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Results for “Extracellular Traps”

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PubMed1990

Evidence for receptor-mediated bivalent-cation entry in A10 vascular smooth-muscle cells.

In fura-2-loaded A10 vascular smooth-muscle cells, 1 nM-vasopressin and 200 nM-endothelin evoked a rapid transient rise in intracellular free Ca2+ concentration [( Ca2+]i), which was then followed by a maintained elevation of [Ca2+]i. The maintained elevation of [Ca2+]i was only partially inhibited by 5 microM-nifedipine, but completely abolished in the presence of 1 mM-EGTA. When extracellular Ca2+ was replaced with 1 mM-Mn2+ (Mn2+ quenches fura-2 fluorescence), both endothelin and vasopressin evoked an Mn2+ quench of the fluorescence from the intracellularly trapped fura-2, even in the presence of 5 microM-nifedipine. These data suggest that both vasopressin and endothelin promote a bivalent-cation influx and provide further evidence for receptor-mediated Ca2+ entry in vascular smooth muscle.

Calcium↗

PubMed1999

Transport of 14C-deferiprone in normal, thalassaemic and sickle red blood cells.

The transport of deferiprone (L1) in normal (N), sickle (S) and thalassaemic (T) red blood cells (RBC) was determined by incubation with 14C-L1 at 37 degrees C. Following incubation with 0.5 mM 14C-L1 for 4 h, the intracellular concentration of L1 in T RBC was 3 times higher than was found extracellularly. In contrast, no concentration gradient across N and S RBC membranes was detected. Efflux studies showed that T RBC released only 17 +/- 2% of 14C-L1 into the extracellular space. We hypothesize that L1 accumulation in T RBC results from their high content of chelatable iron and formation of large, hydrophilic L1-Fe(III) complexes trapped within the cytosol.

Adult↗

PubMed1985

Penetration of C8 and C9 in the C5b-9 complex across the erythrocyte membrane into the cytoplasmic space.

We have developed a technique in which transglutaminase is used to measure the penetration of terminal complement proteins across the erythrocyte membrane into the cytoplasmic space. Penetration of a given terminal complement protein into the cytoplasmic space was assessed by labeling the protein of interest with radioactive iodine, forming the complement channel using the labeled protein, adding transglutaminase to only one side of the membrane, and allowing the enzyme to cross-link the susceptible proteins on that side of the membrane. Cross-linking was assessed by measuring the increase in molecular weight of the appropriate molecule on sodium dodecyl sulfate gels under reducing conditions. The results of these experiments indicate that C8 and C9 are rapidly cross-linked to high molecular weight from either the interior or the exterior of the membrane. In order to determine whether the cross-linking mediated by enzyme on the interior was occurring from within the ghosts and not via enzyme that had leaked into the extracellular medium, experiments were performed with dimethylcasein in the extracellular medium. In the presence of this protein, cross-linking of C8 and C9 from outside was negligible. Hence, if cross-linking occurs when transglutaminase is trapped inside the ghosts, it cannot be due to leakage of enzyme, but must be attributable to cross-linking from the inside. The results show that C9 definitely penetrated across the membrane into the intracellular space. With respect to C8, statistical evaluation indicates that C8 probably penetrated into the intracellular space.

Acyltransferases↗

PubMed1995

Mechanism of clofilium block of the human Kv1.5 delayed rectifier potassium channel.

The effect of clofilium on potassium conductance was studied in excised membrane patches from Chinese hamster ovary cells stably transfected with the Kv1.5/hPCN1 delayed rectifier K+ channel gene. Bath application of clofilium resulted in current inhibition, displaying concentration-dependent acceleration of the apparent channel inactivation in both outside-out and inside-out patches. The steady state half-inhibition concentration in inside-out patches was 140 +/- 80 nM (n = 10), which was less than the half-inhibition concentration of 840 +/- 390 nM (n = 10) observed in outside-out patches. Clofilium accelerated apparent current inactivation but did not influence the kinetics of current activation or deactivation. The rate of onset of channel block induced by clofilium was not voltage dependent. In contrast, the rate of recovery from channel block was slower at more hyperpolarized membrane potentials. Elevation of extracellular K+ levels accelerated recovery from channel block without influencing the rate of onset of block. These data suggest that clofilium may induce channel block by an "activation trap" mechanism. Clofilium may be trapped near the conductivity pore so that permeating K+ ions promote recovery from clofilium-induced block.

