Direct Photoaffinity Labeling Research Articles

Abstract 2140: Cabazitaxel is more active than first generation taxanes in ABCB1(+) cell lines due to its higher intracellular accumulation and reduction in ATPase stimulation

Abstract The taxanes paclitaxel (Taxol) and docetaxel (Taxotere) have substantial clinical activity in breast, ovarian, lung, and other cancers, but their clinical efficacy is limited by preexisting or acquired drug resistance, such as the expression of the ATP-dependent multidrug resistance (MDR) transporter P-glycoprotein (P-gp). Cabazitaxel (Jevtana), the dimethoxy derivative of docetaxel, was identified because of its activity in taxane-resistant models both in vitro and in vivo. Our studies confirm that cabazitaxel is more active in cell variants that express P-gp, and the objective of this study was to investigate cabazitaxel's affinity for the transporter. Taxane accumulation patterns were studied in ABCB1(+) variants using [14C]-radiolabeled docetaxel and cabazitaxel over a time course up to 1 h. Time points were collected and spun (10,000 x g, 1 min) through Nyosil M20 oil thereby terminating uptake, cell pellets lysed with 2% (w/v) SDS, and counts determined by liquid scintillation, with all measurements normalized to protein content. The kinetics of drug accumulation between the two taxanes is different, with the maximum intracellular drug concentration achieved faster with cabazitaxel (5 min) than docetaxel (15-30 min) in all cell lines. ABCB1(+) variants accumulated less taxane, and these levels could be restored to parental levels in the presence of known P-gp inhibitors (2 μM PSC-833, valspodar). However, the MDR cell models tested accumulated twice as much cabazitaxel than docetaxel under identical experimental conditions. Efflux in drug-free medium confirmed that ABCB1(+) variants retained 2.2x more cabazitaxel than docetaxel. We observed a strong association (r2 = 0.91) between the degree of taxane resistance conferred by P-gp expression and the accumulation differences observed with the two taxanes. Several low P-gp-expressing cell models were not cross-resistant to cabazitaxel while demonstrating resistance to docetaxel. Furthermore, we determined ATPase stimulation in membranes isolated from our MDR cell models as an indirect measure of P-gp activity following taxane treatment. We observed a 1.9x reduction in sodium orthovanadate-sensitive ATPase stimulation resulting from treatment with cabazitaxel than with docetaxel (1 μM for 30 min). Future experiments will include direct photoaffinity labeling of P-gp with [3H]-azido-cabazitaxel and docetaxel, and competition assays with known P-gp substrates. Our studies indicate that the improved activity of cabazitaxel in MDR models might be due to its reduced affinity for P-gp compared to docetaxel. Citation Format: George E. Duran, Dietmar Weitz, Dorothée Sémiond, Diego Gianolio, Sandrine Macé, Branimir I. Sikic. Cabazitaxel is more active than first generation taxanes in ABCB1(+) cell lines due to its higher intracellular accumulation and reduction in ATPase stimulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2140.

Probing a putative dantrolene-binding site on the cardiac ryanodine receptor

Dantrolene is an inhibitor of intracellular Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). Direct photoaffinity labelling experiments using [3H]azidodantrolene and synthetic domain peptides have demonstrated that this drug targets amino acids 590-609 [termed DP1 (domain peptide 1)] of RyR1 (ryanodine receptor 1), the skeletal muscle RyR isoform. Although the identical sequence exists in the cardiac isoform, RyR2 (residues 601-620), specific labelling of RyR2 by dantrolene has not been demonstrated, even though some functional studies show protective effects of dantrolene on heart function. Here we test whether dantrolene-active domains exist within RyR2 and if so, whether this domain can be modulated. We show that elongated DP1 sequences from RyR1 (DP1-2s; residues 590-628) and RyR2 (DP1-2c; residues 601-639) can be specifically photolabelled by [3H]azidodantrolene. Monoclonal anti-RyR1 antibody, whose epitope is the DP1 region, can recognize RyR1 but not RyR2 in Western blot and immunoprecipitation assays, yet it recognizes both DP1-2c and DP1-2s. This suggests that although the RyR2 sequence has an intrinsic capacity to bind dantrolene in vitro, this site may be poorly accessible in the native channel protein. To examine whether it is possible to modulate this site, we measured binding of [3H]dantrolene to cardiac SR as a function of free Ca2+. We found that > or =10 mM EGTA increased [3H]dantrolene binding to RyR2 by approximately 2-fold. The data suggest that the dantrolene-binding site on RyR2 is conformationally sensitive. This site may be a potential therapeutic target in cardiovascular diseases sensitive to dysfunctional intracellular Ca2+ release.

