A band at ≈52-kDa is labeled by both [3H]CW12 and [3H]CW14. Photolabeling of the ≈52-kDa band by [3H]CW14 is concentration-dependent.
Figure 6
Figure 6. Blue native-PAGE of [3H]CW 14 photolabeled rat brain membranes
A. Rat brain membranes (400 μg) photolabeled with 3 μM [3H]CW14 were loaded onto a BN-PAGE followed by autoradiography, gel slicing or immunobloting with antibodies to the α1, β2/3 (bd17) or γ2 subunits of the GABAA receptor, to VDAC-1 or to β-tubulin. The photolabeled proteins correspond to GABAA receptors (≈240 and ≈480-kDa) as shown by anti-α1, bd17 and anti-γ2 antibodies; tubulin (≈1236 kDa); and VDAC (≈140 kDa). B. Autoradiogram of blue native-PAGE from rat brain membranes (400 μg) photolabeled with 0.3 and 1 μM [3H]CW14 or the 3β-epimer of [3H]CW14. [3H]CW14 photolabels GABAA receptors (≈240-kDa) in a concentration-dependent pattern. The photolabeling is stereospecific as evidenced by the minimal photolabeling of GABAA receptors and VDAC by [3H]3β-CW14.
Figure 7
Figure 7. CW14 photolabels GABAA receptors expressed in TSA cells
Membranes (400 μg protein) from wild-type TSA cells or TSA cells expressing α1β2 subunits or β3 subunits of the GABAA receptors were photolabeled with 10 μM [3H]CW14. Gel slicing demonstrated photolabeled protein peaks at ≈52-kDa and ≈100-kDa in the α1β2 and β3 containing membranes, corresponding to monomers and dimers of GABAA receptor subunits. No photolabeled peaks were observed in the wild-type membranes.

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