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1.
Cholesterol is an essential component of cell membranes, and is required for mammalian pentameric ligand-gated ion channel (pLGIC) function. Computational studies suggest direct interactions between cholesterol and pLGICs but experimental evidence identifying specific binding sites is limited. In this study, we mapped cholesterol binding to Gloeobacter ligand-gated ion channel (GLIC), a model pLGIC chosen for its high level of expression, existing crystal structure, and previous use as a prototypic pLGIC. Using two cholesterol analogue photolabeling reagents with the photoreactive moiety on opposite ends of the sterol, we identified two cholesterol binding sites: an intersubunit site between TM3 and TM1 of adjacent subunits and an intrasubunit site between TM1 and TM4. In both the inter- and intrasubunit sites, cholesterol is oriented such that the 3‑OH group points toward the center of the transmembrane domains rather than toward either the cytosolic or extracellular surfaces. We then compared this binding to that of the cholesterol metabolite, allopregnanolone, a neurosteroid that allosterically modulates pLGICs. The same binding pockets were identified for allopregnanolone and cholesterol, but the binding orientation of the two ligands was markedly different, with the 3‑OH group of allopregnanolone pointing to the intra- and extracellular termini of the transmembrane domains rather than to their centers. We also found that cholesterol increases, whereas allopregnanolone decreases the thermal stability of GLIC. These data indicate that cholesterol and neurosteroids bind to common hydrophobic pockets in the model pLGIC, GLIC, but that their effects depend on the orientation and specific molecular interactions unique to each sterol.  相似文献   

2.
We have investigated the effect of cholesterol and two abundant phytosterols (sitosterol and stigmasterol) on the voltage-dependent anion-selective channel (VDAC) purified from mitochondria of bean seeds (Phaseolus coccineus). These sterols differ by the degree of freedom of their lateral chain. We show that VDAC displays sensitivity to the lipid-sterol ratio and to the type of sterol found in the membrane. The main findings of this study are: 1), cholesterol and phytosterols modulate the selectivity but only stigmasterol alters the voltage-dependence of the plant VDAC in the range of sterol fraction found in the plant mitochondrial membrane; 2), VDAC unitary conductance is not affected by the addition of sterols; 3), the effect of sterols on the VDAC is reversible upon sterol depletion with 10 μM methyl-β-cyclodextrins; and 4), phytosterols are essential for the channel gating at salt concentration prevailing in vivo. A quantitative analysis of the voltage-dependence indicates that stigmasterol inhibits the transition of the VDAC in the lowest subconductance states.  相似文献   

3.
Cellular cholesterol homeostasis is maintained by Scap, an endoplasmic reticulum (ER) protein with eight transmembrane helices. In cholesterol-depleted cells, Scap transports sterol regulatory element-binding proteins (SREBPs) to the Golgi, where the active fragment of SREBP is liberated by proteases so that it can activate genes for cholesterol synthesis. When ER cholesterol increases, Scap binds cholesterol, and this changes the conformation of cytosolic Loop 6, which contains the binding site for COPII proteins. The altered conformation precludes COPII binding, abrogating movement to the Golgi. Consequently, cholesterol synthesis declines. Here, we identify the cholesterol-binding site on Scap as Loop 1, a 245-amino acid sequence that projects into the ER lumen. Recombinant Loop 1 binds sterols with a specificity identical to that of the entire Scap membrane domain. When tyrosine 234 in Loop 1 is mutated to alanine, Loop 6 assumes the cholesterol-bound conformation, even in sterol-depleted cells. As a result, full-length Scap(Y234A) cannot mediate SREBP processing in transfected cells. These results indicate that luminal Loop 1 of Scap controls the conformation of cytosolic Loop 6, thereby determining whether cells produce cholesterol.  相似文献   