Animals↗

PubMed1994

Ultrastructural findings in the cochlea of AIDS cases.

Neurotologic manifestations associated with the human immunodeficiency virus (HIV-1) are poorly understood. Eight temporal bones of acquired immunodeficiency syndrome (AIDS) cases were dissected and investigated ultrastructurally to determine if pathogens and pathologic changes were present within the cochlea. Extracellular viral-like particles with morphologic characteristics of HIV-1 were identified on the tectorial membrane in three cases. Numerous viral-like particles with cores appeared trapped within lacunae along the tectorial membrane in one specimen. Intracellular viral-like particles, appearing essentially similar to identified HIV-1 particles of infected lymphocyte cultures, were found within the cytoplasm of connective tissue cells. These same cells had gradients of enlarged viral envelopes forming cisterns that contained cores and varying amounts of cistern particulate. Some sectioned profiles of enlarged cytoplasmic cisterns revealed the attachment and inward protrusion of the core from the cistern wall. Other ultrastructural findings included cytoplasmic globular-dense particulate bodies, aggregates of smaller viral-like particles, and mitochondrial changes. This demonstration of prominent viral-like particles and cochlear pathology may help to explain the neurotologic manifestations associated with HIV-1 infection.

Adult↗

PubMedDate not supplied

Rapid detection of protein aggregates in the brains of Alzheimer patients and transgenic mouse models of amyloidosis.

Extracellular and/or intracellular aggregates are pathological features of many, if not all, neurodegenerative diseases. In Alzheimer disease (AD), extracellular aggregates of beta-amyloid (Abeta) and intracellular aggregates of tau or a-synuclein are key diagnostic markers of the disease. We report here a method to rapidly detect these protein aggregates that relies on size exclusion filtration and immunostaining of trapped material, a method termed filter trapping. We demonstrate that aggregated forms of Abeta and tau are readily trapped in 0.22 microm cellulose acetate filter membranes, which can then be immunostained with specific antibodies in a manner similar to the standard immunoblot. Coupling this method with serial dilution permits a rapid assessment of relative aggregate burden.

Alzheimer Disease↗

PubMed2005

Redefining the facilitated transport of mannose in human cells: absence of a glucose-insensitive, high-affinity facilitated mannose transport system.

Current evidence suggests that extracellular mannose can be transported intracellularly and utilized for glycoprotein synthesis; however, the identity and the functional characteristics of the transporters of mannose are controversial. Although the glucose transporters are capable of transporting mannose, it has been postulated that the entry of mannose in mammalian cells is mediated by a transporter that is insensitive to glucose [Panneerselvam, K., and Freeze, H. (1996) J. Biol. Chem. 271, 9417-9421] or by a transporter induced by cell treatment with metformin [Shang, J., and Lehrman, M. A. (2004) J. Biol. Chem. 279, 9703-9712]. We performed a detailed analysis of the uptake of mannose in normal human erythrocytes and in leukemia cell line HL-60. Short uptake assays allowed the identification of a single functional activity involved in mannose uptake in both cell types, with a K(m) for transport of 6 mM. Transport was inhibited in a competitive manner by classical glucose transporter substrates. Similarly, the glucose transporter inhibitors cytochalasin B, genistein, and myricetin inhibited mannose transport by 100%. Using long uptake experiments, we identified a second, high-affinity component associated with the intracellular trapping of mannose in the HL-60 cells that is not directly involved in the transport of mannose via the glucose transporters. Thus, the transport of mannose via glucose transporters is a process which is kinetically and biologically separable from its intracellular trapping. A general survey of human cells revealed that mannose uptake was entirely blocked by concentrations of cytochalasin B that obliterates the activity of the glucose transporters. The transport and inhibition data demonstrate that extracellular mannose, whose physiological concentration is in the micromolar range, enters cells in the presence of physiological concentrations of glucose. Overall, our data indicate that transport through the glucose transporter is the main mechanism by which human cells acquire mannose.

Biological Transport, Active↗

PubMed2006

Production of extracellular superoxide by human lymphoblast cell lines: comparison of electron spin resonance techniques and cytochrome C reduction assay.