Key differences in molecular complexes of the cholecystokinin receptor with structurally related peptide agonist, partial agonist, and antagonist.

The molecular basis of docking of receptor ligands having differences in biological activity and their subsequent effects on receptor conformation represent areas of great interest. In this work, we focus on the sulfated tyrosyl residue in position 27 of cholecystokinin (CCK) and its spatial approximation with the type A CCK receptor residue Arg(197) that has been predicted from mutagenesis experiments. We have examined the requirement for sulfation of this residue in a series of structurally related peptide agonists, partial agonists, and antagonists using assays of receptor binding and biological activity. Whereas sulfation of CCK position 27 was critical for affinity and potency of a full agonist, it had progressively less effect as the biological activity of the ligand was reduced. It had an intermediate effect on the partial agonist and no effect on the antagonist. In addition, photoaffinity labeling was used to determine the spatial approximations between the receptor and residue 27 of the agonist and antagonist in this series. Direct photoaffinity labeling with a full agonist probe confirmed the spatial approximation of ligand residue 27 and receptor residue Arg(197) in the active complex. Of note, the analogous antagonist probe labeled a distinct region within the receptor amino terminus, confirming a key structural difference in active and inactive complexes.

G protein-coupled receptors as direct targets of inhaled anesthetics.

The molecular pharmacology of inhalational anesthetics remains poorly understood. Despite accumulating evidence suggesting that neuronal membrane proteins are potential targets of inhaled anesthetics, most currently favored membrane protein targets lack any direct evidence for anesthetic binding. We report herein the location of the binding site for the inhaled anesthetic halothane at the amino acid residue level of resolution in the ligand binding cavity in a prototypical G protein-coupled receptor, bovine rhodopsin. Tryptophan fluorescence quenching and direct photoaffinity labeling with [(14)C]halothane suggested an interhelical location of halothane with a stoichiometry of 1 (halothane/rhodopsin molar ratio). Radiosequence analysis of [(14)C]halothane-labeled rhodopsin revealed that halothane contacts an amino acid residue (Trp265) lining the ligand binding cavity in the transmembrane core of the receptor. The predicted functional consequence, competition between halothane and the ligand retinal, was shown here by spectroscopy and is known to exist in vivo. These data suggest that competition with endogenous ligands may be a general mechanism of the action of halothane at this large family of signaling proteins.

Transport of leukotriene C4 and structurally related conjugates

Transport proteins control the release of the endogenous glutathione conjugate leukotriene C4 (LTC4) from leukotriene-synthesizing cells as well as its hepatobiliary and renal elimination. The photolabile conjugated triene structure of LTC4 has enabled direct photoaffinity labeling of the multidrug resistance protein 1 (MRP1, symbol ABC C1) in membranes from mastocytoma cells, leading to the identification of the function of this protein as an ATP-dependent export pump for LTC4 and structurally related conjugates. MRP1 is assigned to the C branch of the superfamily of ATP-binding cassette (ABC) transporters and was originally identified by virtue of its association with drug resistance in tumor cells. Besides LTC4, which is a high-affinity substrate, a variety of conjugates of hydrophobic endogenous or xenobiotic substances with glutathione, glucuronate, or sulfate are transported by MRP1. In addition, hydrophobic compounds may undergo cotransport with glutathione. Effective inhibitors of MRP1-mediated transport include structural analogs of LTC4 and of other cysteinyl leukotrienes. The ATP-dependent transport system which transports cysteinyl leukotrienes across the hepatocyte canalicular membrane into bile was cloned and characterized as the second isoform or paralog of the mammalian MRP family, MRP2 (ABC C2). MRP2 is localized to the apical membrane of polarized cells. The overall substrate specificities of MRP1 and MRP2 are similar, despite an amino acid identity of only 48%. The transport proteins mediating the uptake of LTC4 into hepatocytes across the basolateral membrane are members of the organic anion transporter (OATP) branch of the solute carrier (SLC) superfamily and are thus distinct from the ATP-dependent export pumps of the MRP family.

