George H. Addona

The cholesterol dependence of activation and fast desensitization of the nicotinic acetylcholine receptor

When nicotinic acetylcholine receptors are reconstituted into lipid bilayers lacking cholesterol, agonists no longer stimulate cation flux. The kinetics of this process are difficult to study because variations in vesicle morphology cause errors in flux measurements. We developed a new stopped-flow fluorescence assay to study activation independently of vesicle morphology. When receptors were rapidly mixed with agonist plus ethidium, the earliest fluorescence increase reported the fraction of channels that opened and their apparent rate of fast desensitization. These processes were absent when the receptor was reconstituted into dioleoylphosphatidylcholine or into a mixture of that lipid with dioleoylphosphatidic acid (12 mol%), even though a fluorescent agonist reported that resting-state receptors were still present. The agonist-induced channel opening probability increased with bilayer cholesterol, with a midpoint value of 9 +/- 1.7 mol% and a Hill coefficient of 1.9 +/- 0.69, reaching a plateau above 20-30 mol% cholesterol that was equal to the native value. On the other hand, the observed fast desensitization rate was comparable to that for native membranes from the lowest cholesterol concentration examined (5 mol%). Thus the ability to reach the open state after activation varies with the cholesterol concentration in the bilayer, whereas the rate of the open state to fast desensitized state transition is unaffected. The structural basis for this is unknown, but an interesting corollary is that the channels of newly synthesized receptors are not fully primed by cholesterol until they are inserted into the plasma membrane--a novel form of posttranslational processing.

Low chemical specificity of the nicotinic acetylcholine receptor sterol activation site

The nicotinic acetylcholine receptor (nAcChoR) has an absolute requirement for cholesterol if agonist-stimulated channel opening is to occur [Biochemistry 25 (1986) 830]. Certain non-polar analogs could replace cholesterol in vectorial vesicle permeability assays. Using a stopped-flow fluorescence assay to avoid the limitations of permeability assays imposed by vesicle morphology, it was shown that polar conjugates of cholesterol could also satisfy the sterol requirement [Biochim. Biophys. Acta 1370 (1998) 299]. Here this assay is used to explore the chemical specificity of sterols. Affinity-purified nAcChoRs from Torpedo were reconstituted into bilayers at mole ratios of 58:12:30 [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phosphate (DOPA)/steroid]. When the enantiomer of cholesterol was used, or when the stereochemistry at the 3-hydroxy group was changed from beta to alpha by substituting epicholesterol for cholesterol, activation was still supported. The importance of cholesterols planar ring structure was tested by comparing planar cholestanol (5alpha-cholestan-3beta-ol) with nonplanar coprostanol (5beta-cholestan-3beta-ol). Both supported activation. Thus, these steroids support activation independent of structural features known to be important for modulation of lipid bilayer properties. This provides indirect support for a steroid binding site possessing very lax structural requirements.

Dynamics and orientation of N+(CD3)(3)-bromoacetylcholine bound to its binding site on the nicotinic acetylcholine receptor

Dynamic and structural information has been obtained for an analogue of acetylcholine while bound to the agonist binding site on the nicotinic acetylcholine receptor (nAcChoR), using wide-line deuterium solid-state NMR. Analysis of the deuterium lineshape obtained at various temperatures from unoriented nAcChoR membranes labeled with deuterated bromoacetylcholine (BAC) showed that the quaternary ammonium group of the ligand is well constrained within the agonist binding site when compared with the dynamics observed in the crystalline solids. This motional restriction would suggest that a high degree of complementarity exists between the quaternary ammonium group of the ligand and the protein within the agonist binding site. nAcChoR membranes were uniaxially oriented by isopotential centrifugation as determined by phosphorous NMR of the membrane phospholipids. Analysis of the deuterium NMR lineshape of these oriented membranes enriched with the nAcChoR labeled with N+(CD3)3-BAC has enabled us to determine that the angle formed between the quaternary ammonium group of the BAC and the membrane normal is 42° in the desensitized form of the receptor. This measurement allows us to orient in part the bound ligand within the proposed receptor binding site.

Discovery and Pharmacology of a Novel Class of Diacylglycerol Acyltransferase 2 Inhibitors

DGAT2 plays a critical role in hepatic triglyceride production, and data suggests that inhibition of DGAT2 could prove to be beneficial in treating a number of disease states. This article documents the discovery and optimization of a selective small molecule inhibitor of DGAT2 as well as pharmacological proof of biology in a mouse model of triglyceride production.