Superoxide production by NADPH oxidases plays an important role in the development and progression of cardiovascular disease (CVD). However, measurement of superoxide (O(2)(-)), a marker of oxidative stress, remains a challenging task in clinical and translational studies. In this study we analyzed O(2)(-) production in cultured human lymphoblast cell lines by three different methods: (a) superoxide dismutase (SOD)-inhibitable cytochrome C reduction, (b) spin trapping of superoxide with 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), and (c) using electron spin resonance (ESR) with the cell-permeable spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH). Lymphocytes were isolated and immortalized by an Epstein-Barr Virus (EBV)-transformation procedure. Superoxide was measured in cultured lymphoblast cell lines at baseline and upon stimulation with phorbol 12-myristate 13-acetate (PMA). Cytochrome C and the spin traps EMPO and DEPMPO detected two to five times less superoxide compared to CMH. Thus, CMH provided the most quantitative measurement of superoxide generation in human lymphoblast cell lines. Superoxide detection with CMH was linear dependent on cell concentration and was inhibited by SOD but not by catalase. Both cell-permeable polyethylene glycol (PEG)-SOD and extracellular Cu,Zn-SOD inhibited O(2)(-) detection by 90% in PMA-stimulated cells, suggesting a predominantly extracellular O(2)(-) generation in human lymphoblasts. Our study describes a new technique for O(2)(-) measurement in cultured human lymphoblasts using ESR and CMH. A highly sensitive in vitro measurement of O(2)(-) in human cell lines would allow investigators to study genotype/phenotype interactions in translational studies.

B-Lymphocytes↗

PubMed1995

Chemopreventive properties and mechanisms of N-Acetylcysteine. The experimental background.

The thiol N-acetylcysteine (NAC), now under clinical trial for cancer chemoprevention both in Europe (project Euroscan) and in the US (National Cancer Institute), has been shown during the past decade to exert protective effects in a variety of experimental test systems. NAC inhibited spontaneous mutagenicity and that induced by a number of chemical compounds and complex mixtures. Moreover, NAC significantly decreased the incidence of neoplastic and preneoplastic lesions induced by several chemical carcinogens in rodents (mice, rats, hamsters), e.g., in lung, trachea, colon, liver, mammary gland, Zymbal gland, bladder and skin. Our studies provided evidence that multiple mechanisms contribute to NAC antimutagenicity and anticarcinogenicity. They include extracellular mechanisms, such as detoxification of reactive compounds due to the nucleophilic and antioxidant properties of NAC, inhibition of nitrosation products, and enhancement of thiol concentration in intestinal bacteria; trapping and enhanced detoxification of carcinogens in long-lived non-target cells, such as erythrocytes and bronchoalveolar lavage cells; mechanisms working in the cytoplasm of target cells, such as replenishment of GSH stores, modulation of metabolism of mutagens/carcinogens, blocking of electrophiles, and scavenging of reactive oxygen species; and nuclear effects, such as inhibition of DNA adduction by metabolites of carcinogens, inhibition of "spontaneous" mutations, attenuation of carcinogen-induced DNA damage, and protection of nuclear enzymes, such as poly(ADP-ribose) polymerase.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcysteine↗

PubMed1998

Pore accessibility during C-type inactivation in Shaker K+ channels.

Shaker K+ channels inactivate through two distinct molecular mechanisms: N-type, which involves the N-terminal domain and C-type that appears to involve structural modifications at the external mouth of the channel. We have tested pore accessibility of the Shaker K+ channel during C-type inactivation using Ba2+ as a probe. We determined that external Ba2+ binds to C-type inactivated channels forming an extremely stable complex; i.e. there is Ba2+ trapping by C-type inactivated channels. The structural changes Shaker channels undergo during C-type inactivation create high energy barriers that hinder Ba2+ exit to either the extracellular solution or to the intracellular solution.

Barium↗

PubMed1999

Effect of exercise training, selenium and vitamin E on some free radical scavengers in horses (Equus caballus).

