Methanethiosulfonate Research Articles

Abstract 2233: MARCKS-ED peptide targets highly curved vesicles: Potential probe for exosome detection.

Abstract Malignant tumor cells overexpress and release nanosized, lipid-enclosed vesicles referred to as exosomes into body fluids for the primary function of facilitating cell-to-cell communication and transferring vital proteins necessary for survival to other parts of the body, resulting in metastasis. It was found that an increased secretion of exosomes in the peripheral blood is correlated to lung cancer and melanoma metastasis. The hallmarks of exosomes are their highly curved membrane surface (d = ∼30-100 nm) and their enrichment in the anionic lipid phosphatidylserine (PS) in the outer leaflet of the membrane bilayer. These properties may provide unique strategies in designing peptides to selectively target exosomes as potential cancer biomarkers. Some current clinical methods that are being used to classify metastatic patients, such as the biopsy and assays measuring circulating tumor cells, are not reproducible and do not always indicate whether or not treatment is reducing metastasis. Furthermore, these methods involve invasive procedures. We aim to develop a novel, minimally invasive, peptide-based diagnostic tool to detect and measure exosome overexpression in cancer patients. The successful creation of this diagnostic tool for cancer metastasis will help in the proper prognosis and therapy for cancer patients. We rationally chose the effector domain of the intracellular membrane protein myristoylated alanine-rich C-kinase substrate (MARCKS) as our core molecule based on recent reports that this peptide differentially interacts with nanometer size synthetic lipid vesicles. It has also been shown that MARCKS inserts its five hydrophobic phenylalanine (Phe) residues into the membrane of highly curved synthetic lipid vesicles, and its binding affinity increases with increasing vesicle curvature, presumably corresponding to a larger number of defects on the membrane surface. We hypothesize that as vesicle curvature increases, the Phe residues will insert deeper into the membrane due to more lipid defects exposed on the membrane surface in highly curved vesicles. Five analogs of the MARCKS peptide were synthesized, substituting one Phe residue with a cysteine residue for all 5 Phe positions. The cysteine was then labeled with 1-oxyl-2,2,5,5-tetramethyl-▵3-pyrroline-3-methyl) methanethiosulfonate (MTSL), which will be used as a paramagnetic spin label for electron paramagnetic resonance spectroscopy (EPR). The depth parameter of insertion of the MTSL into the lipid vesicle membrane at each of the five cysteine positions will be quantified using EPR and will be reported herein. Citation Format: Sara K. Coulup, Leslie A. Morton, Hang Yin. MARCKS-ED peptide targets highly curved vesicles: Potential probe for exosome detection. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2233. doi:10.1158/1538-7445.AM2013-2233 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Role of the fourth transmembrane domain in proton-coupled folate transporter function as assessed by the substituted cysteine accessibility method

The proton-coupled folate transporter (PCFT, SLC46A1) mediates folate transport across the apical brush-border membrane of the proximal small intestine and the basolateral membrane of choroid plexus ependymal cells. Two loss-of-function mutations in PCFT, which are the basis for hereditary folate malabsorption, have been identified within the fourth transmembrane domain (TMD4) in subjects with this disorder. We have employed the substituted Cys accessibility method (SCAM) to study the accessibilities of all residues in TMD4 and their roles in folate substrate binding to the carrier. When residues 146-167 were replaced by Cys, all except R148C were expressed at the cell surface. Modification of five of these substituted Cys residues (positions 147, 152, 157, 158, and 161) by methanethiosulfonate (MTS) reagents led to reduction of PCFT function. All five residues could be labeled with N-biotinylaminoethyl-MTS, and this could be blocked by the high-affinity PCFT substrate pemetrexed. Pemetrexed also protected PCFT mutant function from inhibitory modification of the substituted Cys at positions 157, 158, and 161 by a MTS. The findings indicate that these five residues in TMD4 are accessible to the aqueous translocation pathway, play a role in folate substrate binding, and are likely located within or near the folate binding pocket. A homology model of PCFT places three of these residues, Phe¹⁵⁷, Gly¹⁵⁸, and Leu¹⁶¹, within a breakpoint in the midportion of TMD4, a region that likely participates in alterations in the PCFT conformational state during carrier cycling.