4.
NPC2 is a small lysosomal glycoprotein that binds cholesterol with submicromolar affinity. Deficiency in NPC2 is the cause of Niemann-Pick type C2 disease, a fatal neurovisceral disorder characterized by accumulation of cholesterol in lysosomes. Here we report the crystal structure of bovine NPC2 bound to cholesterol-3-O-sulfate, an analog that binds with greater apparent affinity than cholesterol. Structures of both apo-bound and sterol-bound NPC2 were observed within the same crystal lattice, with an asymmetric unit containing one molecule of apoNPC2 and two molecules of sterol-bound NPC2. As predicted from a previously determined structure of apoNPC2, the sterol binds in a deep hydrophobic pocket sandwiched between the two beta-sheets of NPC2, with only the sulfate substituent of the ligand exposed to solvent. In the two available structures of apoNPC2, the incipient ligand-binding pocket, which ranges from a loosely packed hydrophobic core to a small tunnel, is too small to accommodate cholesterol. In the presence of sterol, the pocket expands, facilitated by a slight separation of the beta-strands and substantial reorientation of some side chains, resulting in a perfect molding of the pocket around the hydrocarbon portion of cholesterol. A notable feature is the repositioning of two aromatic residues at the tunnel entrance that are essential for NPC2 function. The NPC2 structures provide evidence of a malleable binding site, consistent with the previously documented broad range of sterol ligand specificity.  相似文献   

5.
Selective binding of cholesterol by recombinant fatty acid binding proteins   总被引:3,自引:0,他引:3  
The sterol binding specificity of rat recombinant liver fatty acid binding protein (L-FABP) and intestinal fatty acid binding protein (I-FABP) was characterized with [3H]cholesterol and a fluorescent sterol analog dehydroergosterol. Ligand binding analysis, fluorescence spectroscopy, and activation of microsomal acyl-CoA:cholesterol acyltransferase activity showed that L-FABP-bound sterols. 1) Lipidex-1000 assay showed a dissociation constant Kd = 0.78 +/- 0.18 microM and stoichiometry of 0.47 +/- 0.16 mol/mol for [3H]cholesterol binding to L-PABP. 2) With [3H]cholesterol/phosphatidylcholine liposomes, the cholesterol binding parameters for L-FABP were Kd = 1.53 +/- 0.28 microM and stoichiometry 0.83 +/- 0.07 mol/mol. 3) L-FABP interaction with dehydroergosterol altered the fluorescence intensity and polarization of dehydroergosterol. Dehydroergosterol bound to L-FABP with Kd = 0.37 microM and a stoichiometry of 0.83 mol/mol. 4) Cholesterol and dehydroergosterol decreased L-FABP tyrosine lifetime. Dehydroergosterol binding produced sensitized emission of bound dehydroergosterol with longer lifetime.5) L-FABP bound two cis-parinaric acid molecules/molecule of protein. Cholesterol displaced one of these bound cis-parinaric acids. 6) L-FABP enhanced acyl-CoA:cholesterol acyltransferase in a concentration-dependent manner. In contrast, these assays indicated that I-FABP did not bind sterols. Thus, L-FABP appears able to bind 1 mol of cholesterol/mol of L-FABP, the L-FABP sterol binding site is equivalent to one of the two fatty acid binding sites, and L-FABP stimulates acyl-CoA:cholesterol acyltransferase by transfer of cholesterol.  相似文献   