Long-lived reactive intermediate photogenerated from N-(5-azido-2-nitrobenzoyl)- N′-( d-biotinyl)-1,2-diaminoethane as an affinity reagent to streptavidin

Irradiation of a complex between N-(5-azido-2-nitrobenzoyl)- N′-( d-biotinyl)-1,2-diaminoethane ( I) and streptavidin with light of 313 nm led to the covalent attachment of the photobiotin analogue I to the protein. Streptavidin could also be labelled in the dark with prephotolyzed I. These results indicate that a long-lived reactive intermediate was formed upon irradiation. Moreover, after cleavage of labelled streptavidin with proteinase K this intermediate appears to be covalently attached to the same peptide as the one obtained by direct photoaffinity labelling. An iminosulfurane II derived from the reaction of biotin sulfur atom with aryl nitrene is responsible for the dark-labelling reaction. The photoproduct II converts in an aqueous solution almost completely into N-(5-amino-2-nitrobenzoyl)- N′-( d-( S-oxo)biotinyl)-1,2-diaminoethane (the half-life of II is 10 days).

Direct photoaffinity labeling of cellular retinoic acid-binding protein I (CRABP-I) with all-trans-retinoic acid: identification of amino acids in the ligand binding site.

Cellular retinoic acid-binding proteins I and II (CRABP-I and -II, respectively) are transport proteins for all-trans-retinoic acid (RA), an active metabolite of vitamin A (retinol), and have been reported to be directly involved in the metabolism of RA. In this study, direct photoaffinity labeling with [11,12-(3)H]RA was used to identify amino acids comprising the ligand binding site of CRABP-I. Photoaffinity labeling of CRABP-I with [(3)H]RA was light- and concentration-dependent and was protected by unlabeled RA and various retinoids, indicating that the labeling was directed to the RA-binding site. Photolabeled CRABP-I was hydrolyzed with endoproteinase Lys-C to yield radioactive peptides, which were separated by reversed-phase HPLC for analysis by Edman degradation peptide sequencing. This method identified five modified amino acids from five separate HPLC fractions: Trp7, Lys20, Arg29, Lys38, and Trp109. All five amino acids are located within one side of the "barrel" structure in the area indicated by the reported crystal structure as the ligand binding site. This is the first direct identification of specific amino acids in the RA-binding site of CRABPs by photoaffinity labeling. These results provide significant information about the ligand binding site of the CRABP-I molecule in solution.

Halothane binding to a G protein coupled receptor in retinal membranes by photoaffinity labeling.

General anesthetics have been reported to alter the functions of G protein coupled receptor (GPCR) signaling systems. To determine whether these effects might be mediated by direct binding interactions with the GPCR or its associated G protein, we studied the binding character of halothane on mammalian rhodopsin, structurally the best understood GPCR, by using direct photoaffinity labeling with [(14)C]halothane. In the bleached bovine rod disk membranes (RDM), opsin and membrane lipids were dominantly photolabeled with [(14)C]halothane, but none of the three G protein subunits were labeled. In opsin itself, halothane labeling was inhibited by unlabeled halothane with an IC(50) of 0.9 mM and a Hill coefficient of -0.8. The stoichiometry was 1.1:1.0 (halothane:opsin molar ratio). The IC(50) values of isoflurane and 1-chloro-1,2, 2-trifluorocyclobutane were 5.0 and 15 mM, respectively. Ethanol had no effect on opsin labeling by halothane. A nonimmobilizer, 1, 2-dichlorohexafluorocyclobutane, inhibited halothane labeling by 50% at 0.05 mM. The present results demonstrate that halothane binds specifically and selectively to GPCRs in the RDM. The absence of halothane binding to any of the G protein subunits strongly suggests that the functional effects of halothane on GPCR signaling systems are mediated by direct interactions with receptor proteins.