Cysteine Scanning of CFTR’s First Transmembrane Segment Reveals Its Plausible Roles in Gating and Permeation

Previous cysteine scanning studies of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have identified several transmembrane segments (TMs), including TM1, 3, 6, 9, and 12, as structural components of the pore. Some of these TMs such as TM6 and 12 may also be involved in gating conformational changes. However, recent results on TM1 seem puzzling in that the observed reactive pattern was quite different from those seen with TM6 and 12. In addition, whether TM1 also plays a role in gating motions remains largely unknown. Here, we investigated CFTR’s TM1 by applying methanethiosulfonate (MTS) reagents from both cytoplasmic and extracellular sides of the membrane. Our experiments identified four positive positions, E92, K95, Q98, and L102, when the negatively charged MTSES was applied from the cytoplasmic side. Intriguingly, these four residues reside in the extracellular half of TM1 in previously defined CFTR topology; we thus extended our scanning to residues located extracellularly to L102. We found that cysteines introduced into positions 106, 107, and 109 indeed react with extracellularly applied MTS probes, but not to intracellularly applied reagents. Interestingly, whole-cell A107C-CFTR currents were very sensitive to changes of bath pH as if the introduced cysteine assumes an altered pKa-like T338C in TM6. These findings lead us to propose a revised topology for CFTR’s TM1 that spans at least from E92 to Y109. Additionally, side-dependent modifications of these positions indicate a narrow region (L102-I106) that prevents MTS reagents from penetrating the pore, a picture similar to what has been reported for TM6. Moreover, modifications of K95C, Q98C, and L102C exhibit strong state dependency with negligible modification when the channel is closed, suggesting a significant rearrangement of TM1 during CFTR’s gating cycle. The structural implications of these findings are discussed in light of the crystal structures of ABC transporters and homology models of CFTR.

Structural Basis for Differential KCNQ1 Interactions with KCNE1 and KCNE2 in the Extracellular Juxtamembrane Region

KCNE1 and KCNE2 are both expressed in human heart and can associate with KCNQ1. KCNE1 and KCNE2 share transmembrane topology and sequence homology, yet they differ in KCNQ1 modulation (KCNE1 slows KCNQ1 Closed-to-Open transition, while KCNE2 slows KCNQ1 Open-to-Closed transition) and pharmacology (KCNQ1/KCNE2 is 5 and 10 fold less sensitive than KCNQ1/KCNE1 to niflumic acid, IKs activator, and azimilide, IKs suppressor). Previous work has shown that the extracellular juxtamembrane (EJM) region of KCNE1 interacts with the extracellular surface of KCNQ1 to modulate gating kinetics and to form IKs activator binding sites. This prompts us to compare the EJMs of the two KCNE subunits in terms of KCNQ1 interaction. We apply cysteine (Cys) scanning mutagenesis to EJMs of KCNE1 and KCNE2 and analyze the patterns of functional perturbation when coexpressed with KCNQ1. We use methanethiosulfonate (MTS) reagents to probe the relationship between EJMs of KCNE subunits and KCNQ1. Finally we probe disulfide formation between Cys engineered into the EJMs of KCNE subunits and those engineered into extracellular surface of KCNQ1. The EJM of KCNE1 makes frequent contacts with KCNQ1, so that MTS modification of exposed Cys side chains can affect channel gating. KCNQ1/KCNE2 is largely indifferent to MTS modification of exposed Cys side chains engineered to EJM of KCNE2. However, MTS can slowly access 7 consecutive hydrophobic positions in the beginning of KCNE2 transmembrane domain, as if there is a crevice between KCNQ1 and KCNE2. Disulfide trapping experiments suggest that KCNE2 is leaning more toward KCNQ1 S2 than KCNE1. We propose that KCNE2 interferes with S2-S4 interactions during KCNQ1 Open-to-Closed transition and thus slows deactivation. This weakens sensitivity to niflumic acid and azimilide by affecting binding site directly or by an allosteric mechanism.