6.
The physiological effects of anesthetics have been ascribed to their interaction with hydrophobic sites within functionally relevant CNS proteins. Studies have shown that volatile anesthetics compete for luciferin binding to the hydrophobic substrate binding site within firefly luciferase and inhibit its activity (Franks, N. P., and Lieb, W. R. (1984) Nature 310, 599-601). To assess whether anesthetics also compete for ligand binding to a mammalian signal transduction protein, we investigated the interaction of the volatile anesthetic, halothane, with the Rho GDP dissociation inhibitor (RhoGDIalpha), which binds the geranylgeranyl moiety of GDP-bound Rho GTPases. Consistent with the existence of a discrete halothane binding site, the intrinsic tryptophan fluorescence of RhoGDIalpha was quenched by halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) in a saturable, concentration-dependent manner. Bromine quenching of tryptophan fluorescence is short-range and W192 and W194 of the RhoGDIalpha are located within the geranylgeranyl binding pocket, suggesting that halothane binds within this region. Supporting this, N-acetyl-geranylgeranyl cysteine reversed tryptophan quenching by halothane. Short chain n-alcohols ( n < 6) also reversed tryptophan quenching, suggesting that RhoGDIalpha may also bind n-alkanols. Consistent with this, E193 was photolabeled by 3-azibutanol. This residue is located in the vicinity of, but outside, the geranylgeranyl chain binding pocket, suggesting that the alcohol binding site is distinct from that occupied by halothane. Supporting this, N-acetyl-geranylgeranyl cysteine enhanced E193 photolabeling by 3-azibutanol. Overall, the results suggest that halothane binds to a site within the geranylgeranyl chain binding pocket of RhoGDIalpha, whereas alcohols bind to a distal site that interacts allosterically with this pocket.  相似文献   

7.
Defects in Niemann-Pick, Type C-1 protein (NPC1) cause cholesterol, sphingolipids, phospholipids, and glycolipids to accumulate in lysosomes of liver, spleen, and brain. In cultured fibroblasts, NPC1 deficiency causes lysosomal retention of lipoprotein-derived cholesterol after uptake by receptor-mediated endocytosis. NPC1 contains 1278 amino acids that form 13 membrane-spanning helices and three large loops that project into the lumen of lysosomes. We showed earlier that NPC1 binds cholesterol and oxysterols. Here we localize the binding site to luminal loop-1, a 240-amino acid domain with 18 cysteines. When produced in cultured cells, luminal loop-1 was secreted as a soluble dimer. This loop bound [(3)H]cholesterol (K(d), 130 nM) and [(3)H]25-hydroxycholesterol (25-HC, K(d), 10 nM) with one sterol binding site per dimer. Binding of both sterols was competed by oxysterols (24-, 25-, and 27-HC). Unlabeled cholesterol competed strongly for binding of [(3)H]cholesterol, but weakly for [(3)H]25-HC binding. Binding of [(3)H]cholesterol but not [(3)H]25-HC was inhibited by detergents. We also studied NPC2, a soluble protein whose deficiency causes a similar disease phenotype. NPC2 bound cholesterol, but not oxysterols. Epicholesterol and cholesteryl sulfate competed for [(3)H]cholesterol binding to NPC2, but not NPC1. Glutamine 79 in luminal loop-1 of NPC-1 is important for sterol binding; a Q79A mutation abolished binding of [(3)H]cholesterol and [(3)H]25-HC to full-length NPC1. Nevertheless, the Q79A mutant restored cholesterol transport to NPC1-deficient Chinese hamster ovary cells. Thus, the sterol binding site on luminal loop-1 is not essential for NPC1 function in fibroblasts, but it may function in other cells where NPC1 deficiency produces more complicated lipid abnormalities.  相似文献   