General Anesthetic Binding to Gramicidin A: The Structural Requirements

There is a distinct possibility that general anesthetics exert their action on the postsynaptic receptor channels. The structural requirements for anesthetic binding in transmembrane channels, however, are largely unknown. High-resolution 1H nuclear magnetic resonance and direct photoaffinity labeling were used in this study to characterize the volatile anesthetic binding sites in gramicidin A (gA) incorporated into sodium dodecyl sulfate (SDS) micelles and into dimyristoylphosphatidylcholine (DMPC) bilayers, respectively. To confirm that the structural arrangement of the peptide side chains can affect anesthetic binding, gA in nonchannel forms in methanol was also analyzed. The addition of volatile anesthetic halothane to gA in SDS with a channel conformation caused a concentration-dependent change in resonant frequencies of the indole amide protons of W9, W11, W13, and W15, with the most profound changes in W9. These frequency changes were observed only for gA carefully prepared to ensure a channel conformation and were absent for gA in methanol. For gA in DMPC bilayers, direct [ 14C]halothane photolabeling and microsequencing demonstrated dominant labeling of W9, less labeling of W11 and W13, and no significant labeling of W15. In methanol, gA showed much less labeling of any residues. Inspection of the 3-D structure of gA suggests that the spatial arrangements of the tryptophan residues in the channel form of gA, combined with the amphiphilic regions of lipid, create a favorable anesthetic binding motif.

The multiple nicotinamide nucleotide-binding subunits of bovine heart mitochondrial NADH:ubiquinone oxidoreductase (complex I).

Direct photoaffinity labeling of purified bovine heart NADH:ubiquinone oxidoreductase (complex I) with 32P-labeled NAD(H), NADP(H) and ADP has shown that five polypeptides become labeled, with molecular masses of 51, 42, 39, 30, and 18-20 kDa. The 51 and the 30-kDa polypeptides were labeled with either [32P]NAD(H), [32P]NADP(H) or [beta-32P]ADP. The 42-kDa polypeptide was labeled with [32P]NAD(H) and to a small extent with [beta-32P]ADP. It was not labeled with [32P]NADP(H). The 39-kDa polypeptide was labeled with [32P]NADPH and to a small extent with [beta-32P]ADP. Our previous studies had shown that this subunit also binds NADP, but not NAD(H) [Yamaguchi, M., Belogrudov, G.I. & Hatefi, Y. (1998) J. Biol. Chem. 273, 8094-8098]. The 18-20-kDa polypeptide was labeled only with [32P]NADPH. Among these polypeptides, the 51-kDa subunit is known to contain FMN and a [4Fe-4S] cluster, and is the NAD(P)H-binding subunit of the primary dehydrogenase domain of complex I. The possible roles of the other nucleotide-binding subunits of complex I have been discussed.

Direct Photoaffinity Labeling of Cysteine 211 or a Nearby Amino Acid Residue of β-Tubulin by Guanosine 5′-Diphosphate Bound in the Exchangeable Site

Tubulin with [8-14C]GDP bound in the exchangeable site was exposed to ultraviolet light, and radiolabel was cross-linked to two peptide regions of the beta-subunit. Following enrichment for peptides cross-linked to guanosine by boronate chromatography, we confirmed that the cysteine 12 residue was the major site of cross-linking. However, significant radiolabel was also incorporated into a peptide containing amino acid residues 206 through 224. Although every amino acid in this peptide except cysteine 211 was identified by sequential Edman degradation, implying that this was the amino acid residue cross-linked to guanosine, radiolabel at C-8 was usually lost during peptide processing (probably during chromatography at pH 10). Consequently, the radiolabeled amino acid could not be unambiguously identified.

