Cyclodextrins increase membrane tension and are universal activators of mechanosensitive channels

The bacterial mechanosensitive channel of small conductance (MscS) has been extensively studied to understand how mechanical forces are converted into the conformational changes that underlie mechanosensitive (MS) channel gating. We showed that lipid removal by β-cyclodextrin can mimic membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted Escherichia coli MscS, that MscS activation by CDs depends on CD-mediated lipid removal, and that the CD amount required to gate MscS scales with the channel’s sensitivity to membrane tension. Importantly, cholesterol-loaded CDs do not activate MscS. CD-mediated lipid removal ultimately causes MscS desensitization, which we show is affected by the lipid environment. While many MS channels respond to membrane forces, generalized by the “force-from-lipids” principle, their different molecular architectures suggest that they use unique ways to convert mechanical forces into conformational changes. To test whether CDs can also be used to activate other MS channels, we chose to investigate the mechanosensitive channel of large conductance (MscL) and demonstrate that CDs can also activate this structurally unrelated channel. Since CDs can open the least tension-sensitive MS channel, MscL, they should be able to open any MS channel that responds to membrane tension. Thus, CDs emerge as a universal tool for the structural and functional characterization of unrelated MS channels.

Bacterial mechanosensitive (MS) channels have been extensively used as models of ion channel–mediated mechanotransduction (1, 2). They have continually provided novel insights into the biophysical principles that govern ion-channel mechanosensitivity (3–6). While the structurally unrelated MS channels MscL (mechanosensitive channel of large conductance) (4) and MscS (mechanosensitive channel of small conductance) (7) both respond to changes in membrane tension (8–10), at the molecular level, they seem to employ different strategies to convert membrane forces into the conformational changes that underlie channel gating.Escherichia coli MscS is the archetypal member of a large structurally diverse family of ion channels that are expressed in bacteria (11, 12), archaea (13), some fungi (14), plants (15, 16), and eukaryotic parasites (17). This channel gates as a result of membrane tension (10) in accordance with the “force-from-lipids” gating mechanism (6). In response to increases in membrane tension, MscS exhibits complex adaptive gating kinetics (18–20). These kinetic responses may represent two separable processes, adaptation and inactivation (21, 22). In particular, point mutations within transmembrane domain 3 can instigate phenotypes in which adaptation and inactivation are affected differently (18, 23, 24). These complex kinetics are important for the role of this channel as an osmotic safety valve (25). However, since it is currently unknown whether these electrophysiologically separable processes correlate to structurally distinct states, we will refer to them collectively as “desensitization.” In addition, while some data suggest that MscS desensitization is sensitive to the lipid environment (26), this notion still awaits definitive proof.MscL was the first MS channel to be cloned and functionally characterized in a lipid-only environment (8). Members of the MscL family, unlike those of the MscS family, are almost exclusively expressed in archaea and bacteria. After X-ray crystallography revealed the structure of MscL (27), subsequent studies implicated membrane thinning in response to membrane tension as a major driver of MscL gating (3, 28).To fully understand the structural basis of the gating transitions in MscL and MscS, one must first find a way to apply a gating stimulus to the channels in a lipidic environment that is compatible with structural studies. This is, of course, less challenging when considering ligand-gated channels (29–31), for which the stimulus is a defined molecule