8.
Niemann-Pick C disease is a fatal neurodegenerative disorder characterized by an endolysosomal accumulation of cholesterol and other lipids. One form of the disease is caused by a deficiency in NPC2, a soluble lysosomal glycoprotein that binds cholesterol. To better understand the biological function of NPC2 and how its deficiency results in disease, we have characterized the structural and functional properties of recombinant human protein. Highly purified NPC2 consists of a complex mixture of glycosylated isoforms, similar to that observed in human brain autopsy specimens. Mass spectrometric analysis revealed that of the three potential N-linked glycosylation sites present in the mature protein, Asn-19 is not utilized; Asn-39 is linked to an endoglycosidase H (Endo H)-sensitive oligosaccharide, and Asn-116 is variably utilized, either being unmodified or linked to Endo H-sensitive or Endo H-resistant oligosaccharides. All glycoforms are endocytosed and ameliorate the cholesterol storage phenotype of NPC2-deficient fibroblasts. In addition, the purified preparation contains a mixture of both free and lipid-bound protein. All glycoforms bind cholesterol, and sterol binding to NPC2 significantly alters its behavior upon cation-exchange chromatography. Based on this observation, we developed chromatography-based binding assays and determined that NPC2 forms an equimolar complex with the fluorescent cholesterol analog dehydroergosterol. In addition, we find that NPC2 binds a range of cholesterol-related molecules (cholesterol precursors, plant sterols, some oxysterols, cholesterol sulfate, cholesterol acetate, and 5-alpha-cholestan-3-one) and that 27-hydroxysterol accumulates in NPC2-deficient mouse liver. Binding was not detected for various glycolipids, phospholipids, or fatty acids. These biochemical properties support a direct and specialized function of NPC2 in lysosomal sterol transport.  相似文献   

9.
Structural mechanisms of modulation of γ-aminobutyric acid (GABA) type A receptors by neurosteroids and hormones remain unclear. The thyroid hormone L-3,5,3’-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Here we use functional experiments on α2β1γ2 GABAA receptors expressed in Xenopus oocytes to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces. Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites. In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone.  相似文献   

10.
γ-Aminobutyric acid type A (GABAA) receptors in the brain are located in the outer membranes of brain cells where the concentration of cholesterol is high. Of the 25 available high-resolution structures available for GABAA receptors, none were determined in the presence of cholesterol, but four include resolved molecules of cholesterol hemisuccinate (CHS). Here, a molecular docking procedure is used to sweep the transmembrane (TM) surfaces of the receptors for cholesterol binding sites. Cholesterol docking poses determined in this way match 89% of the resolved CHS when CHS molecules deemed unlikely to represent typical bound cholesterols are excluded. The receptors are pentameric, and their TM surfaces consist of a set of five facets, each including pairs of TM helices from two adjacent subunits. Each facet contains hydrophobic hollows running from one side of the membrane to the other, within which are six potential binding sites for cholesterol, three on each side of the membrane. High-resolution structures of GABAA receptors with bound neurosteroids show that neurosteroids bind in these cholesterol binding sites, so the binding of neurosteroids and cholesterol will be competitive.  相似文献   

11.
The voltage-dependent anion channel (VDAC) is the most abundant protein of the mitochondrial outer membrane (MOM) where it regulates transport of ions and metabolites in and out of the organelle. VDAC function is extensively studied in a lipid bilayer system that allows conductance monitoring of reconstituted channels under applied voltage. The process of switching from a high-conductance state, open to metabolites, to a variety of low-conducting states, which excludes metabolite transport, is termed voltage gating and the mechanism remains poorly understood. Recent studies have implicated the involvement of the membrane-solvated residue E73 in the gating process through β-barrel destabilization. However, there has been no direct experimental evidence of E73 involvement in VDAC1 voltage gating. Here, using electrophysiology measurements, we exclude the involvement of E73 in murine VDAC1 (mVDAC1) voltage gating process. With an established protocol of assessing voltage gating of VDACs reconstituted into planar lipid membranes, we definitively show that mVDAC1 gating properties do not change when E73 is replaced by either a glutamine or an alanine. We further demonstrate that cholesterol has no effect on mVDAC1 gating characteristics, though it was shown that E73 is coordinating residue in the cholesterol binding site. In contrast, we found a pronounced gating effect based on the charge of the phospholipid headgroup, where the positive charge stimulates and negative charge suppresses gating. These findings call for critical evaluation of the existing models of VDAC gating and contribute to our understanding of VDAC's role in control of MOM permeability and regulation of mitochondrial respiration and metabolism.  相似文献   