Physical exercise increases both tissue needs for oxygen and cellular respiration and causes an overproduction of free radicals. When free radical generation exceeds the cell's antioxidant capacity tissue-damage develops due to oxidative stress. Therefore, it appears important to increase the scavenger ability of the tissues. Controlled training and dietary supplements may provide ways of doing this. As a model, we used 3-year-old racehorses (Equus caballus) which underwent a series of different physical exercise trials before and after 70 days of daily training and dietary supplements (vitamin E and selenium). The above treatments were able to increase both red blood cell resistance to the peroxidative stress induced in vitro and the glutathione peroxidase activity in lymphocytes. Moreover, they were also able to decrease malondialdehyde (MDA) concentration in the plasma as well as vitamin E consumption and the mobilisation of low molecular weight antioxidants (total peroxyl-radical trapping) following the physical exercise trials. The results obtained indicated that the training and diet supplements we used were able to significantly increase horse antioxidant defences in both the extracellular fluids and blood cells of our horses, thus decreasing peroxidative phenomena following physical exercise.

Animals↗

PubMed1983

The in vivo tissue disposition and in vitro target-tissue metabolism of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in Syrian golden hamsters.

The tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), labeled with 14C in the carbonyl group, was given i.v. or s.c. to Syrian golden hamsters. Whole-body autoradiography indicated that NNK is distributed evenly in the intra- and extracellular tissue water and reaches all tissues. An accumulation of radioactivity was observed in the stomach contents, probably due to the basicity of NNK and its metabolites, which are trapped in the acid gastric juice. The basicity of these compounds is also responsible for their binding to the eye melanin. A high level of tissue-bound metabolites occurred in the mucosa of the ethmoturbinates, in the lungs and in the trachea, which are the targets for the carcinogenicity of NNK in the hamster. In the lungs and the trachea, the radioactivity was confined to the epithelium of the air-conducting ways (the bronchial, bronchiolar and tracheal epithelium). Microautoradiography of the nasal olfactory mucosa showed the highest labeling over Bowman's glands in the lamina propria mucosae. The liver was the only non-target tissue which retained detectable tissue-bound metabolites in vivo. Levels of in vitro degradation of NNK to tissue bound and unbound metabolites were the highest in the nasal mucosa. This tissue as well as the other target tissues - the lung and the trachea - produced considerable amounts of metabolites which are probably derived from alpha-carbon hydroxylations of NNK. In the liver, on the other hand, the main metabolite was a reduction product of NNK. Our results demonstrate that tumorigenesis is correlated with NNK metabolism and binding in the target tissues and support the hypothesis that alpha-carbon hydroxylation is the mechanism of activation of NNK.

Animals↗

PubMed1996

Sequestration of glutamate-induced Ca2+ loads by mitochondria in cultured rat hippocampal neurons.

1. Buffering of glutamate-induced Ca2+ loads in single rat hippocampal neurons grown in primary culture was studied with ratiometric fluorescent Ca2+ indicators. The hypothesis that mitochondria buffer the large Ca2+ loads elicited by glutamate was tested. 2. The relationship between glutamate concentration and the resulting increase in the free intracellular Ca2+ concentration ([Ca2+]i) reached an asymptote at 30 microM glutamate. This apparent ceiling was not a result of saturation of the Ca2+ indicator, because these results were obtained with the low-affinity (dissociation constant = 7 microM) Ca2+ indicator coumarin benzothiazole. 3. Five minutes of exposure to glutamate elicited concentration-dependent neuronal death detected 20-24 h later by the release of the cytosolic enzyme lactate dehydrogenase into the media. Maximal neurotoxicity was elicited at glutamate concentrations > or = 300 microM. The discrepancy between the glutamate concentration required to evoke a maximal rise in [Ca2+]i and the higher concentration necessary elicit maximal Ca(2+)-triggered cell death suggests that large neurotoxic Ca2+ loads are in part removed to a noncytoplasmic pool. 4. Treatment of hippocampal neurons with the protonophore carbonyl cyanide p-(trifluoro-methoxy) phenylhydrazone (FCCP; 1 microM, 5 min) greatly increased the amplitude of glutamate-induced [Ca2+]i transients, although it had little effect on basal [Ca2+]i. The effect of FCCP was more pronounced on responses elicited by stimuli that produced large Ca2+ loads. Similar results were obtained by inhibition of electron transport with antimycin A1. Neither agent, under the conditions described here, significantly depressed cellular ATP levels as indicated by luciferase-based ATP measurements, consistent with the robust anaerobic metabolism of cultured cells. Thus inhibition of mitochondrial function disrupted the buffering of glutamate-induced Ca2+ loads in a manner that was not related to changes in ATP. 5. Removal of extracellular Na+ for 20 min before exposure to N-methyl-D-aspartate (NMDA) (200 microM, 3 min), presumably reducing intracellular Na+, evoked a prolonged plateau phase in the recovery of the [Ca2+]i transient that resembled the mitochondrion-mediated [Ca2+]i plateau previously observed in sensory neurons. Return of extracellular Na+ immediately after exposure to NMDA increased the height and shortened the duration of the plateau phase. Thus manipulation of extracellular Na+ altered the plateau in a manner consistent with plateau height being modulated by intracellular Na+ levels. 6. In neurons depleted of Na+ and challenged with NMDA, a plateau resulted; during the plateau, application of FCCP in the absence of extracellular Ca2+ produced a large increase in [Ca2+]i. In contrast, similar treatment of cells that were not depleted of Na+ failed to increase [Ca2+]i. Thus Na+ depletion traps Ca2+ within an FCCP-sensitive intracellular store. 7. Glutamate-induced Ca2+ loads are sequestered by an intracellular store that had a low affinity and a high capacity for Ca2+, was released by FCCP, was sensitive to antimycin A1, and was modulated by intracellular Na+ levels. We conclude that mitochondria sequester glutamate-induced Ca2+ loads and suggest that Ca2+ entry into mitochondria may account for the poor correlation between glutamate-induced neurotoxicity and glutamate-induced changes in [Ca2+]i.