Direct Photoaffinity Labeling of the Kir6.2 Subunit of the ATP-sensitive K+ Channel by 8-Azido-ATP

ATP-sensitive potassium channels are under complex regulation by intracellular ATP and ADP. The potentiating effect of MgADP is conferred by the sulfonylurea receptor subunit of the channel, SUR, whereas the inhibitory effect of ATP appears to be mediated via the pore-forming subunit, Kir6.2. We determined whether ATP directly interacts with a binding site on the Kir6.2 subunit to mediate channel inhibition by analyzing binding of a photoaffinity analog of ATP (8-azido-[gamma-32P]ATP) to membranes from COS-7 cells transiently expressing Kir6.2. We demonstrate that Kir6.2 can be directly labeled by 8-azido-[gamma-32P]ATP but that the related subunit Kir4.1, which is not inhibited by ATP, is not labeled. Photoaffinity labeling of Kir6.2 is reduced by approximately 50% with 100 microM ATP. In addition, mutations in the NH2 terminus (R50G) and the COOH terminus (K185Q) of Kir6.2, which have both been shown to reduce the inhibitory effect of ATP upon Kir6.2 channel activity, reduced photoaffinity labeling by >50%. These results demonstrate that ATP binds directly to Kir6.2 and that both the NH2- and COOH-terminal intracellular domains may influence ATP binding.

Direct Photoaffinity Labeling of Individual Cytosolic Domains of Adenylyl Cyclase by [32P]2′-deoxy-3′-AMP and [α-32P]5′-ATP

The susceptibility of purines to form a covalent attachment with proteins upon exposure to UV irradiation was applied to adenylyl cyclase by use of [32P]2'-d-3'-AMP, a dead-end inhibitor that binds to the post-transition configuration of the enzyme. [32P]2'-d-3'-AMP was synthesized enzymatically. It and [alpha-32P]5'-ATP were used for direct photocross-linking to individually expressed cytosolic domains of adenylyl cyclase. Both the C1 domain of the type V isozyme (VC1) and the C2 domain of the type II isozyme (IIC2) were labeled, whether alone or combined, upon photolysis of [32P]2'-d-3'-AMP in the presence of acetone. Labeling of VC1 and IIC2 was greatly enhanced in the presence of PPi, was almost completely suppressed by 50 microM 2',5'-dideoxy-3'-ATP, the most potent reported P-site inhibitor of adenylyl cyclases, but was partially suppressed by 1 mM 3'-IMP, a ligand that does not inhibit the enzyme via the P-site. Neither 3':5'-cAMP nor 5'-ATP had a major effect on labeling by [32P]2'-d-3'-AMP. Direct cross-linking of VC1 with [alpha-32P]5'-ATP was substantially suppressed by 2', 5'-dideoxy-3'-ATP and partially suppressed by 2'-d-3'-AMP, whereas cross-linking of IIC2 was less affected by the 3'-triphosphate. The data imply that either cytosolic domain can interact directly with either substrate or P-site ligand and that subunit interaction modifies the susceptibility of each domain to UV-induced covalent modification by either [alpha-32P]5'-ATP or [32P]2'-d-3'-AMP.

Preferential binding of E7010 to murine beta 3-tubulin and decreased beta 3-tubulin in E7010-resistant cell lines.

N‐[2‐[(4‐Hydroxyphenyl)amino]‐3‐pyridyl]‐4‐methoxybenzenesulfonamide (E7010) is a novel sulfonamide antimitotic agent, which is active against mouse and human tumors. E7010 binds to β‐tubulin and inhibits polymerization of microtubules. In order to clarify the mechanisms of E7010‐resistance, two murine leukemic P388 subclones resistant to E7010, 0.5r‐D and 4.0r‐M, were characterized. The two clones showed approximately 10‐ and 100‐fold resistance to E7010‐induced growth‐inhibitory effects, respectively, compared with the parental cells in 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. These cell lines showed no cross‐resistance to other anticancer agents such as taxanes, vinca alkaloids, mitomycin C, cisplatin and irinotecan hydrochloride (CPT‐11). Increased α‐ and β‐tubulin protein and mRNA levels were observed in 0.5r‐D and 4.0r‐M cells as compared with the parental cells. We examined the isotype‐specific expression of β‐tubulin in these E7010‐resistant cells by a competitive reverse transcription‐polymerase chain reaction method. Although a 50% increase in β5 isotype mRNA levels was observed in 4.0r‐M cells, the levels of β3 isotype message in the two resistant clones were approximately 50% less than the parental cells. To elucidate the binding properties of E7010 with β‐tubulin isotypes, we prepared isotype‐specific fusion proteins of β‐tubulins. Direct photoaffinity labelling of the isotype‐specific fusion proteins with [14C]E7010 revealed that E7010 preferentially binds to the β3 isotype rather than β2, β4, and β5 isotype proteins. Therefore, altered expression of β‐tubulin isotypes, especially β3 isotype, to which E7010 binds with high affinity, may account for the decreased sensitivity of these resistant clones to E7010.