12.
Overexpression of the adipocyte differentiation and determination factor-1 (ADD-1) or sterol regulatory element binding protein-1 (SREBP-1) induces the expression of numerous genes involved in lipid metabolism, including lipoprotein lipase (LPL). Therefore, we investigated whether LPL gene expression is controlled by changes in cellular cholesterol concentration and determined the molecular pathways involved. Cholesterol depletion of culture medium resulted in a significant induction of LPL mRNA in the 3T3-L1 preadipocyte cell line, whereas addition of cholesterol reduced LPL mRNA expression to basal levels. Similar to the expression of the endogenous LPL gene, the activity of the human LPL gene promoter was enhanced by cholesterol depletion in transient transfection assays, whereas addition of cholesterol caused a reversal of its induction. The effect of cholesterol depletion upon the human LPL gene promoter was mimicked by cotransfection of expression constructs encoding the nuclear form of SREBP-1a, -1c (also called ADD-1) and SREBP-2. Bioinformatic analysis demonstrated the presence of 3 potential sterol regulatory elements (SRE) and 3 ADD-1 binding sequences (ABS), also known as E-box motifs. Using a combination of in vitro protein-DNA binding assays and transient transfection assays of reporter constructs containing mutations in each individual site, a sequence element, termed LPL-SRE2 (SRE2), was shown to be the principal site conferring sterol responsiveness upon the LPL promoter. These data furthermore underscore the importance of SRE sites relative to E-boxes in the regulation of LPL gene expression by sterols and demonstrate that sterols contribute to the control of triglyceride metabolism via binding of SREBP to the LPL regulatory sequences.  相似文献   

13.
We compared the abilities of cholesterol versus various oxysterols as substrate and/or as activator for the enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT), by monitoring the activity of purified human ACAT1 in response to sterols solubilized in mixed micelles or in reconstituted vesicles. The results showed that 5 alpha,6 alpha-epoxycholesterol and 7 alpha-hydroxycholesterol are comparable with cholesterol as the favored substrates, whereas 7-ketocholesterol, 7 beta-hydroxycholesterol, 5 beta,6 beta-epoxycholesterol, and 24(S),25-epoxycholesterol are very poor substrates for the enzyme. We then tested the ability of 7-ketocholesterol as an activator when cholesterol was measured as the substrate, and vice versa. When cholesterol was measured as the substrate, the addition of 7-ketocholesterol could not activate the enzyme. In contrast, when 7-ketocholesterol was measured as the substrate, the addition of cholesterol significantly activated the enzyme and changed the shape of the substrate saturation curve from sigmoidal to essentially hyperbolic. Additional results show that, as an activator, cholesterol is much better than all the oxysterols tested. These results suggest that ACAT1 contains two types of sterol binding sites; the structural requirement for the ACAT activator site is more stringent than it is for the ACAT substrate site. Upon activation by cholesterol, ACAT1 becomes promiscuous toward various sterols as its substrate.  相似文献   

14.
The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step of steroidogenesis, delivery of cholesterol to the inner mitochondrial membrane. However, the mechanism whereby cholesterol translocation is accomplished has not been resolved. Recombinant StAR proteins lacking the first N-terminal 62 amino acids comprising the mitochondrial-targeting sequence were used to determine if StAR binds cholesterol and alters mitochondrial membrane cholesterol domains to enhance sterol transfer. First, a fluorescent NBD-cholesterol binding assay revealed 2 sterol binding sites (K(d) values near 32 nm), whereas the inactive A218V N-62 StAR mutant had only a single binding site with 8-fold lower affinity. Second, NBD-cholesterol spectral shifts and fluorescence resonance energy transfer from StAR Trp residues to NBD-cholesterol showed (i) close molecular interaction between these molecules (R(2/3) = 33 A) and (ii) sensitized NBD-cholesterol emission from only one of the two sterol binding sites. Third, circular dichroism showed that cholesterol binding induced a change in StAR secondary structure. Fourth, a fluorescent sterol transfer assay that did not require separation of donor and acceptor mitochondrial membranes demonstrated that StAR enhanced mitochondrial sterol transfer as much as 100-fold and induced/increased the formation of rapidly transferable cholesterol domains in isolated mitochondrial membranes. StAR was 67-fold more effective in transferring cholesterol from mitochondria of steroidogenic MA-10 cells than from human fibroblast mitochondria. In contrast, sterol carrier protein-2 (SCP-2) was only 2.2-fold more effective in mediating sterol transfer from steroidogenic cell mitochondria. Taken together these data showed that StAR is a cholesterol-binding protein, preferentially enhances sterol transfer from steroidogenic cell mitochondria, and interacts with mitochondrial membranes to alter their sterol domain structure and dynamics.  相似文献   