Bulky Substitutions in the Thumb Domain of ASIC1a Restrict Proton-Gating

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation selective ion channels expressed in the nervous system. Each monomer consists of two transmembrane helices with short cytoplasmic tails and a large extracellular region organized in distinct domains named the thumb, finger, knuckle, β-ball, and palm. In this report we investigated the contribution to mouse ASIC1a activation and desensitization of three pairs of acidic residues, Glu238-Asp345, Asp237-Asp349 and Glu219-Asp407. Residues in each pair are located in close proximity and together form an acidic cluster at the interface between the thumb and β-ball domains. We examined the reactivity toward methanethiosulfonate (MTS) reagents of channels carrying individual Cys substitutions at these positions. MTSET-modification of D345C channels shifted the pH of half-maximal activation (pH50) from 6.23±0.06 to 4.48±0.08, and increased the desensitization rate. A similar effect was observed with other positively-charged MTS reagents such as MTSMT, MTSPT and MTSEA, as well as with MTSES, a negatively-charged reagent. Moreover, Lys substitution at position 345 shifted pH50 toward more acidic values. Remarkably, Lys substitution at positions 219, 237 or 238 did not significantly change the pH50, suggesting that acid residues at these positions do not substantially contribute to proton activation. Our results indicate that the introduction of a bulky moiety at position 345 restricts conformational movements required for proton-gating, suggesting that the thumb-β-ball interface undergoes dynamic rearrangements during activation.

Introduction to spin label electron paramagnetic resonance spectroscopy of proteins

An undergraduate laboratory exercise is described to demonstrate the biochemical applications of electron paramagnetic resonance (EPR) spectroscopy. The β93 cysteine residue of hemoglobin is labeled by the covalent binding of 3-maleimido-proxyl (5-MSL) and 2,2,5,5-tetramethyl-1-oxyl-3-methyl methanethiosulfonate (MTSL), respectively. The excess spin label is removed by gel-exclusion chromatography. Changes in the mobility of the reporter groups attached to the protein are monitored by EPR spectroscopy. While the spectral parameters of the rigidly attached 5-MSL provide information on the rotation of the whole spin labeled protein, MTSL bound by a more flexible linkage describes the local environment of the cysteine residue in the interior of the protein structure. Students can study the known crystal structure of hemoglobin in comparison to the results they obtain by analyzing the EPR spectra. Overall, the exercise introduces them to laboratory techniques such as protein labeling, gel filtration, EPR spectroscopy, as well as familiarizes them with the online Protein Data Bank as a research resource and PyMOL software as a structure visualization tool.

Effects of <I>PI3K/AKT</I> inhibitor wortmannin on proliferation and apoptosis of primary porcine preadipocytes

The purpose of this study was to determine the proper concentration of wortmannin that effectively inhibits PI3K/AKT but does not affect the proliferation and apoptosis of primary porcine preadipocytes. Firstly, primary porcine preadipocytes were isolated and their abilities to be induced to differentiation into mature adipocytes were evaluated. The preadipocytes were then treated with different concentrations of wortmannin, and the proliferation of the cells was detected with methanethiosulfonate (MTS). Annexin V- FITC/PI double-staining was used to detect the level of cell apoptosis. The apoptosis-related gene expressions were also quantified by qRT-PCR. At the same time, single cell electrophoresis was used to examine the extent of cellular DNA damage. Our data demonstrated that the primary porcine preadipocytes could differ-entiate into mature adipocytes. Up to 200 nmol/L of wortmannin had no effect on the proliferation ability of primary porcine preadipocytes (P>0.05). Results from the flow cytometry Annexin V- FITC/PI double-staining showed that 200 nmol/L wortmannin significantly induced apoptosis of the primary porcine preadipocytes (P<0.05). QRT-PCR results also showed that the expressions of caspase8, TNFR1, GZMB, and Bcl-x1 were significantly upregulated, while the expression of GZMA and cFLIP were not significantly affected when treated with 200 nmol/L wortmannin. In addition, results from the single cell gel electrophoresis indicated that 100 nmol/L wortmannin did not induce DNA damage. In conclusion, our results col-lectively showed that 100 nmol/L wortmanin can be used to study the role of PI3k pathway on the preadipocytes differen-tion without affecting the cell proliferation and apoptosis.

