Karol S. Bruzik

Specificity of Intersubunit General Anesthetic-binding Sites in the Transmembrane Domain of the Human α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor

Background: General anesthetics of diverse chemical structure potentiate GABAA receptors by binding to unknown sites. Results: A photoreactive barbiturate identifies intersubunit-binding sites distinct from, but homologous to, sites identified by photoreactive etomidate analogs. Conclusion: Propofol, barbiturates, and etomidate analogs bind with variable selectivities to two classes of sites. Significance: This study helps define the diversity of GABAA receptor general anesthetic-binding sites. GABA type A receptors (GABAAR), the brains major inhibitory neurotransmitter receptors, are the targets for many general anesthetics, including volatile anesthetics, etomidate, propofol, and barbiturates. How such structurally diverse agents can act similarly as positive allosteric modulators of GABAARs remains unclear. Previously, photoreactive etomidate analogs identified two equivalent anesthetic-binding sites in the transmembrane domain at the β+-α− subunit interfaces, which also contain the GABA-binding sites in the extracellular domain. Here, we used R-[3H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB), a potent stereospecific barbiturate anesthetic, to photolabel expressed human α1β3γ2 GABAARs. Protein microsequencing revealed that R-[3H]mTFD-MPAB did not photolabel the etomidate sites at the β+-α− subunit interfaces. Instead, it photolabeled sites at the α+-β− and γ+-β− subunit interfaces in the transmembrane domain. On the (+)-side, α1M3 was labeled at Ala-291 and Tyr-294 and γ2M3 at Ser-301, and on the (−)-side, β3M1 was labeled at Met-227. These residues, like those in the etomidate site, are located at subunit interfaces near the synaptic side of the transmembrane domain. The selectivity of R-etomidate for the β+-α− interface relative to the α+-β−/γ+-β− interfaces was >100-fold, whereas that of R-mTFD-MPAB for its sites was >50-fold. Each ligand could enhance photoincorporation of the other, demonstrating allosteric interactions between the sites. The structural heterogeneity of barbiturate, etomidate, and propofol derivatives is accommodated by varying selectivities for these two classes of sites. We hypothesize that binding at any of these homologous intersubunit sites is sufficient for anesthetic action and that this explains to some degree the puzzling structural heterogeneity of anesthetics.

Multiple Propofol-binding Sites in a γ-Aminobutyric Acid Type A Receptor (GABAAR) Identified Using a Photoreactive Propofol Analog

Background: Propofol binding to GABAAR sites of uncertain location potentiates receptor function and produces anesthesia in vivo. Results: A photoreactive propofol analog identifies propofol-binding sites in α1β3 GABAARs. Conclusion: Propofol binds to each class of intersubunit sites in the GABAAR transmembrane domain. Significance: This study demonstrates that propofol binds to the same sites in a GABAAR as etomidate and barbiturates. Propofol acts as a positive allosteric modulator of γ-aminobutyric acid type A receptors (GABAARs), an interaction necessary for its anesthetic potency in vivo as a general anesthetic. Identifying the location of propofol-binding sites is necessary to understand its mechanism of GABAAR modulation. [3H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[3H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (etomidate) and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit-binding sites for general anesthetics in the GABAAR transmembrane domain. Here, we use a photoreactive analog of propofol (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol ([3H]AziPm)) to identify propofol-binding sites in heterologously expressed human α1β3 GABAARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [3H]AziPm photoincorporation into GABAAR subunits maximally by ∼50%. When the amino acids photolabeled by [3H]AziPm were identified by protein microsequencing, we found propofol-inhibitable photolabeling of amino acids in the β3-α1 subunit interface (β3Met-286 in β3M3 and α1Met-236 in α1M1), previously photolabeled by [3H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [3H]AziPm photolabeling of β3Met-227 in βM1, the amino acid in the α1-β3 subunit interface photolabeled by R-[3H]mTFD-MPAB. The propofol-inhibitable [3H]AziPm photolabeling in the GABAAR β3 subunit in conjunction with the concentration dependence of inhibition of that photolabeling by etomidate or R-mTFD-MPAB also establish that each anesthetic binds to the homologous site at the β3-β3 subunit interface. These results establish that AziPm as well as propofol bind to the homologous intersubunit sites in the GABAAR transmembrane domain that binds etomidate or R-mTFD-MPAB with high affinity.