15.
The structure of the NPC1L1 N-terminal domain in a closed conformation   总被引:1,自引:0,他引:1  

Background

NPC1L1 is the molecular target of the cholesterol lowering drug Ezetimibe and mediates the intestinal absorption of cholesterol. Inhibition or deletion of NPC1L1 reduces intestinal cholesterol absorption, resulting in reduction of plasma cholesterol levels.

Principal Findings

Here we present the 2.8 Å crystal structure of the N-terminal domain (NTD) of NPC1L1 in the absence of cholesterol. The structure, combined with biochemical data, reveals the mechanism of cholesterol selectivity of NPC1L1. Comparison to the cholesterol free and bound structures of NPC1(NTD) reveals that NPC1L1(NTD) is in a closed conformation and the sterol binding pocket is occluded from solvent.

Conclusion

The structure of NPC1L1(NTD) reveals a degree of flexibility surrounding the entrance to the sterol binding pocket, suggesting a gating mechanism that relies on multiple movements around the entrance to the sterol binding pocket.  相似文献   

16.
Benzodiazepine (BDZ) is generally thought to bind to site II of human serum albumin (HSA), also known as the indole-BDZ site, which is located at subdomain III A of the molecule. However, differences in the binding characteristics of BDZ drugs with HSA have been reported. The photolabeling profiles of HSA with [(3)H]flunitrazepam (FNZP) in the presence and absence of diazepam (DZP) were shown to be identical, suggesting that each drug primarily binds to different regions. The results of fluorescent probe displacement experiments showed that FNZP failed to decrease the fluorescence of dansylsarcosine to an extent similar to that of DZP. In the photoinhibition experiment, site I and site II ligands failed to inhibit the photoincorporation of [(3)H]FNZP to HSA. In order to evaluate the photolabeling specificity of FNZP, an attempt was made to photolabel alpha(1)-acid glycoprotein (AGP) which also binds BDZ with similar affinity as HSA. The effect of myristate (MYR) and DZP on the FNZP photolabeling of these two major drug binding plasma proteins was examined. Photoincorporation was inhibited when HSA was photolabeled with [(3)H]FNZP in the presence of MYR but not in the presence of DZP. Conversely, DZP inhibited the photolabeling of [(3)H]FNZP to AGP. These results suggest that FNZP interacts with HSA at regions which are not located in the preformed binding pocket of subdomain III A.  相似文献   

17.
The free sterol mixture of the sponge Psammaplysilla purpurea was shown to contain aplysterol as the major constituent. In addition to other sterols such as 5,7-cholestadien-3β-ol, cholesterol, 5α-cholestan-3β-ol, 24ε-methylcholesta-5,22-dien-3β-ol, 24ε-methylcholesterol, 24ε-ethylcholesta-5,22-dien-3β-ol and 24,28-dehydroaplysterol, a new minor sterol was isolated and shown by spectral analysis as well as partial synthesis to be 3β-hydroxy-26,27-bisnorcholest-5-en-24-one. The sterol mixture contains no other short side chain or 24-keto sterols except for small amounts of 3β-hydroxypregn-5-en-20-one and 3β-hydroxy-5α-pregnan-20-one.  相似文献   