Animals↗

PubMed1988

Evidence for Na+/H+ exchange and pH sensitive membrane voltage in cultured bovine corneal epithelial cells.

Two methods were used to investigate cellular ion transport processes in confluent monolayers of cultured bovine corneal epithelial cells: measurements of membrane voltage (V) using conventional microelectrodes, and intracellular pH (pHi) measurements using the pH sensitive absorbance of intracellularly trapped 5 (and 6)-carboxy-4', 5'dimethyl-fluorescein. (1) V averaged -39.2 +/- 0.9 mV (mean +/- SEM, n = 71) with a range of -30 to -59 mV. Increasing extracellular potassium depolarized the cell membrane with a K+-slope of 43.3 mV/decade [K+] (for [K+] between 20 and 80 mM). Intracellular as well as extracellular acidification reversibly depolarized the cell membrane. Depolarization induced by 40 mM K+-pulses was smaller at extracellular pH (pHo) of 6.9 as compared to pHo = 7.9. These findings are compatible with a pH-sensitive K+ conductance. (2) During steady state pHi was 6.96 +/- 0.05 (mean +/- SEM, n = 7). After intracellular acidification, induced by NH4Cl-prepulse technique, pHi was regulated back towards normal steady state pHi. Application of 1 mM amiloride reversibly inhibited pHi recovery. Furthermore, pHi backregulation was inhibited by removing sodium from the extracellular solution. The effect was reversible after readdition of sodium. These findings suggest that a Na+/H+ exchange is present in corneal epithelial cells and participates in pHi backregulation after an intracellular acid load.

Acids↗

PubMed1998

The effects of methamphetamine on the production of free radicals and oxidative stress.

The effects of methamphetamine (METH) on pro-oxidant processes and on the production of reactive oxygen species were examined in vivo in the rat brain. The presence of oxidative damage in striatum, as revealed by the oxidation of lipid, also was assessed via the measurement of the lipid peroxidation product malonyldialdehyde. To elucidate further the mechanisms mediating METH-induced oxidative stress, we studied the possible reversal of the long-term METH-induced decrease in striatal dopamine content by antioxidants through iron chelation and trapping of free radicals. The uric acid concentration in the striata of rats killed 1 hr, but not 24 hr, after a four-injection regimen of METH was increased significantly compared with saline-injected control rats. METH increased the in vivo formation of the hydroxylated products of salicylate and d-phenylalanine, as evidenced by the elevated extracellular concentrations of 2,3 dihydroxybenzoic acid and p-tyrosine, respectively. The local perfusion of the striatum with the iron chelator deferroxamine attenuated the long-term depletions of striatal dopamine content produced by METH. In other experiments, malonyldialdehyde concentrations in incubated striatal homogenates were elevated significantly in METH-treated rats. Finally, pretreatment with the spin trapping agent phenylbutylnitrone before the METH injections attenuated the subsequent long-term depletions in striatal dopamine content. Overall, the results illustrate that METH increases pro-oxidant processes and offer supportive evidence that METH produces oxidative damage. These studies also demonstrate that iron may be involved in mediating the long-term damage to dopamine neurons after repeated administrations of METH.