that can readily be applied to visualize the resulting changes in protein conformation. For MS channels, until recently, only spectroscopic approaches, such as electron paramagnetic resonance spectroscopy (32, 33) and Förster resonance energy transfer spectroscopy (34, 35), were available to provide structural insights into their gating in response to changes in forces in their lipid environment. Other approaches had been confined to the use of activators (36) or mutations (37, 38). We recently demonstrated that lipid removal by β-cyclodextrin can mimic membrane tension in membrane-scaffold protein-based lipid nanodiscs, providing novel insights into the structural rearrangements that underlie MscS channel gating in response to membrane tension (39). The idea was that, as long as the surface area would not change and the lipids would not be replaced, β-cyclodextrin–mediated lipid removal from a membrane would result in the remaining lipids having to cover a larger surface area. This increase in “area-per-lipid” would result in a corresponding increase in membrane tension (40, 41) that would be experienced by integral membrane proteins incorporated in that membrane.Cyclodextrins (CDs) are a family of cyclic glucose oligomers with a cone-like three-dimensional architecture characterized by a polar external surface and a hydrophobic cavity (42, 43). α-, β-, and γ-CD contain six to eight glucose units, respectively. As the number of units increases, so does the diameter of the hydrophobic cavity (5 to 8 Å) (43). These compounds are of broad utility, as the hydrophobic cavity can chelate a plethora of small lipophilic molecules (44, 45). CDs can also form complexes with fatty acids and phospholipids (46). CDs have thus been widely used to remove lipids from native cell membranes (47, 48) and from model membranes (49, 50). This removal of lipids has already been directly linked to increases in membrane tension even in intact cellular environments (51). CDs also exhibit differential lipid selectivity. For example, α-CD has the selectivity profile of phosphatidylserine > phosphatidylethanolamine >> phosphatidylcholine (52). The methylated version of β-CD (mβ-CD) shows selectivity toward cholesterol at low concentrations and has been widely used to selectively remove or add cholesterol to cell membranes (48, 53–55). In addition to the headgroup, CDs also preferentially chelate unsaturated lipids and those containing shorter acyl chains (56, 57).Here, we show that all members of the CD family (α, β, and γ) can activate E. coli MscS in liposomal membranes. Even the methylated version of β-CD, which is widely used for its cholesterol selectivity, can activate E. coli MscS. Congruent with lipid removal increasing tension, the CD amount required for the activation of an MS channel depends on its tension sensitivity. Importantly, as a control, cholesterol-loaded CDs do not activate MscS. Our studies also clearly establish that MscS desensitization is modified by the lipid environment. Moreover, we show that CD-mediated lipid removal causes a concentration- and time-dependent increase in the tension in excised membrane patches and that the resulting tension can become sufficiently high to activate the structurally unrelated MS channel MscL that gates at membrane tensions immediately below the lytic limit of membranes. Two-dimensional (2D) class averages of nanodisc-embedded MscL obtained by cryo–electron microscopy (cryo-EM) indicate that β-CD treatment results in membrane thinning and channel expansion. The fact that CD activates MscL, which opens immediately below the lytic tension of the membrane, suggests that all other MS channels (which are all more sensitive to membrane tension) should also open in response to CDs. These data suggest that CDs will be of broad utility for the structural and functional characterization of structurally diverse MS channels, including Piezo channels (58, 59), two-pore domain K⁺ channels (40, 60), and OSCA channels (61, 62), all of which are known to respond to membrane forces.     