Synthesis of 2,5-Bis(spirocyclohexane)-Substituted Nitroxides of Pyrroline and Pyrrolidine Series, Including Thiol-Specific Spin Label: An Analogue of MTSSL with Long Relaxation Time

The nitroxides of 7-azadispiro[5.1.5.2]pentadecane and 7-azadispiro[5.1.5.2]pentadeca-14-ene series have been prepared, including thiol-specific methane thiosulfonate spin label for site-directed spin labeling. The effect of spirocyclohexane moieties on chemical and spectral properties has been studied. The obtained temperature dependencies of electron spin relaxation parameters demonstrate that new nitroxides may be suitable for PELDOR distance measurements at 80-120 K. Moreover, the new nitroxides demonstrated much higher stability toward reduction by ascorbate than spirocyclohexane-substituted nitroxides of piperidine series and showed 1.3-3.14 times lower reduction rates compared to corresponding 2,2,5,5-tetramethyl nitroxides.

Catalytic Zinc Site and Mechanism of the Metalloenzyme PR-AMP Cyclohydrolase

The enzyme N(1)-(5'-phosphoribosyl) adenosine-5'-monophosphate cyclohydrolase (PR-AMP cyclohydrolase) is a Zn(2+) metalloprotein encoded by the hisI gene. It catalyzes the third step of histidine biosynthesis, an uncommon ring-opening of a purine heterocycle for use in primary metabolism. A three-dimensional structure of the enzyme from Methanobacterium thermoautotrophicum has revealed that three conserved cysteine residues occur at the dimer interface and likely form the catalytic site. To investigate the functions of these cysteines in the enzyme from Methanococcus vannielii, a series of biochemical studies were pursued to test the basic hypothesis regarding their roles in catalysis. Inactivation of the enzyme activity by methyl methane thiosulfonate (MMTS) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) also compromised the Zn(2+) binding properties of the protein inducing loss of up to 90% of the metal. Overall reaction stoichiometry and the potassium cyanide (KCN) induced cleavage of the protein suggested that all three cysteines were modified in the process. The enzyme was protected from DTNB-induced inactivation by inclusion of the substrate N(1)-(5'-phosphoribosyl)adenosine 5'-monophosphate; (PR-AMP), while Mg(2+), a metal required for catalytic activity, enhanced the rate of inactivation. Site-directed mutations of the conserved C93, C109, C116 and the double mutant C109/C116 were prepared and analyzed for catalytic activity, Zn(2+) content, and reactivity with DTNB. Substitution of alanine for each of the conserved cysteines showed no measurable catalytic activity, and only the C116A was still capable of binding Zn(2+). Reactions of DTNB with the C109A/C116A double mutant showed that C93 is completely modified within 0.5 s. A model consistent with these data involves a DTNB-induced mixed disulfide linkage between C93 and C109 or C116, followed by ejection of the active site Zn(2+) and provides further evidence that the Zn(2+) coordination site involves the three conserved cysteine residues. The C93 reactivity is modulated by the presence of the Zn(2+) and Mg(2+) and substantiates the role of this residue as a metal ligand. In addition, Mg(2+) ligand binding site(s) indicated by the structural analysis were probed by site-directed mutagenesis of three key aspartate residues flanking the conserved C93 which were shown to have a functional impact on catalysis, cysteine activation, and metal (zinc) binding capacity. The unique amino acid sequence, the dynamic properties of the cysteine ligands involved in Zn(2+) coordination, and the requirement for a second metal (Mg(2+)) are discussed in the context of their roles in catalysis. The results are consistent with a Zn(2+)-mediated activation of H(2)O mechanism involving histidine as a general base that has features similar to but distinct from those of previously characterized purine and pyrimidine deaminases.