Direct Photoaffinity Labeling of Cysteine-295 of α-Tubulin by Guanosine 5′-Triphosphate Bound in the Nonexchangeable Site

The alphabeta-tubulin heterodimer has two high affinity guanosine 5'-triphosphate binding sites, so that purified tubulin usually contains two molecules of bound guanosine nucleotide. Half this nucleotide is freely exchangeable with exogenous guanine nucleotide, and its binding site has been readily localized to the beta-subunit. The remaining nonexchangeable guanosine 5'-triphosphate can only be released from tubulin by denaturing the protein. We replaced the exchangeable site nucleotide of tubulin with 2'-deoxyguanosine 5'-diphosphate, exposed the resulting tubulin to ultraviolet light, degraded the protein, and isolated ribose-containing peptide derived from the nonexchangeable site. A large cyanogen bromide peptide was recovered, and its further degradation with endoproteinase Glu-C established that cysteine-295 of alpha-tubulin was the major reactive amino acid cross-linked to guanosine by ultraviolet irradiation.

Interaction of estramustine with tubulin isotypes.

The interaction of the antimitotic agent estramustine with bovine microtubule proteins and purified tubulin was investigated. Direct photoaffinity labeling of microtubule protein with [14C]estramustine resulted in the labeling of both alpha- and beta-tubulin, and this was inhibited with unlabeled estramustine in a dose-dependent manner. [14C]Estramustine was incorporated into both the soluble and polymerized forms of tubulin. The affinity constant for estramustine binding to tubulin was determined by equilibrium dialysis to be 23 +/- 5 mM. Estramustine did not affect [3H]vinblastine binding, and vinblastine had no effect on direct labeling with [14C]estramustine. Both rhizoxin and paclitaxel decreased the covalent labeling of tubulin with [14C]estramustine in a dose-dependent fashion and were noncompetitive inhibitors of the binding of estramustine to tubulin. The binding of colchicine to tubulin was not inhibited by estramustine as detected by fluorescence and DEAE filter assays. The estramustine binding site on tubulin is therefore distinct from that of colchicine and vinblastine and may at least partially overlap with the binding site for paclitaxel. In both bovine brain microtubules and cytoskeletal proteins from human prostatic carcinoma cells, the incorporation of [14C]estramustine into the beta III isotype of tubulin was found to occur with a reduced efficiency compared to that of the other beta-tubulin isotypes and alpha-tubulin. Since this isotype is overexpressed in estramustine resistant human prostate carcinoma cells, these results indicate that beta III-tubulin may play a role in the response to the effects of estramustine.

Identification of the multidrug-resistance protein (MRP) as the glutathione-S-conjugate export pump of erythrocytes.

The identification of the multidrug resistance protein (MRP) as a conjugate export pump in several cell types suggested its involvement in the long-known glutathione-S-conjugate transport across erythrocyte membranes. We investigated the ATP-dependent transport of glutathione S-conjugates in human erythrocyte and erythroleukemia cell membrane vesicles using the endogenous conjugate leukotriene C4 (LTC4), known to be a high-affinity substrate for MRP, in addition to S-(2,4-dinitrophenyl)glutathione. The kinetic parameters, including the Km value for LTC4 of 118 +/- 5 nM and the inhibition constants for transport of both substrates for the quinoline-based inhibitor MK 571, were similar to those obtained for transport mediated by recombinant MRP. Direct photoaffinity labeling of human erythrocyte membranes with [3H]LTC4 revealed a major binding protein of about 190 kDa which was immunoprecipitated by an anti-MRP serum. The radiolabeling of this protein was specifically suppressed by the transport inhibitor MK 571. Several additional anti-MRP sera detected the protein of about 190 kDa in human erythrocyte and erythroleukemia cell membranes. These data identify for the first time the glutathione-S-conjugate transporting protein in erythrocyte membranes.