Conformation of the polar headgroup of sphingomyelin and its analogues

The conformation of the polar headgroup of synthetic D-erythro-stearoylsphingomyelin (1), its L-threo-isomer (2) and phosphorothioyl analogues of 1 (3 and 4) has been studied in detail by high-resolution NMR spectroscopy. In both monomeric and aggregated states the phosphocholine function of 1 adopts the synclinal conformation (alpha 5 torsional angle), in analogy with phosphatidylcholine (Hauser, H., Guyer, W., Pascher, I., Skrabal, P. and Sundell, S. (1980) Biochemistry 19, 366-373). The conformation about the C1-C2 bond (theta 1 angle) of the sphingosine backbone is predominantly -synclinal, analogously to the conformation of the crystalline galactosyl cerebroside (Pascher, I. and Sundell, S. (1977) Chem. Phys. Lipids 20, 175-191). In contrast, the L-threo-isomer displays unrestricted rotation about C1-C2 bond. The possibility of the existence of a hydrogen bond between the 3-hydroxyl function and the bridged oxygen atom of sphingosine responsible for the different conformation of 1 and 2 is discussed. The modification of the phosphate function in 1 with sulfur has no significant effect on the conformation of the resulting analogues. The conformation of all studied compounds about the C-O phosphoester bonds (alpha 1 and alpha 4 torsion angles) is mainly antiperiplanar. Similar to other double-chain phospholipids, sphingomyelin shows a preference towards the antiperiplanar conformation about the C2-C3 bond.

Regulation of Ca2+-dependent Cl- conductance in a human colonic epithelial cell line (T84): cross-talk between Ins(3,4,5,6)P4 and protein phosphatases.

1 We have studied the regulation of whole‐cell chloride current in T84 colonic epithelial cells by inositol 3,4,5,6‐tetrakisphosphate (Ins(3,4,5,6)P4). New information was obtained using (a) microcystin and okadaic acid to inhibit serine/threonine protein phosphatases, and (b) a novel functional tetrakisphosphate analogue, 1,2‐bisdeoxy‐1,2‐bisfluoro‐Ins(3,4,5,6)P4 (i.e. F2‐Ins(3,4,5,6)P4). 2 Calmodulin‐dependent protein kinase II (CaMKII) increased chloride current 20‐fold. This current (ICl,CaMK) continued for 7 ± 1.2 min before its deactivation, or running down, by approximately 60 %. This run‐down was prevented by okadaic acid, whereupon ICl,CaMK remained near its maximum value for ≥ 14.3 ± 0.6 min. 3 F2‐Ins(3,4,5,6)P4 inhibited ICl,CaMK (IC50= 100 μM) stereo‐specifically, since its enantiomer, F2‐Ins(1,4,5,6)P4 had no effect at <= 500 μM. Dose‐response data (Hill coefficient = 1.3) showed that F2‐Ins(3,4,5,6)P4 imitated only the non‐co‐operative phase of inhibition by Ins(3,4,5,6)P4, and not the co‐operative phase. 4 Ins(3,4,5,6)P4 was prevented from blocking ICl,CaMK by okadaic acid (IC50= 1.5 nM) and microcystin (IC50= 0.15 nM); these data lead to the novel conclusion that, in situ, protein phosphatase activity is essential for Ins(3,4,5,6)P4 to function. The IC50 values indicate that more than one species of phosphatase was required. One of these may be PP1, since F2‐Ins(3,4,5,6)P4‐dependent current blocking was inhibited by okadaic acid and microcystin with IC50 values of 70 nM and 0.15 nM, respectively.

The synthesis of homochiral inositol phosphates from myo-inositol

Abstract A new synthetic procedure for efficient conversion of myo -inositol into homochiral inositol phosphates is presented, and is illustrated with total synthesis of myo -inositol 1-phosphate, 2-deoxy- myo -inositol 1-phosphate, myo -inositol 3-phosphate, myo -inositol 4-phosphate, myo -inositol 1,4-bisphosphate, myo -inositol 1,4,5-trisphosphate, and myo -inositol 3,4,5,6-tetrakisphosphate. The syntheses start with selfresolving myo -inositol camphanylidene cis -monoacetals 2a and 2a′ , which are obtained in one step from the parent cyclitol and D- and L-camphor dimethyl acetal, respectively, and are harvested conveniently by means of a precipitation driven equilibration. The syntheses feature in the key steps the selective monophosphorylation, selective bissilylation and selective trisacylation of 2a and 2a′ , as well as the use of dibenzyl phosphorochloridate and 2-dimethylamino-5,6-benzo-1,3,2-dioxaphosphepane for effecting mono and polyphosphorylations, respectively. In support of stereochemical assignments an X-ray structure of one of the intermediate fully protected inositol derivatives is also presented.