Animals↗

PubMed2002

The phosphatidylinositol 3-kinase, p38, and extracellular signal-regulated kinase pathways are involved in osteoclast differentiation.

Phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinases (MAPKs) have been implicated in diverse cellular functions, including proliferation, migration, and survival. In this study, we examined the involvement of these kinases in osteoclast differentiation by employing specific inhibitors of the kinases. The osteoclast differentiation was assessed in three different culture systems: a coculture of mouse bone marrow cells with mouse calvarial osteoblasts, a mouse bone marrow cell culture in the presence of receptor activator of NF-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), and a culture of bone-resident osteoclast precursor cells driven by RANKL and M-CSF. LY294002, a specific inhibitor of PI 3-kinase, potently inhibited osteoclast differentiation in all culture systems when assessed by both tartrate-resistant acid phosphatase (TRAP) staining and dentine resorption assays. Inhibition of p38 MAPK by SB202190 resulted in a strong suppression in the exogenous RANKL dependent mouse bone marrow and bone resident precursor cell cultures. Another MAPK pathway inhibitor (PD98059), which blocks the activation of extracellular signal-regulated kinase (ERK) by inhibiting the upstream kinase MAPK-ERK kinase (MEK) 1, exerted an inhibitory effect on osteoclast differentiation only at the highest concentration tested (30 micromol/L) in many cases. Whether the signaling pathways involving these kinases are activated by RANKL was also examined. The RANKL-stimulated phosphorylation of Akt, a downstream target of PI 3-kinase, and that of ERK were observed. RANKL also stimulated the activity of p38. These results suggest that PI 3 kinase, p38, and ERK play roles in osteoclast differentiation, at least in part, by participating in RANKL signaling.

Animals↗

PubMed2001

Neutralization of gating charges in domain II of the sodium channel alpha subunit enhances voltage-sensor trapping by a beta-scorpion toxin.

beta-Scorpion toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3-S4 loop at the extracellular end of the S4 voltage sensor in domain II of the alpha subunit. Here, we probe the role of gating charges in the IIS4 segment in beta-scorpion toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances beta-scorpion toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the beta-scorpion toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from -80 to -140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound toxin. Our results are consistent with a voltage-sensor-trapping model in which the beta-scorpion toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently, neutralization of R850 and R853 removes kinetic barriers to binding of the IIS4 segment by Css IV, and thereby enhances toxin-induced channel activation.

Arginine↗

PubMed2004

Positive selection dictates the choice between kinetic and thermodynamic protein folding and stability in subtilases.

Subtilisin E (SbtE) is a member of the ubiquitous superfamily of serine proteases called subtilases and serves as a model for understanding propeptide-mediated protein folding mechanisms. Unlike most proteins that adopt thermodynamically stable conformations, the native state of SbtE is trapped into a kinetically stable conformation. While kinetic stability offers distinct functional advantages to the native state, the constraints that dictate the selection between kinetic and thermodynamic folding and stability remain unknown. Using highly conserved subtilases, we demonstrate that adaptive evolution of sequence dictates selection of folding pathways. Intracellular and extracellular serine proteases (ISPs and ESPs, respectively) constitute two subfamilies within the family of subtilases that have highly conserved sequences, structures, and catalytic activities. Our studies on the folding pathways of subtilisin E (SbtE), an ESP, and its homologue intracellular serine protease 1 (ISP1), an ISP, show that although topology, contact order, and hydrophobicity that drive protein folding reactions are conserved, ISP1 and SbtE fold through significantly different pathways and kinetics. While SbtE absolutely requires the propeptide to fold into a kinetically trapped conformer, ISP1 folds to a thermodynamically stable state more than 1 million times faster and independent of a propeptide. Furthermore, kinetics establish that ISP1 and SbtE fold through different intermediate states. An evolutionary analysis of folding constraints in subtilases suggests that observed differences in folding pathways may be mediated through positive selection of specific residues that map mostly onto the protein surface. Together, our results demonstrate that closely related subtilases can fold through distinct pathways and mechanisms, and suggest that fine sequence details can dictate the choice between kinetic and thermodynamic folding and stability.

Amino Acid Sequence↗