Characterization of the molecular mode of action of the sulfonylurea, glimepiride, at beta-cells.

Glimepiride was compared with glibenclamide for its insulin secretion stimulating and beta-cell membrane depolarizing activity as well as for its binding kinetics to beta-cell membranes and for its beta-cell membrane binding proteins. Steady state, kinetic and competitive binding studies revealed a 3- to 4-fold lower binding affinity of glimepiride to isolated beta-cell membranes and intact beta-cells compared to glibenclamide. Direct photoaffinity labeling of beta-cell membrane proteins with the radiolabeled sulfonylureas identified a 65-kDa binding protein for glimepiride and a 140-kDa binding protein for glibenclamide which may be the basis for the different binding characteristics of the two sulfonylureas. The inhibition of binding and photoaffinity labeling of glimepiride and glibenclamide to the 65- and 140-kDa protein, respectively, by glibenclamide and glimepiride can be explained with allosteric interactions between two distinct sulfonylurea binding proteins, subunits of a KATP protein complex, each regulating the open/closed-state of a common separate pore-forming subunit by allosteric interactions. Whole-cell patch clamp experiments with RINm5F cells demonstrated a 3- to 4-fold lower depolarization activity of glimepiride compared to glibenclamide which correlates well with the lower binding affinity of glimepiride. The lower binding affinity and depolarization activity of glimepiride were not reflected in vitro in perifused islets and the isolated perfused pancreas of the rat. This discrepancy remains to be elucidated. The binding, depolarisation and insulin releasing characteristics of glimepiride and glibenclamide suggest that different sulfonylureas can interact with different components of KATP.

Minimum structural requirement for an inhalational anesthetic binding site on a protein target

The present study makes use of direct photoaffinity labeling and fluorescence and circular dichroism spectroscopy to examine the interaction of the inhalational anesthetic halothane with the uncharged α-helical form of poly( l-lysine) over a range of chain lengths. Halothane bound specifically to long chain homopolymers (190 to 1060 residues), reaching a stable stoichiometry of 1 halothane to 160 lysine residues in polymers longer than 300 residues. Halothane bound only non-specifically to an α-helical 30 residue polymer and to all of the polymers in their charged, random coil form. The data suggest that halothane binding is a function of supersecondary structure whereby intramolecular helix-helix clusters form in the longer polymers, resulting in the creation of confined hydrophobic domains. Circular dichroism spectroscopy cannot demonstrate changes in poly( l-lysine) secondary structure at any chain length with up to 12 mM halothane, suggesting that extensive hydrogen bond disruption by the anesthetic does not occur.

An inhalational anesthetic binding domain in the nicotinic acetylcholine receptor.

To determine inhalational anesthetic binding domains on a ligand-gated ion channel, I used halothane direct photoaffinity labeling of the nicotinic acetylcholine receptor (nAChR) in native Torpedo membranes. [14C]Halothane photoaffinity labeling of both the native Torpedo membranes and the isolated nAChR was saturable, with Kd values within the clinically relevant range. All phospholipids were labeled, with greater than 95% of the label in the acyl chain region. Electrophoresis of labeled nAChR demonstrated no significant subunit selectivity for halothane incorporation. Within the alpha-subunit, greater than 90% of label was found in the endoprotease Glu-C digestion fragments which contain the four transmembrane regions, and the pattern was different from that reported for photoactivatable phospholipid binding to the nAChR. Unlabeled halothane reduced labeling more than did isoflurane, suggesting differences in the binding domains for inhalational anesthetics in the nAChR. These data suggest multiple similar binding domains for halothane in the transmembrane region of the nAChR.