Approaches to proving there are general anesthetic sites on ligand gated ion channels

(1) There are at least two broad classes of general anesthetic action on the anesthetic-sensitive ligand gated superfamily of ion channels. (2) First, some channels may be inhibited upon opening. Pharmacology, kinetics and site directed mutagenesis all suggest that inhibition is mediated by a site on the acetylcholine receptor probably located in the channel lumen. (3) Second, the agonists concentration response curve may be shifted to the left without affecting the maximum response. (4) This effect does not saturate with anesthetic concentration and might involve partial occupancy of many low affinity sites, mechanism consistent with the observation that the conformation changes accompanying channel gating involve most structural features of the receptor and its surrounding environment.

The Location and Nature of General Anesthetic Binding Sites on the Active Conformation of Firefly Luciferase; A Time Resolved Photolabeling Study

Firefly luciferase is one of the few soluble proteins that is acted upon by a wide variety of general anesthetics and alcohols; they inhibit the ATP–driven production of light. We have used time–resolved photolabeling to locate the binding sites of alcohols during the initial light output, some 200 ms after adding ATP. The photolabel 3-azioctanol inhibited the initial light output with an IC50 of 200 µM, close to its general anesthetic potency. Photoincorporation of [3H]3-azioctanol into luciferase was saturable but weak. It was enhanced 200 ms after adding ATP but was negligible minutes later. Sequencing of tryptic digests by HPLC–MSMS revealed a similar conformation–dependence for photoincorporation of 3-azioctanol into Glu-313, a residue that lines the bottom of a deep cleft (vestibule) whose outer end binds luciferin. An aromatic diazirine analog of benzyl alcohol with broader side chain reactivity reported two sites. First, it photolabeled two residues in the vestibule, Ser-286 and Ile-288, both of which are implicated with Glu-313 in the conformation change accompanying activation. Second, it photolabeled two residues that contact luciferin, Ser-316 and Ser-349. Thus, time resolved photolabeling supports two mechanisms of action. First, an allosteric one, in which anesthetics bind in the vestibule displacing water molecules that are thought to be involved in light output. Second, a competitive one, in which anesthetics bind isosterically with luciferin. This work provides structural evidence that supports the competitive and allosteric actions previously characterized by kinetic studies.

A thermodynamic analysis of the partitioning of cholesterol and related compounds between trioleoylglycerol and egg phosphatidylcholine bilayers

The free energy of transfer of a number of alcohols, including cholesterol, from a bulk isotropic lipid phase, trioleoylglycerol (TG), to an anisotropic lipid phase, egg phosphatidylcholine (PC), was determined. n-Alkane-1-ols partitioned preferentially into the bilayer phase; for example, the free energy of transfer of octanol-1 from TG to PC was about -1.0 kcal/mol. This preference declined with increasing number of carbons at a rate of 40 cal/mol of CH2. Cholesterol had a much stronger preference for the bilayer with a free energy of -1.3 kcal/mol, compared to an extrapolated value of -0.2 kcal/mol for a normal alkane-1-ol with the same number of carbon atoms. Thus, the excess free energy of -1.1 kcal/mol represents the favourable interaction of the cholesterol skeleton with the bilayer phase. This conclusion was confirmed by comparing cholesterol 3-hemisuccinate to oleic acid. Substituting TG for water as the standard state has eliminated the large hydrophobic effect and has permitted us to identify for the first time the subtle binding increment of the steroid ring system.

C-terminal Loop Mutations Determine Folding and SecretionProperties of PCSK9

Human genetics and pharmacologic clinical intervention demonstrate the key role of PCSK9 in cholesterol regulation. To understand the role of the C-terminal domain of PCSK9, two human mutations in this region (S462P and A522T PCSK9) have been profiled. Confirming and extending previous observations, S462P and WT PCSK9 bind to LDLR with equivalent affinity; however, while S462P PCSK9 cleavage is unaffected, its secretion is defective, and association with the ER protein-folding sensor calreticulin, increased. In a similar manner, A522T PCSK9 also exhibits defective secretion and an enhanced association with calreticulin. To assess the in vivo lipid phenotype of the S462P and A522T PCSK9 mutations, Pcsk9-/- mice were infected with AAV8’s encoding the different variants. Although liver transcript levels for all were equivalent, circulating levels of S462P PCSK9, and to a lesser degree A522T PCSK9, were reduced relative to WT PCSK9 correlating with the in vitro phenotype. Further, the extent of reduced circulating S462P or A522T PCSK9 correlated well with increases in mouse liver LDLR and reductions of LDL/ total cholesterol. When interpreted within the context of molecular modeling, it appears that the human non-synonymous polymorphisms S462P and A522T destabilize the C-terminal domain of PCSK9 impacting folding and secretion.