N-Glucuronidation of the antiepileptic drug retigabine: results from studies with human volunteers, heterologously expressed human UGTs, human liver, kidney, and liver microsomal membranes of Crigler-Najjar type II

Retigabine (D-23129), an N-2-amino-4-(4-fluorobenzylamino)phenylcarbamine acid ethyl ester, is a novel antiepileptic drug which is currently in phase II clinical development. This drug undergoes N-glucuronidation. We aimed to identify the principal enzymes involved in the N-glucuronidation pathway of retigabine and compared our findings with those obtained from human liver (a pool of 30 donors) and kidney microsomes (a pool of 3 donors) and with results from a human absorption, distribution, metabolism, and excretion study upon administration of 200 microCi of [(14)C]-D-23129. Essentially, microsomal assays with UGT1A1 produced only one of the 2 N-glucuronides, whereas UGT1A9 is capable of forming both N-glucuronides. The rates of metabolism for UGT1A9, human liver microsomes, and UGT1A1 were 200, 100, and 100 pmol N-glucuronide per minute per milligram of protein, respectively. At the 50 micromol/L uridine diphosphate glucoronic acid (UDPGA) concentration, UGT1A4 also catalyzed the N-glucuronidation of retigabine, the rates being approximately 5 and 6 pmol/(min.mg protein). With UGT1A9, the production of metabolites 1 and 2 proceeded at a K(m) of 38+/-25 and 45+/-15 micromol/L, whereas the K(m) for retigabine N-glucuronidation by human liver microsomal fractions was 145+/-39 micromol/L. Furthermore, a V(max) of 1.2+/-0.3 (nmol/[min.mg protein]) was estimated for human liver microsomes (4 individual donors). We investigated the potential for drug-drug interaction using the antiepileptic drugs valproic acid, lamotrigine, the tricyclic antidepressant imipramine, and the anesthetic propofol. These are commonly used medications and are extensively glucuronidated. No potential for drug-drug interactions was found at clinically relevant concentrations (when assayed with human liver microsomes or UGT1A9 enzyme preparations). Notably, the biosynthesis of retigabine-N-glucuronides was not inhibited in human liver microsomal assays in the presence of 330 micromol/L bilirubin, and glucuronidation of retigabine was also observed with microsomal preparations from human kidney and Crigler-Najjar type II liver. This suggests that lack of a particular UDP-glucuronosyltransferase (UGT) isoform (eg, UGT1A1 in kidney) or functional loss of an entire UGT1A gene does not completely abolish disposal of the drug. Finally, chromatographic separations of extracts from microsomal assays and human urine of volunteers receiving a single dose of (14)C-retigabine provided clear evidence for the presence of the 2 N-glucuronides known to be produced by UGT1A9. We therefore suggest N-glucuronidation of retigabine to be of importance in the metabolic clearance of this drug.     

Cardiopulmonary effects of intravenous prostaglandin E1 during experimental one-lung ventilation

BACKGROUND: One-lung ventilation greatly improves operating conditions during thoracic surgery. Serious disadvantages of one-lung ventilation are hypoxaemia and increased pulmonary vascular resistance. Prostaglandins, like prostaglandin I2 (PGI2), are potent pulmonary vasodilators but may also influence venous admixture and systemic circulation. Since the lung is capable of extensive degradation of prostaglandin E1 (PGE1) but not of PGI2, PGE1 might affect systemic circulation to a lesser degree. Hence, we studied the effects of intravenous PGE1 on systemic and pulmonary circulation and on oxygenation during one-lung ventilation. METHODS: Lateral thoracotomy and cross-clamping of the left main stem bronchus was performed in twelve anaesthetised and ventilated pigs. Animals were cannulated with arterial, central venous and fast response thermodilution pulmonary artery catheters for haemodynamic measurements. PGE1 was administered with infusion rates of 25, 50, and 100 ng x kg (-1) x min (-1) during one-lung ventilation. RESULTS: All doses of PGE1 significantly decreased pulmonary vascular resistance and mean pulmonary artery pressure. However, a comparable significant reduction in systemic vascular resistance and mean arterial pressure was found. Arterial oxygen tension and venous admixture showed a slight but significant deterioration. Oxygen delivery remained unchanged or increased since the cardiac index increased. CONCLUSION: During one-lung ventilation in the pig, infusion of PGE1 significantly decreased pulmonary vascular resistance and pulmonary artery pressure but failed to achieve selective pulmonary vasodilation.     

Dose-dependent induction of IL-6 by plant-derived proteases in vitro

Oral administration of proteases such as bromelain and papain is commonly used in patients with a wide range of inflammatory conditions, but their molecular and cellular mechanisms of action are still poorly understood. The aim of our study was to investigate the impact of these proteases on the release of interleukin-6 (IL-6) and other cytokines in the recently described modified mixed lymphocyte culture (MMLC) test system which is based on the mutual interaction of cells of the innate and adaptive immunity. Bromelain and papain enhanced IL-6 production dose-dependently up to 400-fold in MMLC before and up to 30-fold after neutralization of LPS content of proteases using polymyxin B, indicating that IL-6 induction by protease treatment was attributable to both protease action and LPS content of enzyme preparations. The production of IFNgamma and IL-10 was not altered by bromelain or papain, indicating a selective and differential immune activation. Both proteases impaired cytokine stability, cell proliferation and expression of cell surface molecules like CD14 only marginally, suggesting no impact of these mechanisms on protease-mediated cytokine release. These findings might provide the mechanistic rationale for the current use of proteases in wound healing and tissue regeneration since these processes depend on IL-6 induction.     