Antiparallel Dimers of the Small Multidrug Resistance Protein EmrE Are More Stable Than Parallel Dimers

The bacterial multidrug transporter EmrE is a dual-topology membrane protein and as such is able to insert into the membrane in two opposite orientations. The functional form of EmrE is a homodimer; however, the relative orientation of the subunits in the dimer is under debate. Using EmrE variants with fixed, opposite orientations in the membrane, we now show that, although the proteins are able to form parallel dimers, an antiparallel organization of the subunits in the dimer is preferred. Blue-native PAGE analyses of intact oligomers and disulfide cross-linking demonstrate that in membranes, the proteins form parallel dimers only if no oppositely orientated partner is present. Co-expression of oppositely orientated proteins almost exclusively yields antiparallel dimers. Finally, parallel dimers can be disrupted and converted into antiparallel dimers by heating of detergent-solubilized protein. Importantly, in vivo function is correlated clearly to the presence of antiparallel dimers. Our results suggest that an antiparallel arrangement of the subunits in the dimer is more stable than a parallel organization and likely corresponds to the functional form of the protein.

Role of the Juxtamembrane Region of Cytoplasmic Loop 3 in the Gating and Conductance of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

Opening and closing of the cystic fibrosis transmembrane conductance regulator chloride channel are controlled by interactions of ATP with its cytoplasmic nucleotide binding domains (NBDs). The NBDs are connected to the transmembrane pore via four cytoplasmic loops. These loops have been suggested to play roles both in channel gating and in forming a cytoplasmic extension of the channel pore. To investigate the structure and function of one of these cytoplasmic loops, we have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced into loop 3. We find that methanethiosulfonate (MTS) reagents modify cysteines introduced at 14 of 16 sites studied in the juxtamembrane region of loop 3, in all cases leading to inhibition of channel function. In most cases, both the functional effects of modification and the rate of modification were similar for negatively and positively charged MTS reagents. Single-channel recordings indicated that, at all sites, inhibition was the result of an MTS reagent-induced decrease in channel open probability; in no case was the Cl– conductance of open channels altered by modification. These results indicate that loop 3 is readily accessible to the cytoplasm and support the involvement of this region in the control of channel gating. However, our results do not support the hypothesis that this region is close enough to the Cl– permeation pathway to exert any influence on permeating Cl– ions. We propose that either the cytoplasmic pore is very wide or cytoplasmic Cl– ions use other routes to access the transmembrane pore.

Alternating Access to the Transmembrane Domain of the ATP-binding Cassette Protein Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7)

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.

A Human Disease Mutation (D50N) Reveals Insights on the Mechanisms for Ca2+ Regulation in Human Connexin26 (hCX26) Hemichannels

Connexin hemichannels allow the release of small metabolites, such as ATP and glutamate, which play important roles in autocrine/paracrine signaling in a variety of cell types. Exacerbated hemichannel opening, however, leads to loss of electrochemical gradients and metabolites, causing cell death. Control of hemichannel opening is indispensable, and is achieved by physiological extracellular Ca2+, which drastically reduces hemichannel activity. Here, we explore the molecular mechanism of Ca2+ regulation in hCx26 hemichannels. We found that outward and tail currents are greatly increased with reduction of external Ca2+. The data show that deactivation kinetics are accelerated as a function of Ca2+ concentration suggesting that Ca2+ facilitates closing of the channels. In addition, we found that an Asp to Asn mutation at position 50 (D50N) - a human mutation that causes disease - has lower apparent affinity for Ca2+ compared with wild-type. Strikingly, unlike wild-type channels, the deactivation kinetics of D50N channels were unaffected by changes in Ca2+. To explore the role of Ca2+ interactions with a negatively charged residue at position 50, we substituted this position by a cysteine residue (D50C) and performed chemical modification with positively or negatively charged methanethiosulfonate (MTS) reagents. The D50C mutant hemichannels display similar properties to those of D50N mutants. Chemical modification of D50C with a negatively charged reagent, MTSES, causes the channels to largely return to wild-type sensitivity to Ca2+. Conversely, positively charged MTS reagents have no effect. In the crystal structure of hCx26 channels, position D50 faces the pore lumen and forms an inter-subunit salt bridge interaction with position K61. Because this interaction appears to take place in open conformation, we propose that Ca2+ facilitates the closing of hCx26 hemichannels by disrupting this salt bridge.