Structural and Membrane Binding Analysis of the Phox Homology Domain of Phosphoinositide 3-Kinase-C2α

Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of diverse PI specificities of PX domains, we determined the crystal structure of the PX domain from phosphoinositide 3-kinase C2α (PI3K-C2α), which binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). To delineate the mechanism by which this PX domain interacts with membranes, we measured the membrane binding of the wild type domain and mutants by surface plasmon resonance and monolayer techniques. This PX domain contains a signature PI-binding site that is optimized for PtdIns(4,5)P2 binding. The membrane binding of the PX domain is initiated by nonspecific electrostatic interactions followed by the membrane penetration of hydrophobic residues. Membrane penetration is specifically enhanced by PtdIns(4,5)P2. Furthermore, the PX domain displayed significantly higher PtdIns(4,5)P2 membrane affinity and specificity when compared with the PI3K-C2α C2 domain, demonstrating that high affinity PtdIns(4,5)P2 binding was facilitated by the PX domain in full-length PI3K-C2α. Together, these studies provide new structural insight into the diverse PI specificities of PX domains and elucidate the mechanism by which the PI3K-C2α PX domain interacts with PtdIns(4,5)P2-containing membranes and thereby mediates the membrane recruitment of PI3K-C2α.

Allyl m-Trifluoromethyldiazirine Mephobarbital: An Unusually Potent Enantioselective and Photoreactive Barbiturate General Anesthetic

We synthesized 5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (14), a trifluoromethyldiazirine-containing derivative of general anesthetic mephobarbital, separated the racemic mixture into enantiomers by chiral chromatography, and determined the configuration of the (+)-enantiomer as S by X-ray crystallography. Additionally, we obtained the (3)H-labeled ligand with high specific radioactivity. R-(-)-14 is an order of magnitude more potent than the most potent clinically used barbiturate, thiopental, and its general anesthetic EC(50) approaches those for propofol and etomidate, whereas S-(+)-14 is 10-fold less potent. Furthermore, at concentrations close to its anesthetic potency, R-(-)-14 both potentiated GABA-induced currents and increased the affinity for the agonist muscimol in human α1β2/3γ2L GABA(A) receptors. Finally, R-(-)-14 was found to be an exceptionally efficient photolabeling reagent, incorporating into both α1 and β3 subunits of human α1β3 GABA(A) receptors. These results indicate R-(-)-14 is a functional general anesthetic that is well-suited for identifying barbiturate binding sites on Cys-loop receptors.

General synthesis of phosphatidylinositol 3-phosphates

Uniform synthetic approach to phosphatidylinositol 3-phosphate, 3,4-bisphosphate and 3,4,5-trisphosphate has been elaborated starting from 1D-1-O-tert-butyldiphenylsilyl- and 1,6-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-myo-inositols using regioselective benzoylation at the 3-, 3,4- and 3,4,5-positions of inositol.

Robust photoregulation of GABA A receptors by allosteric modulation with a propofol analogue

Photochemical switches represent a powerful method for improving pharmacological therapies and controlling cellular physiology. Here we report the photo-regulation of GABAA receptors (GABAARs) by a derivative of propofol (2,6-diisopropylphenol), a GABAAR allosteric modulator, that we have modified to contain photo-isomerizable azobenzene. Using α1β2γ2 GABAARs expressed in Xenopus laevis oocytes and native GABAARs of isolated retinal ganglion cells, we show that the trans-azobenzene isomer of the new compound (trans-MPC088), generated by visible light (wavelengths ~440 nm), potentiates the GABA-elicited response and at higher concentrations directly activates the receptors. cis-MPC088, generated from trans-MPC088 by UV light (~365 nm), produces little if any receptor potentiation/activation. In cerebellar slices, MPC088 co-applied with GABA affords bidirectional photo-modulation of Purkinje cell membrane current and spike-firing rate. The findings demonstrate photo-control of GABAARs by an allosteric ligand and open new avenues for fundamental and clinically oriented research on GABAARs, a major class of neurotransmitter receptors in the central nervous system.

Synthesis of the enantiomeric 1,4,5,6-tetra-O-benzyl-myo inositols

Abstract The synthesis of the optically pure 1,4,5,6-tetra-O-benzyl-myo-inositols was achieved in four steps via diastereomeric 2,3-spiro-acetals of myo-inositol with l - and d -camphor as the key intermediates. Camphor dimethyl acetal was used for the acetalation reaction. The diastereomers were benzylated and separated by chromatography, and the structures of the products were determined by 2D- NOESY 1H-n.m.r. spectroscopy. Hydrolysis of the diastereomers then afforded the title products.