Presynaptic plasticity in an immature neocortical network requires NMDA receptor activation and BDNF release

Activity-dependent developmental maturation of the neocortical network is thought to involve the stabilization and potentiation of immature synapses. In particular, N-methyl-d-aspartate (NMDA) receptor-dependent long-term plasticity that is expressed presynaptically appears to be crucial for the selection of functionally adequate synapses. However, presynaptic expression of long-term plasticity in neocortical neurons has mainly been studied indirectly by electrophysiological techniques. Here we analyzed presynaptic plasticity directly by repeated imaging of actively cycling presynaptic vesicles with the styryl dye FM4-64 in cultured neocortical neurons at 34 degrees C. To monitor long-term changes, stimulation-induced saturating FM4-64 staining and subsequent destaining was performed twice with an interval of 1.5 h between stainings and with the first staining serving as a plasticity stimulus. In the vast majority of presynaptic release sites, we found an increase in the mean fluorescence intensity after the second staining indicating an enhanced number of cycling synaptic vesicles. Most intriguingly, we additionally observed the appearance of new active release sites. As demonstrated by the addition of the NMDA receptor antagonist d-2-amino-5-phosphonopentanoic acid (d-AP5), both plasticity phenomena were strictly dependent on NMDA receptor activation. This suggests that a subpopulation of release sites was functionally silent during the first round of staining. Moreover, we studied a potential role of brain-derived neurotrophic factor (BDNF) in this type of presynaptic plasticity by imaging BDNF-deficient neocortical neurons. The increase in fluorescence intensity was strongly inhibited in BDNF-knockout neurons and was absent in wild-type neurons in the presence of BDNF scavenging trkB receptor bodies. These results indicate that BDNF might play an important role as a plasticity-related messenger molecule in neocortical neurons.     

Effect of neoadjuvant anthracycline–taxane-based chemotherapy in different biological breast cancer phenotypes: overall results from the GeparTrio study

In order to explore the effect of neoadjuvant chemotherapy (NACT) on clinical mid-course and pathological complete response (pCR) at surgery in different biological breast cancer subtypes. The GeparTrio study included 2,072 patients with operable or locally advanced breast cancer. After two cycles with docetaxel, doxorubicin and cyclophosphamide (TAC) patients were randomized according to their clinical response. Clinical and biological factors were assessed for predicting clinically mid-course response and pCR at surgery. The overall pCR rate, defined as no invasive residuals in breast and axilla, was 20.5%. The highest pCR rate of 57% was observed in patients below 40 years of age with triple negative or grade 3 tumors. Independent factors for mid-course response and pCR were: young age, non-T4 tumors, high grade, and hormone receptor status, the strongest single predictive factor. Within the biological subtypes, grading was an independent factor to predict pCR for luminal tumors, clinical tumor stage for the HER2 like tumors and age for the triple negative ones. Grading gave independent information for mid-course response within the triple negative group. No factor predicted mid-course response within the other groups. Grading and age can identify subgroups within the luminal and triple negative patients who have an increased benefit from NACT.     

Eye movements during reading of randomly shuffled text

In research on eye-movement control during reading, the importance of cognitive processes related to language comprehension relative to visuomotor aspects of saccade generation is the topic of an ongoing debate. Here we investigate various eye-movement measures during reading of randomly shuffled meaningless text as compared to normal meaningful text. To ensure processing of the material, readers were occasionally probed for words occurring in normal or shuffled text. For reading of shuffled text we observed longer fixation times, less word skippings, and more refixations than in normal reading. Shuffled-text reading further differed from normal reading in that low-frequency words were not overall fixated longer than high-frequency words. However, the frequency effect was present on long words, but was reversed for short words. Also, consistent with our prior research we found distinct experimental effects of spatially distributed processing over several words at a time, indicating how lexical word processing affected eye movements. Based on analyses of statistical linear mixed-effect models we argue that the results are compatible with the hypothesis that the perceptual span is more strongly modulated by foveal load in the shuffled reading task than in normal reading. Results are discussed in the context of computational models of reading.