Declaration of Independence by CFTR's Transmembrane Domains

Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) transporter superfamily in that it functions as an ATP-gated chloride channel. Recent cysteine scanning studies in our lab demonstrate that both the sixth transmembrane segment (TM6) and its C-terminal counterpart, TM12, play pivotal roles in chloride permeation and are also involved in gating conformational changes. Here, to study the functional role of TM1 in gating and permeation, we introduced cysteine residues into this TM and assessed their reactivity towards internally-applied thiol-directed methanethiosulfonate (MTS) reagents. Our initial cysteine scanning experiments (1-2 patches for each cysteine-substituted cysless-CFTR channels) identified four positive hits, including E92, K95, Q98 and L102, where the negatively-charged MTSES reacting with engineered cysteines diminishes ATP-induced current. Surprisingly, according to the CFTR topology based on hydropathy plots, all these positions reside in the external half of TM1- contrary to results with TM6 and TM12. We also found that modification by the positively-charged MTSET enhances macroscopic current in K95C-, Q98C-, and L102C-cysless-CFTR channels. Intriguingly, at the single-channel level, we observed that whereas the mutation L102C appears to destabilize the gate, deposition of the MTSET adduct decreases the single-channel current amplitude but increases the Po. Moreover, channel gating persisted even after a complete removal of ATP, raising the possibility that gating motion in the TMDs of modified L102C channels is independent of that in their NBDs. Finally, MTSET modification of L102C is state-dependent, meaning that L102C-cysless-CFTR channels do not respond to the treatment of MTSET in the absence of ATP. These findings suggest that either position 102 moves during gating or gating motion in other regions of CFTR alters its reactivity towards MTSET. Overall, our preliminary data reveal several interesting yet enigmatic aspects of TM1 that call for further studies.

Time- and state-dependent effects of methanethiosulfonate ethylammonium (MTSEA) exposure differ between heart and skeletal muscle voltage-gated Na+ channels

The substituted-cysteine scanning method (SCAM) is used to study conformational changes in proteins. Experiments using SCAM involve site-directed mutagenesis to replace native amino acids with cysteine and subsequent exposure to a methanethiosulfonate (MTS) reagent such as methanethiosulfonate ethylammonium (MTSEA). These reagents react with substituted-cysteines and can provide functional information about relative positions of amino acids within a protein. In the human heart voltage-gated Na+ channel hNav1.5 there is a native cysteine at position C373 that reacts rapidly with MTS reagents resulting in a large reduction in whole-cell Na+ current (INa). Therefore, in order to use SCAM in studies in this isoform, this native cysteine is mutated to a non-reactive residue, e.g., tyrosine. This mutant, hNav1.5-C373Y, is resistant to the MTS-mediated decrease in INa. Here we show that this resistance is time- and state-dependent. With relatively short exposure times to MTSEA (<4min), there is little effect on INa. However, with longer exposures (4–8min), there is a large decrease in INa, but this effect is only found when hNav1.5-C373Y is inactivated (fast or slow) — MTSEA has little effect in the closed state. Additionally, this long-term, state-dependent effect is not seen in human skeletal muscle Na+ channel isoform hNav1.4, which has a native tyrosine at the homologous site C407. We conclude that differences in molecular determinants of inactivation between hNav1.4 and hNav1.5 underlie the difference in response to MTSEA exposure.