18.
Bupropion, a clinically used antidepressant and smoking-cessation drug, acts as a noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). To identify its binding site(s) in nAChRs, we developed a photoreactive bupropion analogue, (±)-2-(N-tert-butylamino)-3'-[(125)I]-iodo-4'-azidopropiophenone (SADU-3-72). Based on inhibition of [(125)I]SADU-3-72 binding, SADU-3-72 binds with high affinity (IC(50) = 0.8 μM) to the Torpedo nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with 3-fold higher affinity in the desensitized (IC(50) = 1.2 μM) than in the resting state. Photolabeling of Torpedo nAChRs with [(125)I]SADU-3-72 followed by limited in-gel digestion of nAChR subunits with endoproteinase Glu-C established the presence of [(125)I]SADU-3-72 photoincorporation within nAChR subunit fragments containing M1-M2-M3 helices (αV8-20K, βV8-22/23K, and γV8-24K) or M1-M2 helices (δV8-14). Photolabeling within βV8-22/23K, γV8-24K, and δV8-14 was reduced in the desensitized state and inhibited by ion channel blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine (TCP)) state, and this pharmacologically specific photolabeling was localized to the M2-9 leucine ring (δLeu(265), βLeu(257)) within the ion channel. In contrast, photolabeling within the αV8-20K was enhanced in the desensitized state and not inhibited by TCP but was inhibited by bupropion. This agonist-enhanced photolabeling was localized to αTyr(213) in αM1. These results establish the presence of two distinct bupropion binding sites within the Torpedo nAChR transmembrane domain: a high affinity site at the middle (M2-9) of the ion channel and a second site near the extracellular end of αM1 within a previously described halothane (general anesthetic) binding pocket.  相似文献   

19.
Neuroactive steroids modulate the function of gamma-aminobutyric acid, type A (GABA(A)) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3 alpha,5 beta-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABA(A) receptors as evidenced by its ability to inhibit binding of [(35)S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents in Xenopus oocytes and human endothelial kidney 293 cells expressing GABA(A) receptor subunits (alpha(1)beta(2)gamma(2)). [(3)H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of approximately 35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [(3)H]6-AziP concentration of 1.9 microM, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [(3)H]6-AziP. The beta(2) and beta(3) subunits of the GABA(A) receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABA(A) receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABA(A) receptor may be mediated by binding to an accessory protein, VDAC-1.  相似文献   

20.
The dinoflagellates Amphidinium carterae and Amphidinium corpulentum have been previously characterized as having Δ8(14)-nuclear unsaturated 4α-methyl-5α-cholest-8(14)-en-3β-ol (C28:1) and 4α-methyl-5α-ergosta-8(14),24(28)-dien-3β-ol (amphisterol; C29:2) as predominant sterols, where they comprise approximately 80% of the total sterol composition. These two sterols have hence been considered as possible major sterol biomarkers for the genus. Here, we have examined the sterols of four recently identified species of Amphidinium (Amphidinium fijiense, Amphidinium magnum, Amphidinium theodori, and Amphidinium tomasii) that are closely related to Amphidinium operculatum as part of what is termed the Operculatum Clade to show that each species has its sterol composition dominated by the common dinoflagellate sterol cholesterol (cholest-5-en-3β-ol; C27:1), which is found in many other dinoflagellate genera, rather than Δ8(14) sterols. While the Δ8(14) sterols 4α-methyl-5α-cholest-8(14)-en-3β-ol and 4α,23,24-trimethyl-5α-cholest-8(14),22E-dien-3β-ol (C30:2) were present as minor sterols along with another common dinoflagellate sterol, 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol; C30:1), in some of these four species, amphisterol was not conclusively observed. From a chemotaxonomic perspective, while this does reinforce the genus Amphidinium's ability to produce Δ8(14) sterols, albeit here as minor sterols, these results demonstrate that caution should be used when considering Δ8(14) sterols, especially amphisterol, as Amphidinium-specific biomarkers within these species where cholesterol is the predominant sterol.  相似文献   

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