Extracellular Determinants of Anion Discrimination of the Cl−/H+ Antiporter Protein CLC-5

Mammalian CLC proteins comprise both Cl- channels and Cl-/H+ antiporters that carry out fundamental physiological tasks by transporting Cl- across plasma membrane and intracellular compartments. The NO3- over Cl- preference of a plant CLC transporter has been pinpointed to a conserved serine residue located at Scen and it is generally assumed that the other two binding sites of CLCs, Sext and Sin, do not substantially contribute to anion selectivity. Here we show for the Cl-/H+ antiporter CLC-5 that the conserved and extracellularly exposed Lys210 residue is critical to determine the anion specificity for transport activity. In particular, mutations that neutralize or invert the charge at this position reverse the NO3- over Cl- preference of WT CLC-5 at a concentration of 100 mm, but do not modify the coupling stoichiometry with H+. The importance of the electrical charge is shown by chemical modification of K210C with positively charged cysteine-reactive compounds that reintroduce the WT preference for Cl-. At saturating extracellular anion concentrations, neutralization of Lys210 is of little impact on the anion preference, suggesting an important role of Lys210 on the association rate of extracellular anions to Sext.

Differential Roles of Cysteine Residues in the Cellular Trafficking, Dimerization, and Function of the High-Density Lipoprotein Receptor, SR-BI

The scavenger receptor, class B, type I (SR-BI) binds high-density lipoprotein (HDL) and mediates selective delivery of cholesteryl esters (CEs) to the liver and steroidogenic cells of the adrenal glands and gonads. Although it is clear that the large extracellular domain (ECD) of SR-BI binds HDL, the role of ECD in the selective HDL-CE transport remains poorly understood. In this study, we used a combination of mutational and chemical approaches to systematically evaluate the contribution of cysteine residues, especially six cysteine residues of ECD, in SR-BI-mediated selective HDL-CE uptake, intracellular trafficking, and SR-BI dimerization. Pretreatment of SR-BI-overexpressing COS-7 cells with a disulfide (S-S) bond reducing agent, β-mercaptoethanol (100 mM) or dithiothreitol (DTT) (10 mM), modestly but significantly impaired SR-BI-mediated selective HDL-CE uptake. Treatment of SR-BI-overexpressing COS-7 cells with the optimal doses of membrane permeant alkyl methanethiosulfonate (MTS) reagents, positively charged MTSEA or neutral MMTS, that specifically react with the free sulfhydryl group of cysteine reduced the rate of SR-BI-mediated selective HDL-CE uptake, indicating that certain intracellular free cysteine residues may also be critically involved in the selective cholesterol transport process. In contrast, use of membrane impermeant MTS reagent, positively charged MTSET and negatively charged MTSES, showed no such effect. Next, the importance of eight cysteine residues in SR-BI expression, cell surface expression, dimer formation, and selective HDL-derived CE transport was evaluated. These cysteine residues were replaced either singly or in pairs with serine, and the mutant SR-BIs were expressed in either COS-7 or CHO cells. Four mutations, C280S, C321S, C323S, and C334S, of the ECD, either singly or in various pair combinations, resulted in significant decreases in SR-BI (HDL) binding activity, selective CE uptake, and trafficking to the cell surface. Surprisingly, we found that mutation of the two remaining cysteine residues, C251 and C384 of the ECD, had no effect on either SR-BI expression or function. Other cysteine mutations and substitutions were also without effect. Western blot data indicated that single and double mutations at C280, C321, C323, and C334 residues strongly favor dimer formation. However, they are rendered nonfunctional presumably because of mutation-induced formation of aberrant disulfide linkages resulting in inhibition of optimal HDL binding and, thus, selective HDL-CE uptake. These results provide novel insights into the functional role of four cysteine residues, C280, C321, C323, and C334, of the SR-BI ECD in SR-BI expression and trafficking to the cell surface, its dimerization, and associated selective CE transport function.

The antifibrosis effect of adrenomedullin in human lung fibroblasts

ABSTRACTAdrenomedullin (AM) is a regulatory peptide involved in cellular proliferation and protein synthesis. The authors investigated AM and the AM receptor system in the human fetal lung fibroblasts (HFLFs), and assessed whether AM can inhibit proliferation and collagen synthesis in HFLFs under hypoxia. Fibroblasts were exposed to hypoxia (2% O2) after the addition of AM. The effects of AM and transforming growth factor β1 (TGF-β1) on the proliferation of fibroblasts were determined by the methanethiosulfonate (MTS) assay. Total collagen synthesis was determined by [3H]proline incorporation. TGF-β1 levels in the culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). The concentration of intracellular calciumion ([Ca2+]i) in fibroblasts was detected with a laser scanning confocal microscope. AM, adrenomedullin receptor (ADMR), calcitonin receptor–like receptor (CRLR), AM receptor chaperone receptor activity–modifying protein-1 (RAMP1),RAMP2, and RAMP3 were detected in the HFLFs. The hypoxia-induced increases in cell proliferation, collagen synthesis, and TGF-β1 production were inhibited by AM. AM also inhibited proliferation and collagen synthesis in fibroblasts induced by TGF-β1. AM caused a decrease of the hypoxia-induced [Ca2+]i in fibroblasts. This study suggests that AM is produced by HFLFs and AM may function as an antifibrosis factor that protects cells from hypoxic pulmonary damage through its receptors.

Acid Degradable and Biocompatible Polymeric Nanoparticles for the Potential Codelivery of Therapeutic Agents

The synthesis of well-defined functional nanoparticles for the encapsulation of hydrophobic and hydrophilic drugs is described. Nanoparticles were built from amphiphilic copolymers consisting of P(OEG-A) homopolymers chain extended with vinyl benzyl chloride (VBC) and pentafluorophenyl acrylate (PFP-A) comonomers. Subsequently, the pendant chlorine atoms, introduced into the chains by VBC units, were substituted using sodium methanethiosulfonate, yielding copolymer chains with methanethiosulfonate (MTS) pendant functionality. The thiol/MTS exchange chemistry afforded by the MTS groups was then used to introduce different functional groups by reacting with a range of thiols. These copolymers were self-assembled in water yielding nanoparticles with sizes of ∼20 nm. The activated esters in the copolymer were used to cross-link the nanoparticles with difunctional amino compounds (cross-linkers). A cross-linker bearing an acid cleavable bond (ketal) was used to generate pH-sensitive core–shell nanoparticles. D...

Cysteine Scanning Mutagenesis (Residues Glu52–Gly96) of the Human P2X1 Receptor for ATP: MAPPING AGONIST BINDING AND CHANNEL GATING

P2X receptors are ATP-gated cation channels. The x-ray structure of a P2X4 receptor provided a major advance in understanding the molecular basis of receptor properties. However, how agonists are coordinated, the extent of the binding site, and the contribution of the vestibules in the extracellular domain to ionic permeation have not been addressed. We have used cysteine-scanning mutagenesis to determine the contribution of residues Glu52–Gly96 to human P2X1 receptor properties. ATP potency was reduced for the mutants K68C, K70C, and F92C. The efficacy of the partial agonist BzATP was also reduced for several mutants forming the back of the proposed agonist binding site. Molecular docking in silico of both ATP and BzATP provided models of the agonist binding site consistent with these data. Individual cysteine mutants had no effect or slightly increased antagonism by suramin or pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate. Mutants at the entrance to and lining the upper vestibule were unaffected by cysteine-reactive methanethiosulfonate (MTS) reagents, suggesting that it does not contribute to ionic permeation. Mutants that were sensitive to modification by MTS reagents were predominantly found either around the proposed ATP binding pocket or on the strands connecting the binding pocket to the transmembrane region and lining the central vestibule. In particular, ATP sensitivity and currents were increased by a positively charged MTS reagent at the G60C mutant at the interface between the central and extracellular vestibule. This suggests that dilation of the base of the central vestibule contributes to gating of the receptor.